Brain_Slice_Patterned_Illumination

Following on our recent blog post 5 Considerations for selecting the best optogenetics light source to boost your project“, we’ll compare some of the most popular light delivery solutions for in vitro optogenetics activation here, based on the 5 considerations in our recent post. We are proud to be the neuroscience-focused distribution partner for the Mightex range (See below)

LED Light Sources For Widefield Optogenetics Activation

For widefield in vitro activation of optogenetic proteins, LED-driven light delivery systems seem to be the flavour of the day. They tend to deliver homogenous, high intensity light through the microscope’s optical path, are easy to control in terms of intensity and on/off timing, last a long time and are fairly inexpensive and safe to operate compared to laser light sources.

The comparison tables for the different types of light delivery systems are compiled from information available on the websites of the various suppliers.Some users and developers might be familiar with additional features, not necessarily noted here. Please take the time to contribute by adding comments at the bottom of this blog. We’ll update the tables when there are enough updates.

CoolLED Prizmatix Thorlabs Cairn Excelitas X-Cite LED Mightex
Summary of Available Models PE100 units – simple single band LED unit with control unit; PE-4000 offers 16-selectable wavelengths on 4 motorised sliders; PE-2 Unit offers up to 4 colours; The “affordable” Microscope LED range with a wide range of colours; The Ultra High Power LEDs, with a select few high power LEDs, Can add fiber coupling Single band collimated LEDs; Four wavelength solution in combination with the four wavelength controllers; Also LED with fiber coupling; MonoLED: Simple single wavelength solution; OptoLED – Controller will control 2 LED heads; With optical feedback circuit; Optoflash with fiber; NOT AVAILABLE IN THE US; X-cite 120LED Unit with 1-4 colours, with LED light modules mounted on the microscope; XLED1, with LED light modules mounted in the unit, off the microscope; Very modular, with several combinations available. MLS-range LED units with various levels of output. BioLED range with “Intellipulsing” mode, allowing short pulses of significantly (typically x2) increased output. WheeLED range, with up to nine wavelength modules; Super-High-Power range, with increased power for a selected colours;
Advertised intensity wavelength closest to 405nm N/A Ultra High Power LED provides 2000mW of collimated light at the LED output level (405nm) 260-440mW depending on microscope coupler at the LED output level (405nm) N/A N/A 240mW – 2500mW depending on LED power option and control option (405nm)
Advertised intensity wavelength closest to 470nm N/A Ultra High Power 460nm LED provides 1700mW of collimated power at the LED output level 250 – 350mW, depending on microscope coupler at the LED output level (470nm) N/A N/A 200mW – 3300mW depending on LED power option and control option (470nm)
Advertised intensity wavelength closest to 595nm N/A Ultra High Power LED provides 650mW of collimated power at LED output level (595nm); The 630nm LED provides an output of 800mW; 60-80mW depending on microscope coupler at the LED output level (590nm) N/A N/A 65mW – 400mW depending on LED power option and control option (590nm); The 625nm LED provide an output of 1100mW
Coupling options Directly coupled to the microscope for PE-100, or coupled via liquid light guide (optional for PE-100 and standard for PE-2 and PE-4000); PE-100 also offers option for optical fiber coupling; Directly to microscope; Also offers option for optical fiber coupling; Direct coupling to microscope or coupled via liquid light guide or optical fiber; Direct coupling to microscope; X-Cite XLED1 is microscope coupled via liquid light guide; 120LED has the LED housing directly mounted to microscope; Direct to microscope or via liquid light guide; Optical fiber coupling also available;
Available wavelengths 20 different bands, ranging from 365nm to 770nm 23 different bands, ranging from 365nm to 630nm – some of the “Ultra High Power” variety 15 different bands seen on webpage ranging from 365nm to 940nm. The website mentions that alternative wavelengths available upon request. 15 different wavelengths ranging from 355nm to 627nm; 7 wavelength options, ranging from 370nm to 700nm 32 different wavelengths, ranging from 365 to 940nm, with various power output options for some wavelengths
Combining wavelengths For the PE-100 range, Extra LEDs can be added and combined using supplied beam combiner cubes; The 16 Colour PE-4000 offers several useful wavelength combinations mounted on 4 motorised sliders; Wavelengths can be combined using supplied beam combiner cubes Different bands can be combined, or 4-band system in a single housing; Wavelengths can be combined using supplied beam combiner cubes Up to 4 wavelengths can be accommodated by LED housing; Individual modules combined with Beam Combiner Blocks; WheeLED option has LED modules mounted on turret, with 9 available positions.
Pulse capacity TTL for on/off control using a BNC connection on the control pod. PE-4000 includes pulse-generator for optogenetics; TTL with sub-millisecond response time; Pulse generator software TTL with sub-millisecond response time; TTL with sub-millisecond response time; TTL controlled pulsing; also has the “pulse mode”, which allows for pulses of as short as 10us. TTL with sub-millisecond response time; BioLED Control Units come with software where pulse generator protocols can be programmed
Control options and pulse capacity and intensity control PE-100 with wavelength-specific control pod, which features push-button control of intensity in 1% steps from 0-100%, Instant on/off button and intensity display; Also with TTL In; PE-2 and PE-4000 units have similar control, with push-button wavelength selector and PC control via USB, also includes option for analog control; PE-4000 offers control-pod with programmable pulse generator for optogenetics; Control Unit with on/off key-switch, intensity control knob, TTL in and optional analog input; Also Includes software for programming protocols, including pulsing protocols; A variety of universal controllers, ranging from simple LEDD1B control unit with intensity control knob and TTL/Analog BNC input; option to more sophisticated 4 channel more sophisticated control unit with display screen, intensity control knob and on/off switch, TTL trigger inputs.The 4-channel controller comes with a software. Both OptoLED and MonoLED include control unit with on/off switch, TTL and analog in; OptoLED also includes remote control module with sliders for intensity adjustment and on/off switch; Optional manual control unit (otherwise only through software) with touch screen; Manual control unit with optional on/off and remote rotary intensity control knob, for controlling intensity; Software and touch screen offers pulse generator with the option of programming pulse protocols and TTL input for external control; Ranges from single channel driver to controller for driving up to 16 LED channels. With variety of control options with different combinations, including manual rotary know, TTL, Analog in, software for the BioLED controllers, including pulse generator protocol
Price Approximately £1,000/wavelength for the PE-100 range; PE-4000 16-Colour system approximately £7,5k, including 16 wavelengths; N/A Competitive; £500-£1000 per wavelength including the controller; Four channel system (DC4101) approximately £4k; N/A N/A, but feedback is that it’s towards the higher end of the spectrum Competitive; starting at £650 with prices in the low thousands (£) for more sophisticated versions;

Fixed spot illumination for optogenetics activation

The next level up in terms of control and sophistication is the fixed spot illuminators. Some researchers create a spot in the middle of their microscope’s field of view by simply inserting a pinhole aperture (available from Newport or Thorlabs) into the light path, with their regular widefield LED fitted behind it. A DIY solution with inexpensive laser/s coupled into a conjugate plane of the microscope is also fairly easy to put together using Thorlabs or Newport mounts and couplers. The commercially available, laser-based solutions below offer more flexibility, intensity and several other features:

Rapp Opto Andor
Summary of Available Models Aiwon Fixed Spot System – Consisting of collimating optics for coupling Laser, LED or flash lamp into microscope fluorescence illumination optics; Spot fixed in the middle of FOV, MicroPoint Manual Spot Illuminator; System includes tunable laser – spot can be manually moved (using a joystick) from one position to the next in the field of view;
Advertised intensity at the output – closest 405nm/470nm/595nm Depends on Light Source Range of tunable laser between 365 and 656nm – advertised to be intense enough for ablation and uncaging
Coupling options Aiwon unit directly coupled to microscope, with light source coupled directly to Aiwon unit or via liquid or fiber lightguide; MicroPoint optical block directle mounted onto microscope, with laser coupled to MicroPoint via fiber optic
Available wavelengths Wide range, depending on light source type; Lasers, LEDs or Lumencor light engines can be coupled Tuneable between 365 and 656nm
Combining wavelengths Depends on the light source No
Pulse capacity Depends on the light source Opionally includes a software controlled pulse generator or foot pedal for the laser
Control options and pulse capacity and intensity control Depends on the light source Joystick for laser positioning; Optionally includes a software controlled pulse generator or foot pedal for the laser. Includes motorised variable attenuator slide for controlling intensity
Spot Size spot size ranges from sub micron to several microns, depending on optical setup; Near Diffraction limited
Price N/A N/A

Galvo-driven spot illuminators for optogenetics activation

Galvo-based spot illuminators offer the flexibility of being able to manipulate the laser spot’s XY positioning, and it enables researchers to do pseudo-simultaneous activation of several spots on the sample by programming a fast spot sequence. Choosing lasers with enough power in combination coupled to these systems allows for doing laser micro dissections, ablation and uncaging in addition to the optogenetics activation. Here’s a comparison of a couple of  commercially available options:

Rapp Opto Andor
Summary of Available Models UGA-40 Point Scanning Device; Uga-40 GEO, which allows for alternative spot shape; MicroPoint Galvo with tunable laser
Advertised intensity at the output – closest 405nm/470nm/595nm Laser-based; Intensity depends on the power of the laser, options for ablation and uncaging; Only available for wavelengths between 365 and 656nm – bright enough for ablation and uncaging
Coupling options Lasers are directly or fiber coupled into UGA unit. UGA fits onto the fluorescence illuminator of typical microscope models Laser is coupled to MicroPoint Optical block via fiber optic; Micropoint optical block coupled directly to microscope – available for most major microscope models
Available wavelengths Broad selection of laser wavelengths available, but only 2 wavelenghts in the same experiment Single laser input, tuneable between 365 and 656nm
Combining wavelengths Standard system includes two laser coupling ports; Up to four lasers can be coupled; less than 1ms switching time between lasers; No
Upgrade options N/A N/A
Pulse capacity Laser can be pulsed with TTL, controlled from software Can include a pulse generator or foot pedal for the laser
Relevant Software control Features Click and Fire mode; Grids can be programmed and can be executed in sequence or randomly; Programmable sequences with different scan shape geometries; Software coordinates used for laser positioning; pulse generator for pulses
Integration with electrophysiology or other imaging devices Software can run in conjunction with electrophysiology and imaging software Can be controlled by some imaging acquisition software packages from Zeiss, Olympus, Nikon and Leica;
Intensity control Depends on light source Includes motorised variable attenuator slide for controlling intensity
Spot Size spot size ranges from sub micron to several microns, depending on optical setup; Near Diffraction limited
Price N/A N/A

Flexible array-based patterned illuminators for optogenetics activation

These patterned illuminators offer the most flexibility in terms of the size and shape of the activation light. The active component behind the first three solutions listed in the table below is a so-called Digital Light Processor (DLP), also called Digital Micromirror Device (DMD) from Texas Instruments. The chip consists of an array of several hundred thousand mirrors, each mirror only a few micrometers square. These mirrors can be tilted at an angle that will allow light projected onto the rear surface of the array to pass through. It can be switched between the transmission angle (the on-position) and the resting position (the off-position), at kHz frequencies.

The device is commonly used in projectors, and some laboratories have integrated regular projectors into the microscope light path to project patterns onto the sample. Texas Instruments also offer a developer’s kit with a DMD for end-users who are interested in using the technology for projects like patterned illumination for microscopy. The commercially available options include “turn-key” features like optimized collimating optics from the light source/s into the DLP unit and for coupling the illuminator into the microscope, triggers in and triggers out as well as the software to program your experimental protocol.

These light sources offer the capacity to activate any shape within the limits of its spatial resolution constraints, which enables you to

  • target specific cell shapes or sections of cells for activation
  • Change the size of the activation area to achieve the required activation threshold
  • Activate more than one area in the field of view at the same time

The fast switching frequency of the mirrors allows you to manipulate the on/off switching at frequencies that are faster than most optogenetic protein kinetics require, and with some of the commercially available solutions you have flexibility in terms of the light source (i.e. LED/Laser/HBO Lamp) used for projection onto the rear plain of the DMD. This means you can recycle existing equipment and it allows for multiple wavelength options for more complex experimental designs.

A different kind of technology is the micro-LED array developed as part of an EU project grant by a consortium involving a group at Newcastle University. It consists of an array of 16x16 micro-LEDs with a mask of micro lenses to improve the fill factor on the sample. To our knowledge it’s not currently commercially available, but judged by some publications referenced on their website, it’s proven to be efficient for activating ChannelRhodopsin in neuronal cultures.

Mightex Andor Rapp
Summary of Available Models Polygon400 – Digital Micromirror Device instrument range – Wide range of models available, with internal LEDs (up to three colours), or external light source/s which can either be an LED, laser or arc lamp lightsource. These can be liquid or fiber light guide coupled Mosaic, the basic unit; Mosaic Duet, with four light source ports. uMatrix Patterned Illuminator – Digital Micromirror Device instrument range –
Advertised intensity at the output – closest 405nm/470nm/595nm Depends on the light source – lasers, LEDs and arc lamps can be used; For LEDs, 400nm produce 14mW/mm, 470nm produce 15mW/mm2 and 590nm produce 3mW/mm At specimen under a 20 x 0.75NA Olympus objective Depends on the light source – lasers, LEDs and arc lamps can be used; Depends on the light source – light guide coupled diode lasers and Lumencor light engines, laser and LED based
Coupling options internal LEDs (up to three colours), or external light source/s which can either be an LED, laser or arc lamp lightsource. This can be liquid or fiber light guide coupled; The polygon unit is usually inserted into the rear epi-illumination port of an inverted microscope and coupled to an upright microscope via a beam combiner block. Light source can be Light guide or directly coupled to Mosaic unit, which is mounted on the microscope; uMatrix is coupled directly the the fluorescence illuminator of your microscope;
Available wavelengths Several light source options available between 400nm and 700nm Depending on the light source – optics corrected for wavelengths between 360nm and 800nm Depending on the light source – optics corrected for wavelengths between 350nm and 700nm
Combining wavelengths Several light sources can be coupled; Several light sources can be coupled; Up to 3 fiber coupled light sources can be combined
Pulse capacity 250us minimum exposure time; can be pulsed at 4kHz 50-200us minimum exposure time, can be pulsed at 5kHz N/A
Relevant Software control Features Software allows for the Polygon to be calibrated to any digital camera, so no need for buying a new camera to get have this work; Point and fire, user defined arbitrary patterns and pattern sequences, which can be software timed or timed with inputs from external control hardware; Arbitrary patterns can be uploaded and sequentially projected, triggered by software or by external TTL trigger; Point and Fire; User-defined arbitrary patterns; Projection of image sequences; Flexibility with how a sequence is displayed; Analog and digital laser intensity modulation;
Integration with electrophysiology or other imaging devices real-time TTL in TTL out, as well as analog in control for some models; software doesn’t interfere with electrophysiology/imaging software; and Compatible with Nikon’s NIS Elements software; Can be integrated with various imaging hardwares, as well as electrophysiology with real-time trigger-in functionality TTL in and TTL out.
Intensity control Can be controlled directly on the light source or achieved by rapid gating of mirror patterns; Can be controlled directly on the light source or achieved by rapid gating of mirror patterns; Can be controlled directly on the light source or achieved by rapid gating of mirror patterns;
Price Competitively priced – Between £7k and £12k with single wavelength LED; Seen as fairly expensive Seen as fairly expensive
Minimum Spot Size Diffraction limited Diffraction limited N/A

Polygon Grid Scan

 

We’ll continue our blog series on optogenetics in the coming weeks, including a section on fiber-coupled systems for in vivo and in vitro applications. Please participate in the blog discussions and subscribe to the blog for more regular updates.

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