Light Delivery Systems for Optogenetics

Introduction Optogenetics is a technology that allows targeted, fast control of precisely defined events in biological systems, by delivering optical control at the speed (millisecond scale) and with the precision (cell type specific) required for biological processing. Mightex has developed a range of Light Delivery Systems for optogenetics, as described below. Mightex s Light Delivery Solutions Overview (1) Fiber-Coupled LEDs for in vivo Optogenetics Fiber-coupled LEDs are capable of precisely delivering light to specific cell(s) of interest, without disturbing other cells in the vicinity. Moreover, multiwavelength fiber-coupled LEDs enable one to switch wavelengths (e.g. 470nm and 590nm) of the light delivered to the cell(s) through the same fiber, without the need of any mechanical movement or the need to physically switch fibers. This will allow fast excitation and fast inhibition of the cell(s). Mightex s FCS- and WFC-series fiber-coupled LED sources are designed for such in vivo optogenetics applications. Control Signals Manual Analog TTL Software - customer defined. LED Controllers SLA-1000-2 SLC-MA04-MU BLS-SAxx-US Fiber-Coupled LED s - with single or multiple LED s coupled into one (1) output fiber, without any moving parts in the optical path. Up to 8x LED s can be coupled into the same fiber. FCS-series WFC-series (Single LED) (Up to 4x LEDs) (Up to 8x LEDs) Interchangeable fiber (e.g. 50/100/200µm) Standard SMA Connector Rotary Joint Fiber-Optic Cannula 2

(2) Polygon400 Patterned Illuminators for ex vivo or in vitro Optogenetics For ex vivo or in vitro Optogenetics, microscopes are usually used in order to achieve the spatial resolution required to view individual cells, and patterned illuminators are needed in order to precisely control (i.e. activate/silence) the cells of interest without disturbing other cells within the microscope s Field of View (FoV). Mightex s Polygon400 Dynamic Spatial Illuminators are designed for such ex vivo and in vitro optogenetics applications. Control Signals LED Controller Light Sources Software/PC TTL BLS-PLxx-US LEDs Lasers Others * USB TTL Integrated/ Built-in Lightguide Fiber Coupling Mechanism Polygon400 Patterned Illuminators DSI-x-xxx... Φ ~ mm Φ ~ 100m Key Features: DLP based Patterned Illuminator; Simultaneous illumination of multiple areas of interest; Any custom-defined shape/size of illumination pattern; Microscope Adapters For Upright Microscopes For Inverted Microscopes Up to 4000 frames per second; USB2.0 interface, easy to install; TTL trigger input/output; Adapters for various microscopes; DSI-CUBE-xx-UA DSI-RING xx Built-in LEDs, or custom light sources via fiber or lightguide; Intuitive/powerful software for spatial/temporal/spectral control. 590nm 470nm Polygon400 on an Upright Microscope Polygon400 on an Inverted Microscope * A BLS-PLxx-US LED controller is only needed when an external LED source is used with the Polygon400. Integrated/built-in LEDs have their own integrated LED controllers. 3

Mightex s LED Sources Portfolio As far as optogenetics applications are concerned, LEDs are able to simultaneously meet both the speed (~ms) and the spatial precision (i.e. cell/neuron specific) requirements. Compared to lasers, LEDs excel in many respects: they are cheaper, smaller, more reliable, and easier to control. In addition, there are a wide range of LED wavelengths available in the market, making it easy to find the best wavelengths to interact (i.e. excite or inhibit) with neurons infected with various optogenetics tools, such as 470nm for channelrhodopsin (ChR2) and 590nm for halorhodopsin (NpHR). Mightex has developed the most comprehensive LED source solutions in the market. The chart below shows the wavelength portfolio of Mightex s LED s. In order to satisfy the specific needs of various customer applications, Mightex offers an extensive range of beam formats with our LED solutions, including collimated, uniform, focused, divergent, fiber-coupled, and lightguide-coupled LED sources. Microscope adapters are also available, in order to enable customers to mount the LED sources onto various microscopes. Please refer to the diagram below on how to choose the best beam format (and microscope adapters) for your specific applications. If you require multiple wavelengths for your application, Mightex also offers a range of multi-wavelength LED solutions, as below: (1) Wavelength-switchable LED's with collimated output beam (WLS-series). With an optional lightguide adapter, the output beam can also be coupled into a liquid lightguide; (2) Multi-wavelength fiber-coupled LED's (WFC-series); and (3) Multi-wavelength collimated LED's using beam combiners. With an optional lightguide adapter, the output beam can also be coupled into a liquid lightguide. Various microscope adapters are also available for those customer who would like to couple the LED light into microscopes. 4

LED Controllers In optogenetics research, scientists may have the need to control LED s in one or more of the following ways: (1) manually (e.g. with a turning knob); (2) with TTL pulses; (3) with analog signals; and (4) with software. Mightex has developed a range of LED controllers for this purpose, with detailed described below. (1) LED Controller Product Summary and Selection Guide Keys: Manual TTL Analog Software SLA-1000-2 SLC-MA04-MU SLC-AA02-US / SLC-AA04-US BLS-SA02-US / BLS-SA04-US Control Signals Number of Channels (per device) 1 2 4 2 / 4 2 / 4 Minimum Pulse Width ~1ms 1ms 20s 20s Accepts TTL Input to Encode Output Pulse Pattern No No Yes Yes Accepts TTL Input to Trigger Pre-defined Pulse Pattern No No Yes Yes Manual Control via Turning Knobs Yes Yes No No Accepts Analog Input Control Yes No No No Software control via GUI / SDK No Yes Yes Yes Support Arbitrary Output Waveform/Pattern Via external analog input (0~5V) Via custom program based on SDK Easily definable via Mightex s software GUI / SDK Easily definable via Mightex s software GUI/SDK Note: (1) Each channel is capable of independently controlling an LED or a string of LEDs (all in sync with each other). For example, with a four (4)-channel LED controller, one can control up to four (4) LED s INDEPENDENTLY. If however one needed to control more than four (4) LED s, he could use multiple LED controllers. (2) SLA-1000-2 Manual and Analog LED Controller SLA-1000-2 SLA-1000-2 LED controller has two (2) channels, and each can be operated in one of the following two modes: - Manual Mode: The output current (in CW) can be adjusted manually via a turning knob, ranging between 0 and the Maximum Current stated below; - Analog Input Control Mode: The output current can be controlled via a 0~5V analog input. The maximum modulation frequency is ~1KHz. Each channel has a three (3) digit DIP switch (as shown in the picture on the right): the 1st digit is used to set the working mode, while the 2nd and 3rd digits are used to set the Maximum Current, which allows one to limit the maximum current that passes through the LED to match its current rating, in order to prevent LED damage due to overdriving. There are three (3) current limit settings: 350mA, 500mA and 1000mA, respectively. DIP Switches 1 2 3 A B Parameters SLA-1000-2 Number of Channels 2 DIP Switch Setting Instruction A: Control Mode Setting B: Current Limit Setting Maximum Output Current (Settable via DIP Switches) Power Supply (provided by Mightex, 12V) Input Voltage, V dc 350mA / 500mA / 1000mA 9 ~ 24 V 1 Manual Mode 2 3 Imax = 350mA Maximum Output Voltage Analog Input Control Signal Dimensions Weight (V dc - 3.0) V 0~5 V 80mm (L) x 64.3mm (W) x 23.7mm (H) 60g 1 Analog Input Control Mode 2 3 2 3 Imax = 500mA Imax = 1000mA Note: SLA-1000-2 is a UNIVERSAL LED controller designed to drive LED s of any made and model. It is NOT limited to just Mightex s LED s. 5

(3) SLC-MA04-MU Manual and Software LED Controller SLC-MA04-MU Mightex's SLC-MA04-MU four-channel universal LED controller is designed to drive a broad range of LED light sources, and it offers the flexibility for users to operate each LED channel independently, both manually and through software. The software comes with a user-friendly GUI that enables one to drive LEDs without the need to write any code. Furthermore, a full-featured SDK is provided in order for users to write their own software and to integrate the LED controller into their own systems. 1. Manual Mode: Each of the four channels can be operated manually in CW mode using a knob. The LED controller also has a fifth knob (i.e. a Global knob) that enables one to adjust the output current of all the channels at the same time and with the same step size. Therefore, one can first set the intensities for the LED s - independently - using the four (small) knobs, and then can increase/decrease the set intensities of all channels simultaneously using the Global knob. In order to prevent LED damage due to overdriving/overheating, the maximum output current of each LED channel can be set individually, via the software provided with the LED controller, to match the current rating of the LED connected to the channel. 2. Software Mode: In software control mode, each channel can be individually configured by the software to operate in one of the following three modes: To PC a. Disable Mode: The channel is disabled, and its output is completely turned off. b. Normal Mode (or CW Mode): The output current is constant, which can be adjusted (using software) from 0mA to 1,200mA through the USB interface. c. Strobe Mode: A Pulse-Width-Modulated (or PWM) periodic strobe pattern is output from the channel, which can be activated by a software trigger. The strobe pattern may last indefinitely or for a preset number of cycles, depending on the software setting. The maximum frequency of the PWM strobe is 500Hz. Parameters SLC-MA04-MU Number of Channels 4 Maximum Output Current (Settable via software) Power Supply (provided by Mightex, 12V) Input Voltage, V dc Maximum Output Voltage Minimum Pulse Width Timing Resolution Output Current Resolution Output Current Accuracy Output Current Repeatability Interface Up to 1200mA 12 ~ 24 V (V dc - 3.0) V 1ms 0.1ms 1mA ±5 ma or ±1.0%, whichever is larger ±2 ma or ±0.5%, whichever is larger USB2.0 Device Per Computer* 16 Dimensions Weight 180.5mm (L) x 180mm (W) x 34.5mm (H) 400g * The LED controller s software enables up to sixteen (16) SLC-MA04-MU LED controllers to be operated simultaneously by a single computer, leading to a maximum of 64 channels in total. Note: SLC-MA04-MU is a UNIVERSAL LED controller designed to drive LED s of any made and model. It is NOT limited to just Mightex s LED s. 6

(4) SLC-AA02-US and SLC-AA04-US LED Controllers with Software and TTL Triggering SLC-AA02-US or SLC-AA04-US Mightex's SLC-AA02-US and SLC-AA04-US universal LED controllers are designed to drive a broad range of LED light sources, and they enable one to operate each LED channel independently through software with a user-friendly GUI. Furthermore, a fullfeatured SDK is provided in order for users to write their own software and to integrate the LED controller into their own systems. The SLC-AA02-US and SLC-AA04-US LED controllers can also be controlled via TTL trigger pulses. There are two trigger modes (which are settable/selectable via the software): (1) one can pre-define a pulse sequence (or pattern) in software and load/save it to the LED controller s non-volatile memory, and a TTL pulse can then be used to trigger/activate the pre-defined pulse pattern; or (2) one can pre-set the output current (or the LED s intensity), and then use a train of TTL pulses to encode (the timing of) the LED s output pulse pattern. Since in the latter case, the LED s output pulse pattern strictly follows the input TTL pulse patter, it is also called Follower Mode. USB RS232 With an SLC-AA02-US or an SLC-AA04-US LED controller, one can use up to 128 pairs of [current (ma), duration (μs)] data points to define the 'shape' of the waveform. This will allow one to define an 'Arbitrary Waveform' for the LED driving current and consequently the LED's optical output. Details see diagram below. To PC Parameters SLC-AA02-US / SLC-AA04-US Number of Channels 2 / 4 Maximum Output Current (Settable via Software) Power Supply (provided by Mightex, default 12V) Input Voltage, V dc Maximum Output Voltage Minimum Pulse Width Timing Resolution Output Current Resolution 1000mA in CW mode 3500mA in strobe mode 12 ~ 24 V (V dc - 0.5) V Output Current Repeatability ±1mA (or +/-0.2%) Interface (Settable via a Flip Switch on the Back Panel) Arbitrary Waveform Definition Accepts TTL Trigger 20s 20s 1mA USB2.0 or RS232 Up to 128 points of [current (ma), time interval (s)] Device Per Computer* Up to 16 Dimensions Weight Yes 201mm(L) x 147mm (W) x 40mm (H) * The LED controller s software enables up to sixteen (16) SLC-MA04-MU LED controllers to be operated simultaneously by a single computer, leading to a maximum of 32 channels for SLC-AA02-US and 64 channels for SLC-AA04-US, respectively. Note: SLC-AA02-US and SLC-AA04-US are UNIVERSAL LED controllers designed to drive LED s of any made and model. They are NOT limited to just Mightex s LED s. 600g 7

(5) BLS-Series BioLED Light Source Control Modules BLS-SA02-US, BLS-SA04-US, BLS-PL02-US, and BLS-PL04-US Mightex BLS-series BioLED light sources are modularized fully-customizable turn-key solutions for optogenetics, fluorescence excitation, and other biophotonics applications. Precisely-timed and high-intensity light pulses are required in optogenetics experiments to activate channelrhodopsins (ChR2, ChR1 etc.) and halorhodopsins (NpHR) in order to excite and inhibit neurons. To meet these requirements, Mightex has developed a proprietary IntelliPulsing technology to allow BLS-series sources to output significantly higher power in pulse mode than what the LEDs are rated for in CW mode. (Front View) All standard Mightex LED light sources can be integrated into the BLS-series light source system. Furthermore, customers may choose optical heads with different wavelengths and formats to be integrated with the same control module. Multiple control modules can be stacked in software to support more than 4 optical heads. The control module features a linear LED driver design that eliminates light intensity ripples and oscillations often observed when low-cost buckpuck nonlinear drivers are used. Clean and highly repeatable pulses are critical to quantitative experiments. Both CW mode and pulse modes are supported. Time resolution of the control module is 20s and light intensity can be adjusted with 0.1% increments. Each driving channel on the control module has its own TTL trigger input. Rising edge, falling edge, and follower mode are supported in the trigger mode. (Back View) The control module can be operated without being connected to a computer. Once pulse sequences are programmed and stored into the control module (by user through software), the light source can operate alone without a computer. All it needs is a TTL trigger signal to output the user-programmed pulse sequences. Mightex BioLED light sources come with a Windows-based operation software featuring an intuitive yet powerful graphic user interface. A software development kit (SDK) is also provided for user integration into environment such as Labview and Matlab. (Back Panel) BLS-PLxx-US BioLED control modules are specifically designed to synchronize LED s with Mightex s Polygon400 Patterned Illuminators, in order to support synchronization between external LED s with custom pre-defined illumination patterns using external TTL trigger pulses. BLS-SA02-US BLS-SA04-US BLS-PL02-US BLS-PL04-US Synchronization with Polygon No Yes Number of channels 2 4 2 4 Output Intensity Resolution 0.1% Time Resolution External Trigger Trigger Connector Optical (LED) Head Connector Host Interface On-Device Memory 20s TTL BNC 2-pin Aero Connector USB2.0 or RS232, selectable Yes Device Per Computer* Up to 16 Dimensions Weight 213mm (L) x 156mm (W) x 73mm (H) 800g * The LED controller s software enables up to sixteen (16) BioLED controllers to be operated simultaneously by a single computer, leading to a maximum of 32 channels for BLS-SA02-US and 64 channels for BLS-SA04-US, respectively. 8

Fiber-Coupled LED Sources Mightex has developed two (2) catogories of fiber-coupled LED sources: (1) FCS-series single-wavelength fiber-coupled LED s; and (2) WFC-series multiwavelength fiber-coupled LED s. This section describes the fiber-coupled LED selection guide as well as the detailed specifications of the LED sources. (1) Fiber-Coupled LED Product Summary and Selection Guide Features FCS-Series WFC-Series Number of Wavelengths/LEDs 1 Up to 8 Wavelength Range (nm) 240~940nm, or white LEDs 240~940nm, or white LEDs Output Fiber Interchangeable (e.g. 50/100/200µm etc.) Interchangeable (e.g. 50/100/200µm etc.) Fiber Connector SMA SMA Note: For wavelength availability, please refer to Mightex s LED wavelength portfolio. (2) FCS-Series Single-Wavelength Fiber-Coupled LEDs Mightex FCS-series single-wavelength fiber-coupled LED light sources employ the latest high-power LED technologies and a proprietary coupling optics to achieve maximum optical output power. Optical output is coupled into a fiber through a standard SMA fiber adaptor port (SMA fiber patch cords are sold separately). FCS series also features a locking electrical connector for secured connection. The onepiece machined housing features multiple mounting holes. All Mightex LED controllers can be used to drive the FCS-series light sources. FCS - -XXX Wavelength Code (Refer to table below) Configuration Code (default: 000 ) Interchangeable fiber SMA connector For example, FCS-0470-000 is a 470nm fiber-coupled LED with an SMA fiber connector. For optogenetics applications, light intensity (i.e. power per unit area, in mw/mm2) is a more important measure than light power (mw) itself. The table below summarizes the output power (mw) and the intensity mw/mm^2) of typical Mightex s FCS-series single-wavelength fiber-coupled LED s. Peak Wavelength (nm) 1 Description Wavelength Code Output Power (mw) 2 Intensity (mw/mm^2) 365 UV 365nm 0365 4.6 146 385 UV 385nm 0385 4.6 146 400 400nm 0400 5.1 162 420 420nm 0420 2.0 64 455 Royal Blue 0455 5.5 175 470 Blue 0470 6.3 201 505 Cyan 0505 2.7 86 530 Green 0530 1.6 51 590 Amber 0590 0.9 29 617 Red-Orange 0617 5.1 162 625 Red 0625 5.1 162 656 Deep red 0656 5.1 162 680 Deep Red 0680 1.1 35 740 NIR 0740 2.8 89 780 NIR 0780 1.7 54 850 NIR 0850 3.1 99 870 NIR 0870 1.1 35 940 NIR 0940 3.1 99 Output power measured here! Note: (1) Other wavelengths and white LED s are also available; (2) Output power measured at the output tip of a 200µm 0.39NA fiber. Higher output power can be obtained under certain conditions and in Intellipulsing mode with a BLS-SA02-US or a BLS-SA04-US BioLED control module. Please email sales@mightex.com or call +1-925-218-1885 for details. Related Products: LED Controllers: SLA-1000-2, SLC-MA04-MU, SLC-AAxx-US, BLS-SAxx-US Fiber Patch Cords: FPC-0200-22-01SMA, FPC-0200-22-02SMA, FPC-0200-37-01SMA, FPC-0200-37-02SMA 9

(3) WFC-Series Multiwavelength Fiber-Coupled LEDs Mightex s WFC-series multi-wavelength fiber-coupled light sources are enabled by the latest LED technologies and Mightex s proprietary beam combining/coupling optics. Up to eight (8) LEDs are coherently combined into a single multi-mode fiber with the highest efficiency practically possible. Optical output is coupled into a fiber through a standard SMA fiber adaptor port, and customers can use fibers with different core diameters with the WFC module. Each LED in the module can be powered individually or simultaneously, making the WFC-series a new class of light sources with a tunable spectrum. Up to 4x LED s Up to 8x LED s In optogenetics, for example, one might want to launch 470nm to excite ChR2 in a neuron, and then fast (in sub-ms) switch to 590nm to activate NpHR in order to inhibit the same neuron. This can be achieved by using a WFC-H2-0470-0590-000 which enables sub-ms switching between 470/590nm through the same fiber, and this can all be done electronically (through software) and there is no need for any mechanical movement. WFC - X N - XXXX - XXXX -... XXXX - XXX S - Standard configuration H - High-power configuration Number of wavelengths 1st Wavelength code 2nd Wavelength code (repeat if applicable) N-th Wavelength code Internal use For example, WFC-H4-0400-0470-059-0656-000 is a high-power configuration WFC light source with 4 wavelengths of 400nm, 470nm, 590nm, and 656nm. Output power measured here! Wavelength 1 (nm) Wavelength Code Output Power 2 (mw) / Intensity (mw/mm^2) 2-wavelength 3-wavelength 4-wavelength 5-8 wavelength 365 0365 3.8mW (120mW/mm^2) 3.6mW (115mW/mm^2) 3.4mW (108mW/mm^2) 3.1mW (98 mw/mm^2) 385 0385 3.8mW (120mW/mm^2) 3.6mW (115mW/mm^2) 3.4mW (108mW/mm^2) 3.1mW (98mW/mm^2) 400 0400 3.5mW (110mW/mm^2) 3.3mW (105mW/mm^2) 3.1mW (100mW/mm^2) 2.8mW (90mW/mm^2) 420 0420 1.3mW (40mW/mm^2) 1.2mW (38mW/mm^2) 1.1mW (35mW/mm^2) 1.0mW (33mW/mm^2) 455 0455 4.4mW (140mW/mm^2) 4.2mW (133mW/mm^2) 4.0mW (128mW/mm^2) 3.5mW (113mW/mm^2) 470 0470 4.7mW (150mW/mm^2) 4.5mW (143mW/mm^2) 4.2mW (135mW/mm^2) 3.8mW (123mW/mm^2) 505 0505 2.2mW (70mW/mm^2) 2.0mW (65mW/mm^2) 2.0mW (63mW/mm^2) 1.8mW (58mW/mm^2) 530 0530 1.9mW (60mW/mm^2) 1.8mW (58mW/mm^2) 1.7mW (55mW/mm^2) 1.5mW (48mW/mm^2) 590 0590 1.0mW (33mW/mm^2) 0.9mW (30mW/mm^2) 0.9mW (30mW/mm^2) 0.9mW (28mW/mm^2) 617 0617 4.1mW (130mW/mm^2) 3.8mW (123mW/mm^2) 3.7mW (118mW/mm^2) 3.3mW (105mW/mm^2) 625 0625 4.8mW (153mW/mm^2) 4.6mW (148mW/mm^2) 4.3mW (138mW/mm^2) 3.9mW (125mW/mm^2) 656 0656 4.1mW (130mW/mm^2) 3.8mW (123mW/mm^2) 3.7mW (118mW/mm^2) 3.3mW (105mW/mm^2) 740 0740 1.9mW (60mW/mm^2) 1.8mW (58mW/mm^2) 1.7mW (55mW/mm^2) 1.5mW (48mW/mm^2) 850 0850 2.5mW (80mW/mm^2) 2.4mW (75mW/mm^2) 2.3mW (73mW/mm^2) 2.0mW (65mW/mm^2) 870 0870 1.9mW (60mW/mm^2) 1.8mW (58mW/mm^2) 1.7mW (55mW/mm^2) 1.5mW (48mW/mm^2) 940 0940 2.5mW (80mW/mm^2) 2.4mW (75mW/mm^2) 2.3mW (73mW/mm^2) 2.0mW (65mW/mm^2) Note: (1) Other wavelengths and white LED s are also available; (2) Output power measured at the output tip of a 200µm 0.39NA fiber. Higher output power can be obtained under certain conditions and in Intellipulsing mode with a BLS-SA02-US or a BLS-SA04-US BioLED control module. Please email sales@mightex.com or call +1-925-218-1885 for details. Related Products: LED Controllers: SLA-1000-2, SLC-MA04-MU, SLC-AAxx-US, BLS-SAxx-US Fiber Patch Cords: FPC-0200-22-01SMA, FPC-0200-22-02SMA, FPC-0200-37-01SMA, FPC-0200-37-02SMA 10

Polygon400 Spatiotemporal Patterned Illuminators for Optogenetics DSI- x- xxxx- xxxx- xxxx- xxx I - with built-in LEDs G - with fiber/lightguide input 1 st wavelength code 2 nd wavelength code 3 rd wavelength code Reserved for internal use Not applicable for lightguide/fiber based Polygon models For example, DSI-I-0470-0590-000 is a Polygon400 module with two built-in LED sources: 470nm and 590nm. DSI-G-000 is a Polygon400 module with a lightguide as input light source. Spatially patterned illumination with temporal and spectral control enables numerous new techniques in life-science applications such as optogenetics. For example, in optogenetics, selected neurons in a specimen slice can be activated or silenced with a user defined illumination pattern. Mightex Polygon400 multi-wavelength dynamic spatial illuminator (DSI) integrates the state-of-the-art spatial light modulators and high-power LEDs using a proprietary Etendue-preserving optical design to deliver high-intensity illumination patterns with diffractionlimited resolution. A Texas Instruments DLP spatial light modulator is used to display a user defined image pattern. At the heart of the Polygon400 is a unique optical system that carefully delivers light from LED sources to the DLP panel and then through a microscope to the specimen plane. Such a systematic approach makes it possible to achieve maximum optical intensity while maintaining diffraction-limited imaging performance. Temporal performance is a key to many intended applications for Polygon400. With a frame rate of more than 4,000 fps and fastswitching LEDs, Mightex Polygon400 can deliver illumination patterns with micro-second precision. Advanced DSI models with external LED controllers also enable wavelength switching in between illumination patterns at the highest frame rate. A dedicated software allows users to generate illumination patterns as well as control illumination intensity and timing. The software also supports alignment between illumination patterns and images acquired through any digital cameras on a microscope. Projection Area Projection Area (FOV) and Pixel Resolution (at 1X magnification) Common Microscopes Leica Nikon Olympus Zeiss Height (mm) 8.7 8.7 7.8 7.2 Width (mm) 15.5 15.5 13.9 12.7 Diagonal (mm) 17.7 17.7 16.0 14.6 Pixel size (m) 18.0 18.0 16.2 14.8 Note: To calculate illumination area and pixel resolution at the specimen, simply divide the above numbers by the magnification of the objective. For example, under a 10x objective on an Olympus microscope, the illumination area will be 0.78mm x 1.39mm with a pixel resolution of 1.62m. Available LED Wavelengths, and Output Power and Intensity in Pattern Sequence Mode Wavelength (nm) Output Power 1 (mw) Intensity (mw/mm^2) 400 14.0 53 470 15.0 55 530 4.5 17 590 3.0 11 617 14.0 52 630 14.0 52 656 14.0 52 1. These are estimated power at specimen under a 20x 0.75NA Olympus objective. System and communication USB2.0 interface. 5Vdc 3A input power. Windows 7 (recommended), Windows XP Service Pack 2 or greater Screen resolution of 1366x768 or higher 11

Polygon400 Spatiotemporal Patterned Illuminators for Optogenetics (Cont d) Control and Timing Parameters Minimum Maximum Unit Allowed bitmap depth 1 8 bit Exposure time @ 1bit 250 - s Frame rate @ 1bit 4000-1/sec Exposure time @ 4bit 1600 - s Frame rate @ 4bit 625-1/sec Exposure time @ 8bit 8333 - s Frame rate @ 8bit 120-1/sec Input trigger TTL, BNC connector - Input trigger delay TBD TBD s Output trigger TTL, BNC connector - Output trigger delay TBD TBD s Mightex Polygon400 is designed to be easily inserted into the infinity path of a microscope. For inverted microscopes, the preferred inserting point is usually the back port of the microscope where a fluorescence attachment is commonly placed. A filter cube is required to fold the Polygon400 s light path into the microscope. The filter cubes used for fluorescence observation serves this purpose well. For upright microscopes we provide a beam combiner cube to be inserted below the binocular/ trinocular unit. The dichroic or mirror in the beam combiner directs the Polygon400 beam into the microscope light path. (Left - Polygon400 beam combiner cube for upright microscopes; and, right - adapter ring for inverted microscopes.) Polygon400 Adapters for Microscopes Leica Nikon Olympus Zeiss Upright DSI-CUBE-LC-UA DSI-CUBE-NK-UA DSI-CUBE-OL-UA DSI-CUBE-ZS-UA Inverted DSI-RING-NK-TI TBD DSI-RING-OL-IA TBD 12