multichannel systems IFB-C Interface Board Multiboot System User Manual

multichannel systems IFB-C Interface Board Multiboot System

Specifications

• Product Name: CMOS-MEA5000-System
• Manufacturer: Multi Channel Systems MCS GmbH
• Intended Use: Research and laboratory work
• Not intended for medical use on humans

Product Usage Instructions

Hardware Setup:

1. Ensure the device is not exposed to direct sunlight.
2. Do not obstruct the device or place it on top of other heat-producing equipment.
3. Allow for proper air circulation around the device.

Installation of the Software:

1. Follow the software installation guide provided by the manufacturer.
2. Ensure that the system meets the minimum requirements for software installation.
3. Activate the software using the provided license key or activation process.

FAQs

• Q: Can the CMOS-MEA5000-System be used for medical purposes?
  • A: No, the product is not intended for medical use on humans. It is designed for research and laboratory work only.
• Q: What should I do if I encounter malfunctions with the device?
  • A: If you experience any malfunctions that could affect safety, stop using the device immediately and contact qualified technicians for assistance.
• Q: Where can I find the guarantee and liability information for the product?
  • A: The general conditions of sale and delivery, as well as guarantee and liability information, can be found online at http://www.multichannelsystems.com/sites/multichannelsystems.com/files/documents/TermsandConditions.pdf

CMOS-MEA5000-System
USER MANUAL

CMOS-MEA5000-System · Publication 20231220 · www.multichannelsystems.com

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IMPRINT
IMPRINT
Information in this document is subject to change without notice. No part of this document may be reproduced or transmitted without the express written permission of Multi Channel Systems MCS GmbH. While every precaution has been taken in the preparation of this document, the publisher and the author assume no responsibility for errors or omissions, or for damages resulting from the use of information contained in this document or from the use of programs and source code that may accompany it. In no event shall the publisher and the author be liable for any loss of profit or any other commercial damage caused or alleged to have been caused directly or indirectly by this document.
© 2022 Multi Channel Systems MCS GmbH. All rights reserved.
Printed: 13.07.2022
Multi Channel Systems MCS GmbH Aspenhaustraße 21 72770 Reutlingen Germany Phone +49-71 21-909 25 – 0 Fax +49-71 21-909 25 -11 sales@multichannelsystems.com www.multichannelsystems.com
Microsoft and Windows are registered trademarks of Microsoft Corporation. Products that are referred to in this document may be either trademarks and/or registered trademarks of their respective holders and should be noted as such. The publisher and the author make no claim to these trademarks.

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SAFETY AND WARRANTY

IMPORTANT SAFETY ADVICE

Warning: Make sure to read the following advice prior to installation or use of the device and the software. If you do not fulfil all requirements stated below, this may lead to malfunctions or breakage of connected hardware, or even fatal injuries.
Warning: Always obey the rules of local regulations and laws. Only qualified personnel should be allowed to perform laboratory work. Work according to good laboratory practice to obtain best results and to minimize risks.
The product has been built to the state of the art and in accordance with recognized safety engineering rules.
The device may only
be used for its intended purpose; be used when in a perfect condition.
Improper use could lead to serious, even fatal injuries to the user or third parties and damage to the device itself or other material damage.
Warning: The device and the software are not intended for medical uses and must not be used on humans. MCS assumes no responsibility in any case of contravention.
Malfunctions which could impair safety should be rectified immediately.
Grounding
This product is grounded through the grounding conductor on the power cord. To avoid electric shock, the grounding conductor must be connected to earth.
Orient the Equipment Properly
Do not orient the equipment so that it is difficult to manage the disconnection device.
High Voltage
Electrical cords must be properly laid and installed. The length and quality of the cords must be in accordance with local provisions.
Only qualified technicians may work on the electrical system. It is essential that the accident prevention regulations and those of the employers’ liability associations are observed.
· Each time before starting up, make sure that the power supply agrees with the specifications of the product. · Check the power cord for damage each time the site is changed. Damaged power cords should be replaced immediately and
may never be reused.
· Check the leads for damage. Damaged leads should be replaced immediately and may never be reused. · Do not try to insert anything sharp or metallic into the vents or the case. · Liquids may cause short circuits or other damage. Always keep the device and the power cords dry. Do not handle it with wet
hands.
Requirements for the Installation
Make sure that the device is not exposed to direct sunlight. Do not place anything on top of the device, and do not place it on top of another heat producing device, so that the air can circulate freely.
Explanation of the Symbol used

Caution / Warning

DC, direct current

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SAFETY AND WARRENTY
Guarantee and Liability
The general conditions of sale and delivery of Multi Channel Systems MCS GmbH always apply.
They can be found online at http://www.multichannelsystems.com/sites/multichannelsystems.com/files/documents/Terms and Conditions.pdf Multi Channel Systems MCS GmbH makes no guarantee as to the accuracy of any and all tests and data generated by the use of the device or the software. It is up to the user to use good laboratory practice to establish the validity of his / her findings.
Guarantee and liability claims in the event of injury or material damage are excluded when they are the result of one of the following:
· Improper use of the device. · Improper installation, commissioning, operation or maintenance of the device. · Operating the device when the safety and protective devices are defective and/or inoperable. · Non-observance of the instructions in the manual with regard to transport, storage, installation, commissioning, operation
or maintenance of the device.
· Unauthorized structural alterations to the device. · Unauthorized modifications to the system settings. · Inadequate monitoring of device components subject to wear. · Improperly executed and unauthorized repairs. · Unauthorized opening of the device or its components. · Catastrophic events due to the effect of foreign bodies or acts of God.
Operator’s Obligations
The operator is obliged to allow only persons to work on the device, who
· are familiar with the safety at work and accident prevention regulations and have been instructed how to use the device; · are professionally qualified or have specialist knowledge and training and have received instruction in the use of the device; · have read and understood the chapter on safety and the warning instructions in this manual and confirmed this with their
signature.
It must be monitored at regular intervals that the operating personnel are working safely. Personnel still undergoing training may only work on the device under the supervision of an experienced person.

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INTRODUCTION

INTRODUCTION Welcome to the CMOS-MEA5000-System
Multi Channel Systems is proud to present the CMOS-MEA5000-System. Based on the complementary metal-oxide semiconductor technology, it opens up new possibilities in electrophysiological research. With more than 4000 recording sites, each of them sampled at up to 25 kHz, the chip allows extracellular recordings at a very high spatio-temporal resolution. By including amplification on the chip itself, noise is minimized and a high signal quality is guaranteed. Stimulation is provided via 1024 stimulation sites included in the chip and the stimulus generator in the headstage. The CMOS-MEA5000-System consists of three components, which are all designed to be efficient and powerful, while maintaining a small footprint. CMOS-Chip The chip is based on complementary metal oxide semiconductor (CMOS) technology, facilitating fast, high resolution imaging of electrical activity. The chip is equipped with a culture or slice chamber to house your sample, while allowing the use of a microscope. Headstage The core of the system is the headstage. It samples the data coming from the chip at 25 kHz per channel. Besides A/D conversion and amplification, the headstage also houses a three-channel stimulator. You can freely design the stimulation patterns via software and select each of the 1024 stimulation sites. Interface Board The interface board IFB-C offers the USB 3.0 interface to transfer the recorded data to a computer. Moreover, it has analog and digital in- and outputs for synchronization with other instruments. Connect the headstage via iX cable to the interface board. Connect the interface board via USB-C cable to the data acquisition computer. Connect the interface board to the power outlet. Ground the interface board if necessary. Please use the ground socket on the rear panel of the IFB-C. Computer with Software The software CMOS-MEA-Control and CMOS-MEA-Tools are programmed specifically for the CMOS-MEA5000-System. It facilitates a real- time activity overview on the complete chip with the ability to zoom in and various tools to analyze the data.

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HARDWARE

HARDWARE Headstage
There are three hardware components for the CMOS-MEA5000-System available, the headstage, the interface board and the data acquisition computer. If necessary, you can use a temperature controller additionally. For setting up and connecting the CMOS-MEA5000-System, please read the next chapter “Hardware Setup” on page 14. The headstage samples data coming from the 4225 sensors on the chip at 25 kHz per channel. Besides A/D conversion and amplification, the headstage also houses a three-channel stimulator. You can freely design three stimulation patterns via software and select each of the 1024 stimulation sites. Data transfer is provided via iX cable from the headstage to the interface board. The USB-C cable connection arranges fast data transfer from the interface board to the data acquisition computer. Connect a temperature controller TC to the headstage, if necessary. Insert the test model probe or the CMOS-MEA-Chip in correct orientation into the provided area in the headstage. The round edge of the probe or MEA has to be in the front on the left side, when looking directly to the open headstage. This way the CMOS- Control software displays the electrodes in columns and rows as shown on the scheme below. Electrode No 1 is placed on the left lower round edge of the chip and electrode No 4225 is placed in the upper right edge.
Chip Orientation inside the Headstage If you are stimulating with light or extra electrodes from outside the chip, or if the orientation of the tissue is important, please be aware that the line of vision is from the headstage side where the connector to the IFB is located.

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HARDWARE
CMOS-MEA Chip

The chip is based on complementary metal oxide semiconductor (CMOS) technology, facilitating fast, high-resolution imaging of electrical activity. The chip is equipped with a culture chamber to house your sample, while allowing the use of a microscope.
The CMOS-MEA array is an active device, in contrast to the passive MEAs. It needs to be powered up and down properly, or it will be damaged.
Warning: Before opening the CMOS-MEA headstage for removing the CMOS-MEA chip, it is necessary to power the chip down, otherwise the chip will be destroyed!
CMOS sensor arrays are light sensitive. During recording the sensors need constant light conditions. This is easy to obtain by covering the CMOS array with an appropriate dark chamber “CMOS-DC”.

CMOS-DC

CMOS-TH

Please use the CMOS tissue holder “CMOS-TH”to keep acute slices in place. Available for CMOS-MEA chip SCG and CCM.

The CMOS-MEA chip has a 65 x 65 layout and is available with 16 m or 32 m interelectrode distance (center to center). The electrode diameter is always 8 m. Between the recording electrodes, there is a grid of 32 x 32 bigger stimulation sites. Summarizing, you can record from 4225 electrodes and stimulate your sample at 1024 sites. The chip is coated with a planar oxide, similar to glass, enhancing
the biocompatibility and biostability. Please see the electron micrograph of the CMOS-MEA chip surface (NMI Reutlingen, Germany) and
the schema of the chip below.

The 16 m interelectrode distance chip offers the highest resolution. With the high number of electrodes, you can record from a large surface (1 mm² @ 16 m distance, 4 mm² @ 32 m distance). Thereby, you can see the signals from every single cell and even the signal propagation along an axon, while still getting an overview on your complete sample.
Your data is sampled at up to 25 kHz per channel. Thus, no signal is lost – even axonal spikes are displayed and recorded thoroughly. Together with the A/D conversion at 14 bit, the system ensures accurate and precise data.

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HARDWARE
At the moment two types of culture chambers are available for CMOS-MEAs: One for cell cultures and one for acute slices. In both type of chips a ground electrode is already integrated.
CCM Culture chamber for cell cultures with MEA-MEM lid.
SCG Slice chamber. A CMOS-MEA chip with a SCG Slice chamber with advanced layout for laminar flow. The warranty of a CMOS-MEA chip is six months from the date of delivery.

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HARDWARE
Interface Board IFB-C
The multiboot interface board IFB-C facilitates operation of all MCS in vitro and in vivo headstages within the entire 2100 amplifier solution suite. This suite includes: MEA2100-HS, Multiwell-MEA-HS, CMOS-MEA-HS, MEA2100-Beta- Screen-HS, W2100-HS and ME2100-HS. The modular 2100 amplifier solution suite design makes it easy to modify your lab equipment generally with modest hardware upgrade investments. Front Panel
SYNC Out / In Two or more interface boards IFB-C can be daisy-chained by use of the SYNC Out / SYNC In connectors. All daisy-chained interface boards run on the same clock ­ to allow perfectly synchronized recordings in large settings. Analog Channels Up to eight Analog In channels are available via 10-pin connector. Please read chapter 10-Pin Connector for Analog IN in the Appendix for more information about the pin layout. The additional analog inputs are intended for recording additional information from external devices, for example, for recording patch clamp in parallel to the MEA recording. Analog Channels 1 and 2 Two of these eight analog channels (Analog In No 1 and No 2) are separately available via Lemo connectors on the front panel of the interface board IFB-C. Two Status LEDs The status LEDs indicate the link status of HS 1 and / or HS 2. They light up when one or both headstages are connected to the IFB-C interface board via iX-industrial cable.

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HARDWARE
Digital IN / OUT A Digital IN / OUT for 16 digital in- and output bits is available (Honda-PCS-XE68LFD) on the rear panel of the interface board. On the front panel four Digital IN and four Digital OUT bits are also accessible via Lemo connector (DIG IN bit 0 to bit 3 and DIG OUT bit 0 to bit 3). The Digital OUT delivers TTL pulses with 3.3 V or 5 V. The voltage can be switched between these two voltages with the software IFB-Control, please read chapter “IFB- Control” in the Appendix.

If access to more bits of the DIG IN / OUT channel is required, it is necessary to connect a Digital IN / OUT extension Di/o board with a 68-pin standard cable. This Di/o board is available as optional accessory. Ground If an additional ground connection is needed, you can connect this plug with an external ground using a standard common jack (4 mm).
Rear Panel
Toggle Switch On / Off Toggle switch for turning the device on and off. The CMOS-MEA5000-System is switched to status “ON” when the toggle switch is switched to the left. The device is switched “OFF” when the toggle switch is switched to the right. If the system is “ON”, and the device is connected to the power line, the Power LED on the front panel of the interface board should light up. If not, please check the power source and cabling. Power Connect the power supply unit here. This power supply powers both, the headstage and the interface board of the CMOS-MEA5000System. The device needs 24 V and 2.5 A / 65 W. Ground If an additional ground connection is needed, you can connect this plug with an external ground using a standard common jack (4 mm).

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HARDWARE
Digital IN / OUT
A Digital IN / OUT for 16 digital in- and output bits is available via Honda- PCS-XE68LFD connector. Please read chapter Digital IN / OUT Connector in the Appendix for more information about the pin layout of the connector. The Digital IN / OUT connection accepts or generates standard TTL signals. The Digital OUT delivers TTL pulses with 3.3 V or 5 V. The voltage can be switched between these two levels with the software IFB-Control, please read chapter “IFB-Control” in the Appendix.
TTL stands for Transistor-Transistor Logic. A TTL pulse is defined as a digital signal for communication between two devices. A voltage between 0 V and 0.8 V is considered as a logical state of 0 (LOW), and a voltage between 2 V and 3.3 V or 5 V means 1 (HIGH).
The Digital OUT allows generating a digital signal with up to 16 bits and read it out, for example, by using a Digital IN / OUT extension Di/o board. You can utilize this digital signal to control and synchronize other devices with the MEA2100-Beta-Screen-System.
Bit 0 to 3 of the Digital OUT are separated and available as Lemo connector DIG OUT 0 to 3 on the front panel of the interface board. So, the Di/o extension is only necessary if more than four trigger inputs or outputs are needed.
The Digital IN can be used to record additional information from external devices as a 16 bit encoded number. The Digital IN is most often used to trigger recordings with a TTL signal. The 16 bit digital input channels is a stream of 16 bit values. The state of each bit (0 to 15) can be controlled separately. Standard TTL signals are accepted as input signals on the digital inputs.
Warning: A voltage that is higher than +3.3 Volts or +5 Volts or lower than 0 Volts, that is, a negative voltage, applied to the digital input would destroy the electronics. Make sure that you apply only TTL pulses (0 to 3.3 V or 5 V) to the digital inputs.
Auxiliary Channels
Two reserve auxiliary channels are available for future use. They have no function at the moment.
Audio OUT
The “Audio OUT” function is not available for the CMOS-MEA5000-System.
DSP JTAG Connector
The JTAG connector is used to program the digital signal processor DSP for real-time feature. This feature is not in use in CMOS-MEA5000Systems.
HS / SCU
Sockets for connecting up to two CMOS-MEA-System headstages via iX-industrial cable(s), type B.
USB-C Connectors B and A
Both USB-C connectors are used to transfer the amplified and digitized data from all data channels and the additional digital and analog channels to any connected data acquisition computer via USB-C cable. Connector A corresponds with connector iX input 1, and connector B with iX input 2. If both iX inputs are used, also both USB-C connections must be used. Both USB cables must be connected to different USB-C ports of the computer, do not use an USB hub! Only use high grade USB-C cables, as provided with the system.
Important: It is recommended to connect the USB-C cable direct to the USB 3.0 port of the computer. Do not use an USB hub!
Data Acquisition Computer
The software CMOS-MEA-Control was programmed specially for the CMOS- MEA5000-System. It facilitates a real-time activity overview on the complete chip with the ability to zoom in and various tools to analyze the data. Please read chapter “CMOS-MEA-Control” for information about the software.
The data acquisition computer is provided by Multi Channel Systems MCS GmbH. The operation system Windows ® 10 or 8.1 is necessary.
Due to the huge amount of recorded data, a computer with low performance may lead to performance problems; therefore, Multi Channel Systems provides an up- to-date computer with USB 3.0 connection and Intel chip set. Use a SSP hard drive for recording and a second hard drive for backup. At full frame sampling with maximum sampling rate the currently used 1TB SSD drive will hold about one hour of continuous recording only!

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HARDWARE SETUP
HARDWARE SETUP Please follow the instructions: 1. Connect the CMOS-MEA5000 amplifier via iX-industrial cable, type B to the IFB-C interface board. Please use the plug in labelled
with “1” on the back of the interface board. 2. Connect the IFB-C via power unit to the power outlet. 3. Connect the IFB-C via USB-C to the computer. Use the USB-C plug in labelled with “A” on the backside of the interface board.
4. Connect the IFB-C via USB-C cable to the backside of the computer. It is mandatory to use the designated USB 3.0 port, please see the picture below!
Important: It is necessary to connect the interface board IFB-C of the CMOS- MEA5000-System to an Intel ® USB 3.0 port. Otherwise, you risk data loss.
Please consider the error message when starting the CMOS-MEA-Control software for the first time. Important: Make sure that all paths for recording files go to the SSD drive otherwise no recording is possible! 5. Connect the temperature controller TC via USB 2.0 high speed cable to one of the USB 2.0 ports and via power unit to the
power outlet. 6. Connect the heating element of the CMOS headstage via provided cable to the temperature controller.

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HARDWARE SETUP
Installation of the Software
System Requirements
If you have purchased the CMOS-MEA5000-System with PC everything is preinstalled and tested. If you purchase a system without a PC please make sure that the computer meets our specifications relating processor, memory, hard disk, etc. Please contact Multi Channel Systems MCS GmbH or your local retailer.
Software: One of the following Microsoft Windows ® operating systems is required: Windows 10 or 8.1, 64 Bit (English and German versions supported) with the NT file system. Other language versions may lead to software errors.
Due to the amount of recorded data, a computer with low performance may lead to performance problems; therefore, Multi Channel Systems MCS GmbH recommends an up-to-date computer. Please contact MCS or your local retailer for more information on recommended computer hardware specification.
Important: Because of the huge amount of acquired data (up to 220 MByte per second), make sure that all paths for recording files go to the SSD drive, otherwise no recording is possible!
Please note that there are sometimes hardware incompatibilities of the data acquisition system and computer components; or that an inappropriate computer power supply may lead to artefact signals.
Recommended Operating System Settings
The following automatic services of the Windows operating system interfere with the data storage on the hard disk and can lead to severe performance limits in CMOS-MEA-Control. These routines were designed for use on office computers, but are not very useful for a data acquisition computer.
· Deselect “Windows Indexing Service” for data SSD and HD disks, the system hard drive is not included. · Switch off the sleep mode for displays and HD disks. · Power Options: Power scheme: High performance. Never turn on system standby. · Turn off “Optimize hard disk when idle”, the automatic disk fragmentation. · Turn off the Screen Saver and do not use a virus scanner during experiment. · It is also not recommended to run any applications in the background when using CMOS-MEA-Control. Remove all applications
from the “Autostart” folder.
Important: Please make sure to have full control over your computer as an administrator. Otherwise, it is possible that the installed software does not work properly.
Doubleclick the CMOS-MEA-Control.exe on the installation volume. The installation assistant will show up and guide you through the installation procedure. Follow the instructions of the installation assistant.
Note: During installation, all drivers are installed and if necessary, the firmware of the CMOS-MEA5000 hardware is updated. Please do not interrupt the firmware update.

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TESTING THE CMOS-MEA5000-SYSTEM
Testing the CMOS-MEA5000-System Functional Tests of the CMOS-MEA5000-System with the Test Model Probe
Please read also the datasheet “Test-CMOS-MEA” in the Appendix. Insert the “Test Model Probe” in correct orientation and close the headstage. The round edge of the Test-CMOS-MEA or the CMOS-MEA chip has to be in the front on the left side when looking directly to the open amplifier. The provided test model probe simulates a CMOS-MEA chip with a resistor of 100 k and a 10 p capacitor between ground and each row of the 65 x 65 electrodes in the grid. It can be used for testing the noise level of a CMOS-MEA5000-System, for a test of calibration and for testing the internal stimulators. CMOS-MEA test model probes and CMOS-MEA chips are active devices and must be switched on and shot down properly, otherwise they might suffer damage.
Please click the MEA array icon in the “Data Source” window. CMOS chips must be calibrated before each use. The calibration runs automatically and usually takes two to three minutes. Please do not interrupt the process, until the final message “Finished automatic system calibration” appears. The “Test CMOS- MEA” simulates the calibration of the chip. The ,,ODD” cable supports the odd- numbered channels and the ,,EVEN” cable supports the even-numbered channels. For the intention to check the noise level of a CMOS-MEA5000-System without external signals, please connect the cable soldered to the ,,ODD” connector to the input connector of the even-numbered channels and the cable soldered to the ,,EVEN” connector to the ,,BATH” input. Connected in this way, the signal input supports all 65 rows of the 65 x 65 layout of the CMOS-MEA chip at a time and simulates also the calibration of the bath. Disconnect the “ODD” connector to see the “EVEN” numbered electrodes only and vice versa to see the “ODD” numbered electrodes only.

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TESTING THE CMOS-MEA5000-SYSTEM
Test of the internal Stimulators The Test-CMOS-MEA probe is equipped with three additional connectors to test the internal stimulation, ,,STG1″, ,,STG2″ and ,,STG3″. The stimuli are color coded in the CMOS-MEA-Control software: Stimulus 1 is indicated in green color, Stimulus 2 is indicated in blue and Stimulus 3 in red color.
For testing a stimulator, please connect the cable of the “ODD” connector to the “EVEN” connector and the open cable to one of the “STG” plug ins. Define a respective stimulus pattern in the “Stimulation” window, for example a ramp with 100 ms on stimulator 1 (green), as shown on the screenshot above. CMOS- MEA Chip
Please fill the CMOS-MEA chip with PBS. CMOS sensors are light sensitive. To record a stable baseline the CMOS-MEA array must maintain under stable light conditions, which can easily be provided by covering the CMOS-MEA with the convenient dark chamber “CMOS-DC”. Otherwise, the signal will drift out of range by the effect of light.

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GENERAL SOFTWARE FEATURES

General Software Features The chapter “General Software Features” describes some of the CMOS-MEA-Control and CMOS-MEA-Tools software features on base of examples. Numeric Up-Down Box
Adjust a value in the numeric up-down box either by clicking on the arrow buttons or by clicking into the window and moving the mouse wheel. Turn the wheel forward and the level increases, turn the wheel backward and the value decreases in fast steps. Use the arrow buttons for fine tuning the adjustment. The third possibility is a replacement of the number in the window by overwriting it, if the value is predefined, for example. Zoom In and Zoom Out Zoom Buttons
Click the “Adjust to signal Min/Max” button. The scaling of the y-axis is set to the minimum and maximum of all visible samples in the channel.
Click the “Zoom” buttons. Zooming in cuts the scaling of the respective axis in a half and zooming out doubles the scaling.
Zoom by Mouse-click Additionally, to the zoom buttons you can freely zoom into a region of interest by moving the mouse inside a display from the left to the right while pressing the left mouse button. Move the mouse from the right to the left for zooming out.

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GENERAL SOFTWARE FEATURES
Display Floating
Design your own display with the floating feature. Decouple a dialog display of your choice and place it wherever you want.
Dock the window again by clicking with the right mouse button on top of the window. Hide a window with the “Auto-Hide” option. Creating Regions of Interest ROIs
Selecting ROIs manually by drawing rectangles is identical even in the “Activity” or “Sensor Array Tool” window. Please keep the left mouse button pressed to draw a rectangle in the “Activity” window to create a region of interest. A dashed line in blue indicates the borders of the ROI. The color of the rectangle turns to black and the ID of the ROI appears in the upper right edge. Or create a ROI by clicking on one of the activity maxima. Modify a region of interest be clicking on the borders until a double arrow appears to move the border. Delete a ROI by clicking on the ROI again. The sensor channels included in a region of interest are immediately shown in the “ROI” window beside.

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CMOS-MEA-CONTROL SOFTWARE

CMOS-MEA-CONTROL Introduction The software for controlling the CMOS- MEA5000-System includes two parts, CMOS-MEA-Control for online recording, CMOS-MEA-Tools for offline analysis. The CMOS-MEA-Control software is explicit designed for online data recording with the CMOS-MEA5000-System. It facilitates a real-time activity overview on the complete chip with the ability to zoom into the raw data and various tools to visualize the activity. The CMOS-MEA-Control software controls the CMOS-MEA5000-System, the experimental procedure, the data selection for recording to hard disk and similar functions like the control of stimulation, the online spike detection, the saving of spikes with or without raw data to save disk space or the selection of regions of interest selection for data reduction and many additional functions.
Main Window

The default window of the main menu is divided in three parallel sections which are framed by the menu bar above:
1. Left: Control section to control the CMOS-MEA5000-System hardware, the data acquisition, the experimental procedure, the recording of data, the streaming of detected spikes and a section to monitor the system load.
2. Middle: Tools section with tools to set sensor array properties, to monitor the acquired data online, to detect spikes, to stimulate and to extract events from a digital input signal.
3. Right: Data view section with detailed views of the raw data or of detected spikes. You can zoom into the data in a region of interest above and in a detailed single view of one recording electrode below.

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CMOS-MEA-CONTROL SOFTWARE
Menu Bar File
Menu to open and to save templates and to “Exit” the program. Settings
Menu to activate the “Tools”, “Activity Tool” and “Spike Tool”. Switch the tools on via check box in order to perform their respective tasks or off to reduce the system load. When the “Activity Tool” is switched off, no raw data will be displayed, but of course processed and stored. When the “Spike Tool” is switched off, no spike detection is performed, and no spikes are stored. Note, that the spike detection on so many sensors is an expensive task and needs an appropriate computer. Menu to set “Default Paths”, to “Save as Defaults” and to “Reset Defaults”. Click “Application Settings” to open the “Application Settings” dialog. Define the paths for the “Default Paths” for “Raw Data”, “Template” and “Stimulation Files” in the “Application Settings” dialog.

Important: Make sure that all paths go to the SSD drive otherwise no recording is possible!
Click the check box “Save Sensor Calibration” in “Diagnostics” section.
Click the check box “Save Sensor Calibration” in “Diagnostics” section to save calibration data to a “hdf5” file. Each time a calibration is performed, a file with calibration data is created in the “Raw Data” folder, containing the raw data and the conversion factors calculated from this data.
During the system calibration process the sensors are calibrated as well. For the calibration a voltage is applied to the bath chamber. The signals measured at the sensors are fitted to the calibration stimulus. If both signals fit well, the conversion factor is calculated, and the ADC values are saved in the raw data file.
The file names start with “Calibration” followed by the recording date and the extension: Calibration-2019.08.01-13.44.30.cmcr. The file format is the common raw data file format so that you can load it for inspection into the “CMOS- MEA-Tools” software.

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CMOS-MEA-CONTROL SOFTWARE
Lab Book
Please use entries in the three tabs of the lab book for later analysis of the experiment. Click the button “Set Default” for keeping the information as template. All notes will be saved in the raw data file. Setup
Click “Device” to change the connected device. If hardware is available, choose the “MCS Device”. If no hardware is available, please use the “Simulator” or load a “Data File” to test the software without hardware attached. When replaying a data file, you have the advantage to see “real” data meanwhile the data of the “Simulator” are virtual.
To select a data file from a folder, please choose “Data File” in the “Setup” menu. The “Data Source” window displays “Simulation” beside the CMOS- MEA5000-System button. Then click on the CMOS-MEA chip button to select the file you want to replay from the browser dialog. Click “CMOS System” to open the “Setup CMOS System” dialog.

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“Setup CMOS System” dialog provides the manual control for the offset and calibration settings for the CMOS chip. There is an automatic routine for the chip start up and calibration, the manual settings are usually not needed. Please read chapter “Operating the CMOS-MEAControl Software” for detailed information. Open the “Device Filter” dialog to change filter settings.
Adjust the settings of the “High Pass” and “Low Pass” filter on hardware level during the software is running, but not recording. Choose “Bessel” or “Butterworth” filter “Family” in 1 or 2 “Order” from the drop-down menus. Switching “Off” the “Cutoff Frequency” of the high pass filter enables DC signals. If filter settings should be saved for later experiments, please click the “Set Permanent” button. If needed, please set the “High Pass” filter to option “Off”, that means to DC, direct voltage. No offset will be applied, all raw data are displayed without any filter. Help
The “Help” menu is for opening the online help and to “Check for Update”, if necessary. Click the “About” option for software and firmware information. Click “Online Help” to see the last version of the CMOS-MEA5000-System manual. Click “Check for Updates” to see whether the software is up to date, or a new version is available on the Multi Channel Systems website.
When starting the CMOS-MEA-Control software this pop-up “Software Update Available” appears, when a new version of the software is available. Click on the note to open the “Check for Updates” dialog to have direct and fast access to the MCS web site.

If the software is not up to date, click the button “Visit Web site” and download the newest software version.

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Click option “About”.
Please see technical specifications of the device and the version number of the CMOS-MEA-Control software. Control Section The “Control Section” includes three sections, the “Data Source”, the “Recorder” and the “Load” control. Data Source

The “Data Source” window allows controlling the data source and the CMOS chip. The chip is an active device and has to be powered before you are able to start an experiment with the “Start” button.

Click the “Settings” icon

for the “Set Device” dialog. When using a CMOS-MEA5000-System for the first time, please define which

CMOS-MEA “Device” is currently in use and define the “Sample Rate” from the drop-down menus. Selecting “Analog Channels” and the

digital “Digital Channel” enables the selected channels. Decide in the “Recorder” window whether you want to record from these channels

or not, please see the next screenshot.

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Selecting a high “Sample Rate” of 50 kHz, 100 kHz or 200 kHz, you have to accept following consequences: Please choose one region of interest ROI only, to allow the recording of such a huge amount of data in a defined region of the electrode array. The small window beside the “Sample Rate” drop-down menu specifies the selected ROI additionally.

Recorder

Control the “Recorder” parameter settings in this window. Start and stop the recorder and see the types of data available. Press the

“Settings” icon

to change recorder settings. Please read chapter “Operating CMOS-MEA-Control” for detailed information about

programming the recorder.

Start and stop the recording manually with the “ON / OFF” icon.

When the recorder is running, the recording time and the path where to store the data file is displayed. Define this data path in the “Recorder Settings” dialog. Define additionally the “File Name” and the “Prefix” and “Suffix”.

Start and stop the recorder in three different modes: In “Manual” mode, via “Timer” or in dependency of an “Event”. Use the modes in combination for “Start” and “Stop”. Please read chapter “Operating CMOS-MEA-Control” for detailed information.

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Spike Server
The “Spike Server” allows to stream detected spikes and events to other applications on the same computer or to another computer in the network. The client receives the data and can process it to the needs of the customer. We provide source code for such a client in C#, Matlab and Python. Load

Monitor the current state of the capacity of the data acquisition computer via “Load” window. See the CPU load in “CPU Usage”, and most important the available “Disk Space” on the drive used for recording.
Note that the CMOS-MEA5000-System generates up to 220 MB per second when recording with maximum sample rate!
The “Data Source” and “Recorder”, the “Activity Tool”, the “Spike Tool” and the “Spike Server” window allow to see which components of the software are generating which amount of processor load at the moment. Each tool has its own queue of data packages to process. If the load of the processor is high tools may not be able to process its data and the data queue of the tool grows. This state of the queue is shown for each tool separately.
Sensor Current

See the “Sensor Current” of the CMOS chip. The sensor current is measured as the sum of all current over all 4225 sensors. The total current should not be larger than about 300 mA, otherwise the chip get damaged.

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Log

The “Log” file window displays the ongoing experimental proceedings online. In the “Log” file all software operations during an experiment

are documented. The “Log” can be saved and exported as “*.xml” file by pressing the disc

icon.

Data Display
Activity
Due to the high number of channels, it’s impossible to show raw data of 4225 channels simultaneously. Therefore, activity is visualized in time bins in a false color code. This allows the user to identify active areas and to focus on them in regions of interest. Display the “Activity” data in three modes: “Max Amplitude”, “Mean Amplitude” and “Spike Count”.

See the complete CMOS-MEA electrode array in the “Activity” window. The “Activity” plot shows all 65 x 65 sensors of the CMOS chip as one pixel each. Select as much “Regions of Interest ROIs” as needed to display the most exciting regions of the CMOS array only. In the screenshots below regions of interest are displayed. Offset Correction Use the checkbox “Offset Correction” to put all electrode levels to zero.
Creating Regions of Interest ROIs
Please keep the left mouse button pressed to draw a rectangle in the “Activity” window to create a region of interest. A dashed line in blue indicates the borders of the ROI. The color of the rectangle turns to black, and the number of the ROI appears in the upper right edge. Or create a ROI by clicking on one of the activity maxima. Modify a region of interest be clicking on the borders until a double arrow appears to move the border. Delete a ROI by clicking on the ROI again. Regions of interest are displayed in separate tabbed pages. To switch from one ROI to another, please use the tabs under the “Single View” of the “ROI” window. Important: It is not allowed to overlap regions of interest! The sensor channels included in a region of interest are immediately shown in the “ROI” data window beside.

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The toolbar of the “Activity” is divided into two parts: One part above and one part below the “Activity” display. Upper Toolbar of the “Activity” Window

Click the “Parameter Selection Icon” icon to decide which parameter mode of the sensor activity should be displayed: The maximal “MaxAmplitude” or the “MeanAmplitude or the “SpikeCount”. In dependency of your choice, the toolbar beside changes.
The selected parameter will be shown in the activity window as false color plot in time bins. Define the color of the map in the “Sensor Array View Settings” dialog.

“Reset Offset”

Use the checkbox “Offset Correction” to put all electrode levels to zero, if necessary. Click the button to zero the electrode levels after drifting again.

Important: The “Offset Correction” will not change the raw data, which are recorded! This feature only influences the data displayed!

For the “SpikeCount” parameter, spikes are detected individually for each channel by a threshold crossing. The threshold is calculated as standard deviation of the noise for that channel, as a user defined factor. The factor can be changed with the up-down box next to the parameter selection icon. The calculation of the standard deviation can be updated at any given time with the icon “Update the Std.

Dev. Measure” .

Open the “Activity Tool Settings” dialog by clicking the icon . The basic parameters, like update of the time bins and the detection dead time can be changed in the “Activity Tool Settings” dialog.

The “Update” parameter defines the size of the time bins and the refresh rate of the display. “Accumulate” and “Detection Dead Time” are important if the spike count is displayed. Spike count will be accumulated for the selected time, events crossing the detection threshold within the selected “Detection Dead Time” after a previous event will be ignored. This means that when looking at spike count, the display will begin to show data with a few seconds delay after starting the data acquisition, depending on the “Accumulate” setting.

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Lower Toolbar of the “Activity” Window

If the mouse pointer is positioned on the map, the coordinates of the area the mouse is pointing at is shown at the left side of the toolbar below the plot window.

Define the color of the “Activity Map” by clicking the second setting icon

in the toolbar below. The “Sensor Array View Settings”

dialog appears. Enable the check box “Invert” to invert the colors of the activity map.

By default, one region of interest (ROI) is defined on the “Activity” display. The raw data of all sensors within the ROI is displayed in detail. The size and position of the ROI can be changed by dragging with the mouse. It is possible to define more than one region of interest. Click the buttons for adding or removing additional ROIs. Please draw a rectangle with the mouse over the respective sensor array to define the area of a ROI. If more than one region of interest is available, they are independently displayed in tabbed pages. Please read also chapter “Creating Regions of Interest ROIs” above.
Optionally it is possible to define the area of a ROI with the “Set ROI Cursor” dialog. Define the “Position”, “Size”, and “Color” of the ROI cursor. Show or hide the cross hairs via “Show Cross Hairs” check box below and “Enable” the cursor via the check box above.

Define the value of the color map in microvolt with the up-down box “Range” close to the maximum amplitude or spike frequency of the expected signals.

. The maximum value should be

The color depicts the selected parameter in the “Activity” display. As seen in the image below, activity is shown as colored spots.

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Sensor Array Tool

The “Sensor Array Tool” integrates three functions which are related to the control of the CMOS-MEA chip.
First of all, you can manually recalibrate your sensors, if necessary. Secondly, regions of sensors can be selected manually or automatically to focus only on interesting regions. Thirdly, when necessary, the operating point of the sensors can be resetted by setting the appointed gate voltage once or in repeated mode anew. At the end of the system calibration run the conversion factors of all sensors which are displayed in order to evaluate the quality of the sensor chip. Please select one of the three modes from the drop down menu: “Calibration”, “Sensor Selection” and “Sensor Reset”.
Calibration

As mentioned above the condition of the sensor chip can be estimated by the conversion factors obtained during the system calibration. Each sensor’s calibration value is represented by a color coded dot. The exact values depend on the sensor chip. The pattern of this values should be a kind of random noise pattern.

Any structures within this random noise pattern may indicate a problem. For example, a white area may point to a group of defective sensors. Or a straight horizontal line across the whole chip indicates a bad contact to the sensor chip.

Please stop the experiment and clean the contact pins of the headstage. White pixel point to a defective single sensor, where calibration

was not possible. Try to repeat the process by clicking the “Calibrate” icon command as in the “CMOS-MEA Diagnose” dialog.

for the calibration of the CMOS sensors. This is the same

For calibration a sine signal with 70 Hz and 3 mV is set against a sine signal from the internal stimulator.

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Sensor Selection
The “Sensor Selection” module allows the user to select regions of interests manually or automatically. It is useful to reduce the amount of data transferred to the computer and to hard disk when the activity is restricted to one or several locations of the sensor array. Fetch the activity distribution obtained by the “Activity” or “Spike Tool” and use it to define the regions of interest.

Upper Toolbar of the “Sensor Array Tool” Window
Please download your selection of regions of interest with the “Download the Selected Sensors to Data Acquisition” button to the computer. Use the “Fetch Spike Count from Spike Tool” button to import data from the “Spike Tool” and click the “Fetch Activity from Activity Tool” button to import data from the “Activity” tool. Lower Toolbar of the “Sensor Array Tool” Window
Select the complete array with the “Select complete Sensor Array” button. Use the “Automatic Sensor Selection” button if you are not going to select the regions of interest manually. The automatic tool selects areas around activity peaks. Please define the dimension of the region of interest from the drop- down box, “Size 3 x 3” sensors or “Size 5 x 5” sensors. Deselect all regions of interest with the “Remove selected Sensors” button. Additionally, to the automatic selection of ROIs you are able to add ROIs or to modify or to remove sensor arrays manually. Please read in chapter “General Software Features” on page 18 paragraph “Creating Regions of Interest ROIs”.

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Sensor Reset
For very long recordings or for experimental conditions which causes strong potential drifts of the sensors it may be necessary to reset the sensors operating point. The reset of the sensors can be controlled manually or automatically at user defined intervals. The timing of the reset is acquired and saved as channel data and also as events together with the sensor data. This information can be used to exclude the reset phases from analysis. To reset the sensors operating points, select the “Sensor Reset” module from the “Sensor Array Tool” toolbar. Press the start button to reset the sensors. The duration of the reset can be set with a numerical control. Check the “Repeat” control to automatically repeat the sensor reset with a given interval. The “Sensor Reset” tool raises the floating signals back to the operation point. This short period is visible in the raw data as a potential step. The exact time of the reset is recorded together with the raw data as channel data or as events. Example: “Sensor Reset” without suppression

The example above on the left shows a sensor reset with an interval of 1 ms and no suppression. A filter of 10 Hz is applied.

The example above on the right shows a sensor reset with an interval of 1 ms and no suppression without filter.

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Example: “Sensor Reset” with suppression

The example above on the left shows a sensor reset with an interval of 1 ms, with 10 Hz filter and with suppression. The example above on the right shows a sensor reset with an interval of 1 ms, without filter and with suppression. The effect of the suppression and of the 10 Hz high pass filter is obviously to see in comparison of the four screenshots above. Click the checkbox “Artefact Suppression” to filter out artefacts.
Spike Tool

The “Spike Tool” is used for the online determination of spike parameters to allow an activity dependent recording. This is useful when active phases are embedded in long inactive phases. Using this feature just the interval around active phases are stored on disk.
Spike Tool upper Toolbar

Define the “Threshold” for the spike detection from the drop-down box: “Positive”, “Negative” or “Absolute”. Select the standard deviation “StdDev” from the numeric up-down box. Use the “Update StdDev Measure” feature, if necessary.

Use the checkbox “Offset Correction” to put all electrode levels to zero, if necessary. Click the button “Reset Offset” the electrode levels after drifting again.

to zero

Important: The “Offset Correction” will not change the raw data, which are recorded! This feature only influences the data displayed!

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Example: Data display “Spike Overlay” with “Offset Correction” on the left and without “Offset Correction” on the right.

Click the “Open Settings Dialog” button

to open the “Spike Explorer Settings” dialog.

Spike Detection
Please define various parameter for the spike detection. The noise is used as a base to calculate the threshold of the spikes which should be detected.

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Noise Measure Section “Noise Measure” parameter: Select whether you want to set the “Standard Deviation” or the “Median / Absolute Deviation” for noise measuring. “Timing”: Three parameters are available to define the timespan in which the noise should be measured for using it as a base for the threshold. The “Continuous” measuring is opposed to the interval measuring. Use a “Single Interval” or “Repeat Interval”. Continuous measuring of the noise as base for the spike threshold is necessary if the raw data are drifting. This way, the threshold for the spikes will be adapted. To repeat the noise measure in intervals is also reasonable for ongoing experiments. Please define the number of “Repeat(s)” and the “Duration” of the interval in milliseconds from the up- down boxes.
Detection Section “Detection” parameter: Define the “Threshold Type” from the drop-down box, “Positive”, “Negative” or “Absolute”. It is also possible to define this parameter in the tool bar of the “Spike Tool” display. Select the “Threshold” from 0 to 99 from the up-down box. Define the “Detection Dead Time” in ms.
Spike Cutout Section “Spike Cutout” parameter: Click the check box “Extract Waveform” if necessary. Define the “Waveform Alignment” from the drop-down box, if the check box is enabled. Define the “Pre and Post Interval” in milliseconds via up-down boxes. Spike Tool lower Toolbar
See the coordinates of the courser in the display as usually. Select the sensors automatically with the “Automatic Sensor Selection” button or remove the sensor by clicking the “Remove Sensors” button. Define the “Minimum Spike” rate and the “Range” via up-down boxes. Click the “Open Sensor Selection” button to open the dialog.
Please define the “Extraction Method”, the “Smoothing” and the “Minimum Spikes”.

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Click the “Open Settings Dialog” button to open the dialog.
Define the parameter for the sensor array: The “Update” of the array and the accumulation “Accumulate” in ms and the color of the display in “Color Map”. Spike Tool Event Window
See the spike rate in dependency of the time in the “Spike Tool Event” window and adjust the threshold to trigger events by moving the red bar with the mouse. When the spike rate crosses the threshold in positive direction, a specific positive “Spike Rate Threshold” event is created and dispatched. When the spike rate crosses the threshold in negative direction a specific negative “Spike Rate Threshold” event is created and dispatched. Events are shown as triangles in orange (start event) and blue (stop event) at the bottom of the traces in the “Spike Tool Event” view. Furthermore, they are sent to the “Recorder” for optional use. The spike rate was calculated either of all sensors or for a subset of sensors. If no sensors are selected all sensors are used. To make an analysis more specific, just select the sensors which show the behavior you are interested in. Restricting the analysis to a few sensors of a distinct temporal spike pattern you can reduce the “noise” originating from all other sensors with uncorrelated activity. Please also read chapter “Recording” in “Operating CMOS-MEA-Control Software”.

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Stimulation
Please read chapter “Stimulation” for detailed information. The in the headstage of the CMOS-MEA5000-System integrated stimulator provides the possibility of stimulating via three different stimulation patterns on user defined stimulation electrodes. Analog Channels
Eight analog “Analog Channels” are available to record additional analog signals. Start and stop the analog displays separately. Use the toolbar to customize the zoom factor and the timeframe for the data. Please read also chapter “General Display Features”. Digital Port Events

Digital events can be generated on TTL pulses coming in on the “Dig In” ports on the interface board. In the “Event” box, select the event number. For each event number, a “TRUE” condition can be defined. The condition can be a TTL on any of the 16 digital input bits, either the rising or falling flank. It can also be a combination of different bits, which can be combined by “AND or OR” condition. In the example shown above, an event No 4 will be generated on the rising flank of a TTL coming in on bit 2 or bit 3 of the digital channel.
Only TTL signals are accepted as input signals on the digital input bits. Use the drop-down menu of the tool bar below to customize the timeframe for the displayed data. Clear all events with the “X” button. Start and stop the display separately.

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Detailed View: Region of Interest

Define one or more regions of interest in the “Activity” display on the left side of the main menu. If more than one ROI is available, please see the tabbed pages in the detailed view. Do not choose too many sensors for the detailed view, because displaying the single sensors needs very much computer performance.

On the right side of the main window, you have a detailed look to the electrodes of the currently selected region of interest. Use the toolbar to customize the zoom factor and the timeframe for the displayed data. Single Sensor View

Select one of the electrodes of the ROI for a closer look.
Start and stop the “Single Sensor View” separately to save computer performance or to have a closer look. Use the toolbar to customize the zoom factor and the timeframe for the data. Please read also chapter “General Display Features”.

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OPERATING CMOS-MEA-CONTROL SOFTWARE Set Device

If a device is not recognized automatically after starting the CMOS-MEA- Control Software, please click the “Settings” icon

in the

“Data Source” window for the “Set Device” dialog and select the connected CMOS device. This can happen at the first use on a specific

data acquisition computer, or after upgrades.

Choose the “Sample Rate” from the drop-down menu. The region of interest is limited while recording with high sample rates, more than 25 kHz. Select one to eight “Analog Channels” and the “Digital Channel”, if needed.
Next step is to power up the active CMOS-MEA chip.

Please click on the CMOS-MEA icon in “Data Source” to power the chip. The type of the chip, the distance from the center to the center of the recording electrodes and the serial number ID are identified, for example “nMOS16”. “n” indicates the type of the chip, “n” means negative, “p” is a positive doped chip type.

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Calibration of the CMOS-MEA5000-System Place the CMOS-MEA chip in the headstage and cover it with a dark chamber to eliminate the influence of the light. Every time a CMOS-MEA chip is placed inside the amplifier and the chip is powered up, it needs to be calibrated. Usually, the integrated automatic calibration protocol is sufficient. In rare cases, the automatic calibration fails. Then it is also possible to calibrate the CMOS-MEA chip manually. Both options are described in more detail below.
Automatic Calibration CMOS-MEA chips are active devices and must be switched on and off properly, otherwise they might suffer damage. Please click the MEA array icon in the “Data Source” window to power the chip up or down. CMOS-MEA chips must be calibrated before each use. The calibration runs automatically. Usually, it takes two or three minutes.

After the chip is powered the automatic system calibration dialog is shown. Start the automatic system calibration or close the dialog to calibrate the system manually.

The following important steps for calibration are performed automatically. Please see the protocol log file to monitor the process.
The system calibration consists of four consecutive steps:
1. Set sensor operation point. 2. Wait until the signals stop drifting. 3. Adjust ADC offset. 4. Calibrate sensors.
Start and stop the calibration via the “Automatic System Calibration” dialog with the respective buttons. Press the double arrow icon button to skip one of the calibration steps. In the field below all calibration steps will be logged.
Calibration Step by Step
1. Optimizing the Sensor Operation Point

The “Operating Point” is different in dependency of the type of the CMOS-MEA chip. It will be set automatically.

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2. Control of the Sensor Drift
After the CMOS-MEA chip was powered and the operation point was set, the sensor signals may drift for a while. During the “Sensor Drift” step, the signals of all sensors are measured, and the slopes are calculated. When the slope median of all sensors go below a predefined threshold the signals are considered stable, and the next step can start. The sensor signals will be monitored until they stabilize. If the drifting of all sensors is low, the next calibration step can take place. The duration of the sensor stabilization can vary between CMOS-MEA chips and depends on the condition of the chip. Usually, this step should not take longer than a few minutes.
3. Adjust the ADC Offset In the following calibration step the ADC (Analog to Digital Converter) offset value is adjusted to find the optimal offset for all or most sensor signals. The signals should be close to zero. If the median of all sensors falls below a predefined threshold, the “Adjust ADC Offset” is finished.

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4. Calibration of the Sensors
In the final step of the sensor calibration, a reference voltage signal (sine wave) is applied to the bath via the reference electrode. If this signal is not detected by the sensors, and hence not visible in the single channel view, this means that either the electrodes are damaged or that the bath reference electrode is broken. Manual Calibration Click the option “Setup” in the main menu to open the “Setup CMOS System” dialog. The “Setup CMOS System” dialog allows to control each step of the sensor calibration as described above manually.

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Header
Start the recording via “Start” button. Display of the type of the CMOS-MEA chip. High Pass Filter Use the high pass filter during recording. Important: For diagnosis purposes, please deactivate the high pass filter to observe the drift and the offset! Chip Power Window
After powering the CMOS-MEA chip, you will find information about the voltage applied to the chip, displayed in the up-down boxes. The user is not allowed to change these voltage levels. Set Sensor Operating Point Window

Please set the sensor operating point in mV. Standard values for the “Source- Drain” and the “Source-Gate” for conventional chips are -600 and -650 mV. For “Low Noise” CMOS-MEA chips the standard values are: “Source-Drain” -800mV, “Source-Gate” -950mV. Click the “Set” button to download the settings to the device. Set AD Converter Window
Adjust the voltage for the ,,Input Offset” in mV manually with the numerical up down box. Click the “Adjust Offset” button to download the setting to the device. Update
Click the button “Update” to update the dialog. Close the dialog with the “Close” button.

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The offset for each single sensor should be approximate to zero. The voltage needed for this ADC offset is displayed in mV and can be manipulated with the “Input Offset” up-down box.

You can observe the process in the ROI display and in the single view. Minimize Floating Artefact Window

Define the voltage in “Source-Bulk” in mV via up-down box. Download the settings to the amplifier with “Set”. For recording data, you need the setting “Gate is floating”. “Floating” means the sensor measures the bath only. “Gate to VOP” means to apply a voltage to the gate to define a working point. “Gate to VOP” means that a voltage is applied to the gate to define a working point. Bath Stimulation Window
Independent from the stimulation of electrodes it is possible to stimulate the bath itself. Use this feature to stimulate the complete tissue or culture on the CMOS-MEA chip. Define the sine wave pulse via up down boxes “PP Amplitude” in mV and “Frequency” in Hz. Click the button “Stimulate” to start and stop the bath stimulation and observe the effect in the single view, for example. If the sine wave is not visible in the single channel view, this means that no signals are detected by the CMOS-MEA sensors. This indicates that either the sensors are damaged or that the bath reference electrode is broken.

Sensor Calibration Window

The input voltage of each transistor corresponding to the sensors are different. The “Sensor Calibration” function sets the input voltages to a common level. The stimulation of the bath is the first step, then the response signal is measured and evaluated.

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Recording The CMOS chip includes 4225 recording electrodes and 1024 stimulation sites. The 16 m interelectrode distance chip offers the highest resolution. With the high number of electrodes, you can record from a large surface (1 mm² @ 16 m distance, 4 mm² @ 32 m distance). Thereby, you can see the signals from every single cell and even the signal propagation along an axon, while still getting an overview on your complete sample. The data is sampled at 25 kHz per channel. Together with the A/D conversion at 14 bit, the system ensures accurate and precise data. Thus, CMOS-MEA-Control produces a huge amount of data within very short time segments. Multi Channel Systems MCS GmbH recommends an up to date computer with high memory capacity. Nevertheless, it is useful to limit the recording times thoroughly, otherwise the disk space of the data acquisition computer will overflow soon. CMOS-MEA- Control offers several possibilities to limit the data volume and to set recording times exactly to the periods of interest.
1. Please select the data streams to record. 2. Define the options how to start and to stop the recording. 3. Options: Manual, timer, events (stimulator: on/off/marker, digital event tool). 4. Examples
Please select the data streams you are going to record.

The “Set Device” dialog appears after clicking the “Settings” button

in the “Data Source” window. Select the “Device” and

the “Sample Rate” from the drop-down menu. Select the “Analog Channels” and the “Digital Channels” provided from the hardware.

Whether you record from these channels or not is defined in the “Recorder” window via the respective check box. Please see below.

Start and stop the recorder independent from starting and stopping the system. Please use the drop-down menu to select the mode for starting the recorder on one hand and stopping the recording on the other hand. The combination of different modes offers a lot of possibilities to record data focused on critical periods.

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Starting and Stopping the Recording

“Manual”.

Please click the “On” / “Off” button

on top of the “Recorder” dialog manually.

Starting and Stopping the Recording via

“Timer”.

Select the “Start” time and the recording time in seconds from the up-down box. Enable the check box “Repeat” if necessary. Start the recorder by clicking the button “On”. Now the recorder is “Armed”. In this mode the recorder is disposed and waits for starting via timer or event. Same “Armed” recorder situation may occur if breaks between recording phases are programed.

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Starting and Stopping the Recording via

“Event”.

Using an “Event” as start and stop trigger postulates to build this event before. An “Event” may be a digital TTL trigger coming from external or an internal TTL marker signal programed with a stimulation pattern.

If a synchronization of the CMOS-MEA-System with other devices from external is necessary, please use the “Digital In” ports for triggering with a TTL pulse. Create an digital event in the “Digital Port Events” dialog.

In this example the trigger “Event” appears, if bit 2 or bit 3 changes from low (0) to high (1).
Please read chapter “Stimulation” for detailed information about creating a stimulus pattern. Program a TTL marker signal in the “Set Marker Signal” dialog. The marker signal will be recorded and stored in the data file for a precise comparison of data and marker.

The stimulation pattern appears like this, for example. The marker signal is the orange trace, shown in the lower display of the complete stimulus pattern. Please define a “Marker Port” for delivering the TTL signal.

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Spike Rate triggered Recording The functionality of the “Spike Tool” and the “Recorder” allows an activity dependent recording. This is useful when active phases are embedded in long inactive phases, for example for epilepsy research. Using this feature just the interval around active phases are stored on disk.
The “Recorder” interacts with the events created and dispatched by the “Spike Tool”. The events are displayed as small triangles on the base of the time axis in the “Spike Tool Event” view. The spike rate is calculated either of all sensors or for a subset of sensors. If no sensors are selected all sensors are used.

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To make the analysis more specific just select the sensors which show the behavior you are interested in.
Restricting the analysis to a few sensors of a distinct temporal spike pattern you can reduce the noise originating from all other sensors with uncorrelated activity. The threshold (red line) can be moved by mouse. When the spike rate crosses the threshold in positive direction (gets larger) a specific “Spike Rate Threshold (pos)” event is created and dispatched. When the spike rate crosses the threshold in negative direction (smaller values) a specific “Spike Rate Threshold (neg)” event is created and dispatched. Example: How to do Spike Rate triggered Recording To start a recording by the spike activity measured on the CMOS chip just select the “Start by Event” as start mode and select the “Spike Rate Threshold (pos)” option in the associated drop down box. To set an “Offset” value in milliseconds which is added to the time stamp of the start event needs the requirement of a buffer, which stores all data for a certain amount of time. Negative values mean that the recording starts before the event time stamp (that means in the past), positive values starts the recording after the event time stamp. For a value of zero recording starts exactly at the event time stamp. To stop a recording also by the spike activity measured on the CMOS chip select the “Spike Rate Threshold (neg)” event as the stop mode event. If a recording was started and ongoing, an additional spike event will be ignored until the recording is stopped. So, an overlap of spike triggered recordings is not possible. Other useful combinations may be a start by “Spike Rate Threshold” and a stop by “Timer” or a start by “Stimulus Marker” and a stop with “Spike Rate Threshold”.

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Stimulation Warning: Only positive voltages should be applied to the CMOS arrays. Negative voltages will damage the chip. Changing voltages will generate a current. Default pulse form is therefore a positive voltage ramp. Warning: Do not use voltages higher than 3.4 V, or you will damage the CMOS array. Stronger voltages will lead to a breakdown of the isolation layer. This is optically visible on the surface of the CMOS chip. Please see the scheme:
Important: The resolution of the stimulator is 10 µs in time axis and 105 µV in voltage. The stimulus generator is not able to release pulses shorter than 10 µs or lower than 105 µV, therefore such pulses are skipped! Important: About nine stimulation pads (3×3) minimum are needed for an effective stimulation. This empirical value is valid for retina, no data is available yet for cell cultures. In contrast to regular MEAs, neighboring recording sensors will usually not saturate during stimulation, so recording in very close proximity to the stimulation site is possible.
Stimulation Window

Tool Bar

The tool bar in the “Stimulation” window is divided into three sections: A position indicator, controls for single stimulus patterns, and controls that affect all three stimulus patterns. The function of all icons is explained via tool tip, when you move the mouse over the icon.
First section: The position indicator helps to navigate through the CMOS-MEA electrodes. It shows the exact position of the mouse.
The numbering of CMOS-MEA electrodes in the 65 x 65 grid follows the standard numbering scheme for square grids: The first digit is the column number and the second digit is the row number. For example, electrode 23 is positioned in the second column and the third row.

Second section: Click one of the three color coded “Select Stimulus” icons

to select the respective stimulus pattern

for further processing. You can define the location of the selected stimulus by clicking on electrodes in the electrode panel or by drawing

rectangles over the desired area. Each stimulation electrode accepts one stimulus pattern at a time. It is not possible to overlap patterns,

but to overwrite them with a new selection or to clear the sites.

Clicking the “Define Stimulus” icon

will open a new dialog enabling the definition of the stimulus pattern as explained on the

following pages. To start stimulation with the currently selected pattern only, click the “Start” icon in the second section of the tool bar.

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To remove the currently selected stimulus from all sites, please use the “Clear selected stimulus sites” of the selected pattern will be removed.

button. All created sites

For downloading and starting the stimulus, please click on the next button . To remove all stimulation sites, click the “Remove all

stimulation sites button. Download, if necessary and start all stimuli together with the “Download and start all Stimuli” button.

For displaying the “Activity Map” window, please use the “Display Activity Map” button.

Third section: To clear all sites, click the “Clear all stimulus sites” icon. Start all stimulus patterns simultaneously via the “Start” icon in this section.

To open the “Define Stimulus 2” dialog, click the “Select Stimulus 2” icon

. The “Define Stimulus” icon

in the

“Stimulation” window will be available. Each of the three stimulus patterns 1 (green), 2 (blue) or 3 (red) can be defined independently.

The dialog for the settings of the stimulus patterns are build analog, please see, for example, the “Define Stimulus 2” dialog.

The “Define Stimulus” dialog is divided in an upper and a lower section. The upper part includes the “Primitives”, that means, the icons for the provided stimulus patterns, such as flat line, ramp or sine waves. The last icon represents ASCII files generated by a different software, for example MC_Stimulus II, which can be imported into the CMOS-MEA-Control software. The lower section of the dialog shows the

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“Complete Stimulus Pattern” and a tool bar. The settings in the tool bar influence the complete sequence of the stimulus patterns which are immediately displayed in the window for the “Complete Stimulus Pattern”. The white menu bar “Building a Stimulus” in the middle of the dialog is for creating the desired stimulus pattern from the provided default stimulus patterns, called “Primitives” or for the import of external created stimulus patterns. You can modulate each “Primitive” pattern via individual “Parameter Settings” and the modulation will be immediately displayed in the “Single Pattern” view and also in the view for the “Complete Stimulus Pattern”.
Building a Stimulus Pattern
On the left side of the upper section, you can choose the provided default stimulus pattern. Move the primitive icon via drag and drop into the white field in the middle of the dialog for creating a user defined stimulation pattern. Add as much primitives as needed. It is possible to change the sequence of the primitives by drag and drop. For deleting one of the pattern, please drop it into the bin. Setting a Marker Signal

Additionally, it is possible to set a marker signal. Open the “Set Marker Signal” dialog by clicking the check box “Set Marker”

in the window for parameter settings. Click the check box “Set Marker” and click “Repeat” if you want to repeat the marker signal at each cycle of the stimulation pattern. Adjust the “Offset” in s and the “Duration” in s of the marker signal via the up-down boxes. The orange-colored marker signal will be displayed in the window for the “Complete Stimulus Pattern”.

Modulation of the provided Stimulation Patterns

Initially the provided stimulation patterns are default. To adjust a primitive to your requirements, please click onto the icon, which will be highlighted in pale blue. In the upper part of the dialog the setting parameters for this pattern appears on the left and a picture of the actual shape of the pattern on the right. Immediately after changing a pattern, the modulation is visible in the “Single Pattern” view and in the “Complete Stimulus Pattern” view. Save the stimulus pattern as default by clicking

the “Save Primitive as Default” icon

beside the “Marker” check box.

Most of the parameters have to be adjusted with up-down boxes. Please click into the up-down box and move the wheel of your mouse for quick adjustments in wide steps. Use the arrows for fine tuning. The modulation is immediately displayed in the single pattern and in the complete pattern window. Overwrite the letter in the up-down box, the modulation will be displayed after confirming the value with “Enter” or after clicking into another box. Playing with the different possibilities for adjustment allows to create each shape of pulse which could be necessary. Additionally, it is possible to modulate the shape, when repeating pulses in relationship to each other.

Multi Pattern Mode
The “Multi Pattern Mode” allows the automated application of a list of predefined stimulation electrode patterns. The patterns are applied one after another after the start signal. The pattern list may be repeatedly applied depending on the “Loop” setting. The same stimulation pulse is applied to each of the patterns.

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The ,,Multi Pattern Mode” can be toggled via the ,,Enable Stimulator Multi Pattern Mode” check box in ,,Tools” of the ,,Application Settings” dialog. If enabled, a panel is visible to the right of the electrode layout.

Each entry in the list defines one pattern and can contain a single or multiple selected stimulation electrodes, for one or more stimulators, symbolized in green, blue, and red. The list shows entries in numbered order with the number of selected electrodes for each stimulator in parenthesis. When an entry in the list is selected the electrode pattern for all stimulators is shown. Due to restrictions of the internal memory, the number of list entries is limited to 256.

The functions of the ,,Stimulator Multi Pattern Mode” interface are as follows:
· Clone: Add an entry in the list with the same pattern as the currently selected. · Add New: Add a new entry to the end of the list with an empty electrode pattern. · Remove: Removes the currently selected entry from the list. · Save/Load: Saves the pattern list to a file or load from a file (*.lmp). Only pattern files with the same MEA layout can be loaded.
Loading overwrites the current list entries.
· Loop: Determines the switching to the next pattern in the list. o turned off: only one pattern is applied, until the next trigger occurs o 1: all the patterns in the list are applied once, one after the other o 2,3: all the patterns in the list are applied one after the other, then the list is repeated two or three times, respectively o infinite: the pattern list is applied until the stimulation is stopped
· Restart: Resets the current pattern to the first entry in the list.

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Deactivated/Activated The multi patterns are automatically downloaded to the device, while the button ,,Deactivated” is red. Click onto the button and the device is armed, the button turns to green ,,Activated”. Start the stimulation via the ,,Download and Start” button in predefined modes.
If ,,Loop” is turned off, the stimulator is halted after the stimulation pattern has ended until the next start trigger occurs. The next electrode pattern in the list is then preselected and gets applied by the next trigger. If ,,Loop” is turned on, the patterns in the list are automatically applied one after each other (see above). There must be at least one electrode selected, otherwise the switching to the next pattern doesn’t occur. If the stimulator is stopped manually, then no switch to the next electrode pattern occurs. The handling of the stimulus pattern is not changed by the ,,Multi Pattern Mode”, with one exception: A stimulation pattern with ,,inifinite” loop is incompatible with the ,,Multi Pattern Mode” function. A pattern with infinite duration would prohibit the switching of the electrode patterns.

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Stimulus Pattern: Flat Line

The first provided primitive is a flat line stimulus. To adjust the duration, please use the respective up-down boxes: Hour, minute, second, millisecond and microsecond. Define the value of the pattern in the “Amplitude (mV)” up-down box.
Stimulus Pattern: Sine Wave Warning: Negative voltages will damage the CMOS arrays. Sine waves must be shifted with an offset to positive range! Modulate the amplitude “PP Amp (mV)”, the period “Period (s), the shift “Shift” and the phase “Phase (‘) of the sine wave pattern via up-down boxes. Setting the number of cycles to more than one enables the inter stimulus interval “ISI (s)” up-down box. Additionally, the “Arrow” button will be enabled.

When using more than one cycle you can click onto the arrow symbol for additional options: A highlighted parameter field appears to modulate the amplitude and the period of the sine waves in relationship to each other. In this example increases the amplitude of each sine wave at 16 mV in comparison to the sine wave before. Choosing a negative value from the up-down box, the amplitude will decrease in comparison to the wave before. Analogous you can adjust the value of the period in s in relation to the wave sequences. The window “Single Pattern” shows the sine waves in an overlay plot. The lower window “Complete Stimulation Pattern” shows the combined sequence of all stimulus pulses.

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Stimulus Pattern: Ramp The equivalent of a biphasic current pulse is a triangular voltage ramp. About 9 stimulation pads (3×3) minimum are needed for an effective stimulation (valid for retina, no data yet for cell culture). In contrast to regular MEAs, neighboring recording sensors will usually NOT saturate during stimulation, so recording in very close proximity to the stimulation site is possible. Modulate the amplitude “Amplitude (mV)” and the duration “Duration (s)” of the ramp pulse via up-down boxes. To adjust the steepness of the arms of the pulse, please use the three up-down boxes for the ascending part, the plateau and the descend arm independent from each other. Setting the number of cycles to more than one enables the inter stimulus interval “ISI (s)” up-down box. Additionally, the “Arrow” button will be enabled.

When using more than one cycle you can click onto the arrow symbol for additional options: A highlighted parameter field appears to modulate the amplitude and the duration of the ramp pulse in relationship to each other. The window “Single Pattern” shows the ramp pulse in an overlay plot. The lower window “Complete Stimulation Pattern” shows the combined sequence of all stimulus pulses.
Stimulus Pattern: Biological Pulse via ASCII Import Warning: Negative voltages will damage the CMOS arrays. Sine waves must be shifted with an offset to positive range! Important: The resolution of the stimulator is 10 µs in time axis and 105 µV in voltage. The stimulus generator is not able to release pulses shorter than 10 µs or lower than 105 µV, therefore such pulses are skipped! If you like to use, for example, a biological signal as stimulus pulse or you want to create an arbitrary pattern, you can import signals by clicking the “Import” button. The imported file must mandatorily have the following format:

Timestamp Voltage Value Timestamp Voltage Value Timestamp Voltage Value …
The unit of the timestamp is “µs” and the time value has to be in ascending order. The unit of the voltage value is “µV”. The units are not part of the file. CMOS-MEA-Control software accepts integers only and commas, tabulators or spaces to separate the timestamp and the voltage value. Please remove a possible header, avoid blanc lines and use a new line for each integer pair. Save the file without extension or with any extension “.dat”, for example, or “.csv”, when using a comma separated file.

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If the import file does not meet the requirements, an error message is shown. Stimulus Pattern: Biological Pulse Example: Import of the following file as stimulation pattern:

Note: Because the slope of the signal determines the strength of the induced current, please add ramps to the start and stop of the biological signal to avoid stimulation artefacts.

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Stimulation Pattern: Loop

Place any stimulus pattern between a “Loop” primitive and the patterns can be repeated as often as needed. Please select the number of looping “Cycles” from the up-down box. The “Loop” primitive is not movable in the sequence of the stimulus patterns. Drop it into the bin and start again to change the position of the “Loop” pattern. It is possible to create convoluted patterns.

To place a primitive between the “Start Loop”

and “Stop Loop”

bracket, please drag and drop the respective primitive first

before or behind the brackets and then to the left or to the right over one of the bracket buttons between them.

Tool Bar
The settings in this tool bar influence the complete sequence of the stimulus patterns. Modulate the “Amplitude (%)” and the “Offset (mV)” of the complete pattern via up-down boxes.

Clicking the “Loop” button enables you to infinitely repeat the defined stimulus pattern. Load a previous created stimulus pattern, save or delete the pattern. When using marker signals, you have to define which one of the “Marker Port” (digital out) should show the corresponding TTL signals. Select one of the marker ports from the drop-down menu and connect a TTL signal source to the port.

Start the stimulation manually.
Download and start the created stimulus pattern via the “Download” icon. If downloading is not necessary, the icon is not available. This way you have optical feedback for the download.

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CMOS-MEA-TOOLS Introduction The software for controlling the CMOS- MEA5000-System includes two parts, CMOS-MEA-Control for online recording and CMOS-MEATools for offline analysis. With Multi Channel DataManager software you can export data to third party applications. Main Window

The default window of the start menu is divided in two main sections: The “Control Window” and the “Data Display or Settings Window”. The left side contains control functions: The “Instrument Tree” view and the “Activity Summary” window. See the temporal overview of the complete file in two tabbed pages: The “Explore Activity” tab shows all spikes, in the “Event” tab the stimulator events are listed. The third window shows the “Spatial Distribution”. The right part includes data displays or settings windows and has four tabs in correlation to the instruments listed in the tree view: “Filter Pipeline”, “Raw Data Explorer”, “Spike Explorer” and “STA Explorer”. Both windows are framed by the menu and tool bar in the header and the file information in the footer. File
Menu to import raw data for analysis: “Open Raw Data File” with the extension “.cmcr” created with “CMOS-MEA-Control” software for analysis and to “Exit” the program. Menu to “Load Result File” with the extension “.cmtr” for reanalysis and to save result files. Menu to “Exit” the program.

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Following file formats are available for CMOS-MEA-Tools:
· .cmcr: CMOS-MEA-Control Raw Data · .cmct: CMOS-MEA-Control Templates · .cmtr: CMOS-MEA-Tools Results · .cmtt: CMOS-MEA-Tools Templates · *.cmte: CMOS-MEA-Tools Export
Note: Doubleclick a “CMOS-MEA-Control Raw Data” or “CMOS-MEA-Tools Results” file to open it directly from a folder without starting the CMOS-MEA-Tools software before. Analyze
Menu for analysis of the imported file. Start the analysis with the “Process Segment” command for a segment and with the “Process File” command for the complete file. Click “Batch Analysis” to analyze more than one file in a batch. Settings
Menu to set “Application Settings” and to load and save templates. Menu to open the “Labbook” dialog.

Dialog with three tabs for general information about the experiment. Fill in data referring to your experiment in “Study” tab, “General” notes about the scientist and institution and tags and free text in “Tags and Notes” tab. The “Labbook Settings” will be stored in the “CMOS-MEA-Tools” result file “*.cmtr”. Help
Menu to open the “Online Help” and to “Check for Update” and see information in “About”.

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The option “Check for Update” provides fast access to MCS website.

If the CMOS-MEA-Tools software is not up to date, click the button “Visit Website” and download of the newest software version or use the “Download” button to download the update directly.
Click “About” to open the dialog.

The dialog shows basic information about the CMOS-MEA-Tools software version. Note: Please keep in mind that the information of this dialog is necessary in case of support!
Toolbar

Open Raw Data File
Click “Open Raw Data File” in the “File” menu or click the “Open Raw Data” icon Data File” appears.

in the toolbar. The dialog “Select CMOS-MEA-Raw

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Click “Open Raw Data File” to open the dialog “Select CMOS-MEA Raw Data File”.

Select the input path. The program is scanning the folder for analyzable files: “.cmcr” CMOS-MEA-Control Raw data and “.cmtr” CMOS-MEA-Tools Result files. This search can take a little bit time, please see the bar in the “Scanning Input Folder” dialog.

Available files are listed in the upper part of the dialog. Select a data file from the list and find information about “File Name, Date, Duration, Events, Size, SW Version, Chip ID and Chip Info”. A selected file is highlighted in blue and the entries of the lab book of the respective file are shown in the lower part of the dialog.

Open a file with a double click on the selected file or by pressing the “Open File” “Search recursively” in the headline of the dialog, if necessary.

button. “Refresh” the list or click the check box

Open Result File

Click the “Load Result” button to open a result file

and the “Select CMOS-MEA-Results File” dialog opens. Result files have the

extension “*.cmtr”. Reanalyze a result file and save it again under a new name.

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Batch Analysis
This feature allows to analyze a number of files all at once, which is helpful in case of a huge amount of data files.
To process many files in a single run you can use the “Batch Analysis” built- in feature of the CMOS-MEA-Tools. The selected raw data files are processed one after another using a predefined set of tools and settings. For each raw data file, a separate result is stored at a predefined location. This allows to run a time consuming analysis of many files without constant supervision.

Click “Batch Analysis”

to open the dialog for the selection of the files for one batch.

Please select the “Input Path” in the header. The input path is the root directory used for the search for raw data files. If the check box “Search recursively” is selected, all subfolders of the input path are searched through. The files of each folder are shown as a group. Select all files by clicking the “Select/Deselect All” button or toggle the selection of a file by pressing the “Select/Deselect” button in the first column in front of the file name. The output path defines the root directory for the result files. If the raw data files are located in different folders, the same folder structure is generated for the result files. Start “Batch Analysis” by pressing the “Start” button. The selected files are processed from top to bottom and the status of the processing is depicted by progress bars. The batch analysis can be stopped by pressing the “Stop” button.
Analysis Recommended Workflow
1. Summary 2. Explore 3. Process File 4. Batch Analysis When loading a raw data file, the software will start in “Explore” mode. This allows to examine portions of the raw data in the “Raw Data Explorer”. An overview over the temporal and spatial spiking activity in your data set can be gained with the “Summary Tool”. Besides visualizing the raw data, the main use of the “Explore” mode is to test and adapt different settings, such as filter or spike detector parameters. Once you are satisfied, you may analyze the whole file with these settings using “Process File”. Finally, the “Batch Analysis” mode can be used to analyze multiple raw data files with one or more analysis parameter sets.

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Recommended Workflow for Results Files When loading a result file, the software will start in “Process File” mode and display the results contained in the file. As for raw data files, you can switch to the “Explore” mode to test new parameter sets on portions of the data and to view the raw sensor data. However, please be aware that switching to the “Explore” mode will discard the current results shown in the CMOS-MEA-Tools software. Pressing “Process File” will reanalyze the raw data file associated with the result file using the current parameter set. The progress of the data analysis is displayed immediately in the instrument tree view. Information of the actual analyzed file are displayed in the footer.

1. Summary

The “Summary” tool provides a rough temporal and spatial overview of the activity in the file by analyzing the whole file with a simple spike detection method. It can be very useful to determine regions of interest for a more detailed analysis with the “Explore” mode.

Press the button “Open Settings Dialog”

to optimize the spike detector and the visualization settings.

Adjust the settings for the analysis. It may be necessary to play a little bit with the settings until you find the best settings.
2. Explore Mode
The “Explore” mode works by loading a time segment of the raw data into memory which can then be visualized in the “Raw Data Explorer” or used to test different analysis parameters.
The loaded time segment is highlighted in light blue in the “Explore Activity” tab. Its duration is limited to a maximum of 3 seconds to not exceed the memory capacity. If other analysis tools are active, they will analyze the loaded data segment as well. Because running a full spike sorting analysis on the loaded data segment might take a long time, the “Spike Sorter” will only perform the ROI detection in “Explore” mode.
Please use the “Process File” or “Batch Analysis” mode to run a full spike sorting analysis.

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3. Process File After setting the analysis parameters, for example after optimizing them via the “Explore” mode, press “Process File” to analyze the complete file. For analysis, the data is loaded in short time segments and processed by all active analysis tools. The analysis progress is visualized in the “Explore Activity” view. If the “Spike Sorter” and / or the “STA Explorer” tool are active, it will take more than one pass through the file to finish the analysis. The “Raw Data Explorer” is disabled in “Process File” mode, because it may not be possible to load the full raw data file into memory for visualization. The amount of time it takes for a complete analysis of a file depends on the length of the recording and the performance of the computer. Please be aware that running the spike sorting analysis can take a considerable amount of time.
4. Batch Analysis The “Batch Analysis” mode can be used to analyze one or more raw data files with different analysis sets. Please read the chapter “Batch Analysis” above for more information. The amount of time it takes for the batch analysis depends on the length of the recordings and the performance of the computer. Please be aware that running the spike sorting analysis can take a considerable amount of time.
Control Window

The “Control Section” on the left side of the display consists of three windows: The “Instrument Tree” of the available instruments above, the temporal “Activity Summary” window and the spatial distribution “Summary” window below.
Toolbar

Click the “Create Summary” icon calculation.

or use menu “Analyze” for loading the data in the RAM. Now the raw data are available for

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Click the “Open Settings Dialog”

and the following dialog appears. Adjust the settings for the analysis.

Please define the threshold. Select the “Threshold Type” from the drop-down menu, “Positive”, “Negative” or “Absolute”. Choose the “Threshold” value and the “Detection Dead Time” in ms with the up-down boxes. Select the “Bin Size” in ms with the up-down box. Choose the color of “Color Map”. To invert the colors, please click the check box “Invert”. Instrument Tree
The “Tree View” shows all available instruments and the actual status. Click any of the instruments in the tree view to adjust any parameter. Open the respective tabbed page of that instrument on the “Data Display or Settings” window, for example “Spike Sorting” as shown in the screenshot below. Please read chapter ,,Spike Sorter Tool” on page Fehler! Textmarke nicht definiert. for detailed information.

When clicking the “Explore Data Segments” button

, all data selected in the current data segment of the “Activity Summary”

window are analyzed. Or click “Process File” button

to start analysis for the complete file. During analysis processes you

can observe the progress indicated by the data flow, highlighted in pale blue.

After successful analysis the involved instruments show a check mark.

In cases with huge amounts of data it may be not possible to load a complete file into the RAM and the analysis has to be repeated or if the analysis was stopped or was interrupted for any reason, the respective instrument has no check mark. This way it is easy to control the analysis.

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Activity Summary

Both windows display a summary of the detected spikes of the complete file. The diagram above shows the temporal overview, the map below shows the spatial overview.
Explore Activity

The temporal overview shows the number of detected spikes per bin added up from all channels for the complete time span of the file. So you can see the most active time period during the recording at a glance.
Choose a time span you want to analyze in the “Time” diagram. Click the icon “Explore manually defined interval” to set the time span manually.
Click the blue bars and move them via drag and drop to the desired positions. The time between the blue bars, which will be calculated is highlighted in blue. The spikes per bin are calculated against the time. Zoom into the data by moving the mouse from the left to the right and to zoom out from the right to the left.
Events

Click in the “Activity Summary” window the tabbed page “Events” to list all digital events from the stimulator, recorded in that file with the respective time stamp in the scale. The different stimulator events are color coded. Select one or more than one event which is then highlighted in green and use these events for navigation through the file in the “Explore Activity” tab.

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The button “Explore Event based”

and the tool bar beside is now available.

The selected events are visible color coded below the data. Jump via arrow buttons “Go to next / previous Event” from event to event, the actual time segment indicated by the blue bars as usual. Please define the time segment “Pre” and “Post” the digital event in ms with the up-down boxes. While jumping from event to event the data displays in the “Raw Data Explorer” window on the right side are adapted immediately.

In future versions of this software, the event based analysis will be available. Please check the MCS web site for news.

Spatial Distribution
The spatial overview shows the number of detected spikes per channel for the complete file color coded in the map. It is easy to observe the most active areas in the array.

The numbers in brackets are the coordinates of the mouse in the spike distribution map.
Adjust the “Range” with the up-down box. The range is based on the number of spikes per sensor. The lower the range, the higher the spatial distribution of the sensors with spikes added up to the “Range” value. The color of the map indicates the centers of activity additionally.

Define the color of the map in the “Summary Tool Settings”

dialog.

Data Display and Settings Window This window has five tabbed pages in relation to the instruments in the “Instrument Tree”. Filter

Three types of filters are provided: “Time Filter” like “Band-Pass”, “High- Pass”, “Notch” and “Low-Pass”, “Space Filter” and “Artefact Filter”. Click the “Filter” icon in the “Instrument Tree” and the filter setting icons are available.

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Filter Pipeline Create a filter pipeline in the “Filter” control window. Click onto the desired filter symbol and move it via drag and drop into the header queue.
The selected filter is highlighted in blue and the respective settings parameter or descriptions are available in the window. See a preview of “Raw” and “Filtered” data in the small windows below, if available.

Filter Toolbar

Click the “Process Data” icon please click the bin.

for loading the selected data in the RAM. To clear the filter queue,

Store the filter chain and open it later.

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Raw Data Explorer Load a raw data file and click “Explore” button in the tool bar of the main menu.
Select the “Raw Data Explorer” tab in the “Data Display or Settings” window.

The “Raw Data Explorer” tab displays spike activity: The “Overview” for all electrodes above and the “Single View” of one electrode below. Zoom into a detailed view in the “ROI Region of Interest”. The “Raw Data Explorer” replays the activity of the raw data file as well in overview as detailed in a region of interest or in the single view. The “Activity” window shows voltage values per time bin as a false color image. Spikes appear as blue or red dots, usually on several neighboring pixel in the map simultaneously.
Raw Data Explorer Tool Bar

Define the “Frame” for the data sample with the up-down box. Click the “Step Backward” and the “Step Forward” buttons to replay the data step by step in each direction. For replaying the data continuously, click the “Start / Pause Movie” button. To “Slow Down” the velocity of the data movie, please use the up-down box. The higher the selected digit the slower the movie will run, because the entered number

will be added in ms after each frame. Set the range of the movie with the bars and loop the movie. Arm the video recording with the

“Record Video” button

and name and save the video in “*.mp4” format. Change video settings by clicking the “Open Settings Dialog”

button

in the “Video Recorder Settings” dialog.

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Select the “Resolution” and the “Frame Rate” for the video from the drop down menus. Enable the “Include Timestamp” check box, if necessary.

Start or stop the video with the “Start / Pause Movie”

button. Open the video with a media player.

Single View
After clicking the “Set Movie Range” button, two blue sliders appear in the “Single View” below. Move them with the mouse via drag and drop to define the time segment you want to analyze in the movie. The green bar indicates the actual position of the “Activity” display.

Please see also chapter “General Software Features” for adjusting the displays.
The tool bar below the “Activity” window show the coordinates of the cursor position on the left side. Move the complete ROI with the left mouse button while a symbol of a hand appears. Change the size of the ROI when an arrow symbol appears. Or click the “Set ROI” button
to open the following dialog. If more than one ROI is available, the actual cursor will be adjusted.

Adjust the “Position (X/Y)” and the “Size (W/H) of the ROI with the up-down boxes. Select the color of the cross hair from a color scale, if the “Show Cross Hairs” check box is activated.

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Select the “Threshold” for spike detection and the “Range” from the up-down boxes. The smallest unit of the range is “nV”.
Click the “Setting” button for the color map in the “Activity” window.
Select the colors for the map from the drop-down menu. To “Invert” the colors activate the check box. Zoom Buttons in the “Detailed View” and “Single View” Windows
Click the “Adjust to signal Min/Max” button. The scaling of the y-axis is set to the minimum and maximum of all visible samples in the channel.
Click the “Zoom” buttons. Zooming in cuts the scaling of the respective axis in a half and zooming out doubles the scaling. Export data from the “Single View” as image (“.bmp”, “.jpg”, “png”) or as ASCII in “.csv” format. Spike Explorer Window Select the “Spike Explorer” tab in the “Data Display or Settings” window. Use the “Spike Explorer” for the definition of the spike parameter and the visualization of the detected spikes.

The “Spike Explorer” main window is divided in three sections, the spike activity in overview, and two windows for regions of interest ROIs. One regions of interest shows overlay plots, the other the temporal distribution of the spikes. Select from the “Spike Activity Overview” on the left side one or more regions of interest. The overlay plots of the detected spikes are displayed on the right side. The window below shows the temporal distribution in time stamps for each selected ROI.

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Spike Explorer Toolbar

Click the “Process Data” icon

for starting the analysis by processing the data loaded in the RAM.

Click the “Export Data” button

. Select whether to export the spikes “Complete” or “Selected” via drop down menu. Data can be

exported in “HDF5” or “CSV” format.

Click the “Spike Explorer Settings” button Detection” windows.

to define the “Spike Detection” in the “Detection”, “Spike Cutout” and “Activity Peak

Spike Detection
Please set the “Detection” parameter from the upper window: The “Threshold Type” and the “Noise Measure” from the drop-down menus, the “Threshold” and the “Detection Dead Time” in ms from the up-down boxes. Define the “Spike Cutout” parameter from the lower window: The “Waveform Alignment” from the drop-down box, the “Pre and Post Intervals” from the up-down boxes.
Activity Peaks Detection
Click the check box “Auto Extract while process File” in the “Spike Explorer Settings” dialog if you do not set the extraction parameter manually.

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Spike Activity Overview

The coordi

References

• The HDF Group - ensuring long-term access and usability of HDF data and supporting users of HDF technologies
• www.multichannelsystems.com | Innovations in Electrophysiology

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