Advantages and Limitations
The interesting features of lasers for optogenetics are:
- their high power (interesting for compensating for poor coupling, doing beam splitting etc...).
- the possibility to easily manipulate laser beams (launching into optical fibers, beam splitting, wavelength division etc).
- Most DPSS systems can be modulated to only deliver a fraction of the maximal output power in steady state conditions. This is usually called analog modulation and is often set by the value of an input voltage (where 0V = 0% and 5V = 100%). But the relation between this analog modulation voltage and the emitted power is not necessarily linear. Therefore it is necessary to map the output of your laser using a power meter. See Light Delivery > Light Sources > Measuring Optical Power.
- Although most affordable DPSS systems have fast (1-5 kHz) on/off TTL modulation, the resulting pulses have unexpected (but reproducible) power and shape. Let's say for example that you set your laser to deliver 20% of its maximal power in steady state operating conditions. Triggering the laser on/off using a TTL input will allow you to produce millisecond scale light pulses but 1) the maximal power reached during the pulse will not be 20% (it can be more or less than that), and the pulse will have strange temporal characteristics (slow onset and offset, oscillation etc...). The only way to know what your laser is giving during short pulses is to measure them! See Light Delivery > Light Sources > Measuring Optical Power.
Principle of the Diode-pumped Solid-State (DPSS) Laser
A high power semi-conductor laser diode is electrically stimulated. Its emission is used to "pump" (see optical pumping) a lasing media, which in the case of the DPSS system is a solid crystalline material. This crystal in turn emits an increased amount of photons in a coherent manner which can be:
- optically manipulated (diverged, converged, collimated, etc).
- secondarily altered by wavelength (second harmonic generation, third harmonic generation, high harmonic generation, sum frequency generation, difference frequency generation, and others) via non-linear crystals and beam amplifiers, oscillators, rectifiers, and conversion techniques.
Laser Safety and Maintenance
Laser safety entails much more than "don't point it in someone's eye." All rooms where lasers are used should be clearly marked and, if possible, should have a sign that indicates when a laser is in use. Always use proper eye protection when laser beams are exposed. Every laser in your lab should be stored with at least one pair of goggles that attenuate light for the proper range of wavelengths.
If your institution offers a laser safety course, take it as soon as possible. It's also recommended to undergo an eye examination prior to working with lasers, as it's possible to sustain retinal damage that is undetectable under everyday conditions.
Maximizing the lifespan of your DPSS laser requires proper mounting practices. According to Scott Browes, Technical Sales Executive at Laserglow Technologies, DPSS lasers should always have their bottom surface in contact with an aluminum breadboard or other large heat sink (see Launching for a detailed image). Simply mounting a DPSS laser on four posts and leaving the base exposed to air prohibits thermal transfer. Even though the laser module may not feel warm, the laser's thermo-electric cooling circuitry may become permanently damaged if heat cannot escape quickly enough. This can cause your laser to become unreliable or even unusable with time.
Other best practices include turning the power source off before switching the safety key to the "off" position, and allowing the laser to warm up for at least 10 minutes (or as specified for your particular model) before using.
- Optical pumping: a process in which light is used to raise ("pump") electrons from a lower energy level to a higher one.
- Solid state: The solid state reference for diode pumped solid state lasers (DPSS) is due to the use of solid materials (versus gases, and other media) for 'lasing' and optical manipulation such as secondary, tertiary, and quadruple harmonic generation, or non-linear optical beam path results. The reference to the solid state electronics controlling many laser types is the application of solid materials (versus gases, vacuum, and other media) being used to create the circuitry required for delivering energy to the diode, electronic feedback, and optical feedback, as well as cooling and other control (modulation etc) within the laser power controller (often called a power supply). This means there is a double meaning to the term solid state in many lasers as they are both electronically and optically using solid materials.
Links and References
- Open Access Encyclopedia for Photonics and Laser Technology from RP Photonics.
- DPSS lasers on Wikipedia
- Optical pumping on wikipedia
- Solid-state laser on Wikipedia
- Laser Fundamentals I (MIT Understanding Lasers and Fiberoptics) by Pr. Shaoul Ezekiel.
- Laser Fundamentals II (MIT Understanding Lasers and Fiberoptics) by Pr. Shaoul Ezekiel.
- Laser Fundamentals III (MIT Understanding Lasers and Fiberoptics) by Pr. Shaoul Ezekiel.
- Laser Fundamentals III (cont.) (MIT Understanding Lasers and Fiberoptics) by Pr. Shaoul Ezekiel.
Once you've acquired a laser for optogenetic stimulation, you must figure out the best way to deliver its light to the region of interest, whether this region is part of a cell culture, brain slice, or in vivo preparation.
There are two primary options for routing your laser light:
1. Delivery through a fiber optic cable
- Allows light to travel long distances in a shielded manner
- As little as a single alignment step
- Major advantage: fibers are flexible enough for use in freely moving animals or temporary experimental rigs
- Drawback: some coupling loss is inevitable, although this is usually not a limiting factor
2. Delivery through a system of mirrors
- Light travels through open air, unless an enclosure is built
- No coupling loss, although some light will be lost with each reflection step
- Major advantage: scanning galvo mirrors can be used to deliver light with incredibly high spatiotemporal resolution
- Drawbacks: difficulty of aligning multiple mirrors
Coupler on the laser
Often the simplest solution to fiber coupling is to have a fiber adapter mounted directly on the laser. Laser can be purchased with connectors pre-mounted, the most common connector types being FC/PC (Ferrule Connect/Positive Contact) or SMA (Sub-Miniature A). The primary advantage of such a setup is ease of use, as everything is housed in a single module with minimal alignment issues. The drawback, of course, is a lack of flexibility: adding additional components between the laser and the fiber is impossible.
Coupler outside the laser (free space launching)
For maximum flexibility, moving the coupling system away from the laser is recommended. Examples of useful additional components include:
- Neutral density filters, for adjusting power output in systems without analog modulation
- Shutters, for controlling light output in a manner more reliable than TTL pulses
- Beamsplitters, for coupling multiple lasers into a single fiber, or a single laser into multiple fibers
- Photodiodes, for online measurement of laser power
The image below shows an example of a setup with the coupler outside of the laser. A neutral density filter and a beamsplitter lie between the laser and the coupler. All parts (except the laser) are readily available from Thorlabs. The entire system is mounted on an aluminum breadboard with 1" hole spacing. Note the presence of an additional small breadboard beneath the laser, which helps dissipate heat. If the bottom of the laser is exposed to air, the buildup of heat can cause electrical and/or optical failures, which will cause your laser to become unreliable. If the MB6 base plate from Thorlabs is used, additional holes must be drilled to accommodate the laser, as most DPSS laser packages adhere to metric spacing. Alternatively, table clamps (such as Thorlabs CL3) can be used to secure the laser to the base plate.
The system is based on the PAF-SMA-11-A collimator from Thorlabs, which is also available in an FC/PC version. Although more costly than other styles of collimators, this model offers 5 set screws for precision alignment. This allows high-efficiency coupling to be achieved with little effort, or for light power to be adjusted without the need for a neutral-density filter or analog modulation.
Such a setup takes up more space, and is more difficult to align, than one with a coupling system on the laser itself, but the added versatility may be worth the extra effort.
One minute laser-fiber alignment procedure
Delivery via mirrors
Using a free-space DPSS laser beam onto a galvonometric mirror landing one of the galvo positions on a fibre coupler ('dumping' the other position) works as both a modulation device as well as fibre launch device. There are limitations on speed (modulation parameters of pulse duration and frequency) based on the galvo mirrors you choose, as well as size of the beam and galvo mirrors since there is mechanical movement, therefore keeping the mirrors and their movement to an accurate, small as possible size assists in this aspect of the system. The transverse profile of the laser as well as the input size of the fibre coupler are all important considerations in aligning the system.
Typical anomalous DPSS signal responses are then avoided since the laser runs CW at a power determined by the user and is modulated to provide the optical delivery routine of your choosing. Laserglow Technologies has a turnkey system in beta testing for launch in mid-2011 which can be retro fit to existing laser systems or added to a new laser purchase for a complete optogenetics system.
What it’s good for
Analog modulation allows you to smoothly vary the power output of your laser. This is useful for characterizing the stimulus parameters necessary to elicit spikes or other physiological events, or for delivering time-varying light stimuli other than on/off pulse trains (for example, the ramped stimulation used by Adesnik & Scanziani (2010) .
How to do it
Any laser beam can be passed through a neutral-density filter (such as Thorlabs NDC-50C-2M) to manually adjust the power output. This is a simple solution that would work well for systematically varying light power, but is not viable when temporal precision is required. It is also possible to buy computer-controlled filter wheels (such as Thorlabs FW102C), which can be loaded with a small number of neutral-density filters. This eliminates the need for manual adjustment, but only allows modulation at discrete intervals.
Some—but not all—lasers come with power supplies that allow laser power to be smoothly modulated. In this case power output is controlled by a dial on the power supply, an external source, or both. The external source can be the output of a DAQ board, a signal generator, or anything that can create waveforms in the specified voltage range (usually 0-5 V). These lasers are usually more expensive than models without analog modulation capabilities.
If neither of these solutions are available, analog modulation can be simulated by adjusting the duty cycle of the laser. For example, a laser can be pulsed “on” for 50 microseconds and “off” for 50 microseconds which should, in theory, give you half the power output of a continuous “on” pulse. In practice, however, things will not be this simple, due to the nonlinearities of laser power modulation (see below). Be sure to measure the power output of your laser for different duty cycles before running any experiments.
What to watch for
The input-output curve for direct analog modulation of lasers is often nonlinear, even within the specified modulation range. Scaling the input voltage will not necessarily scale the laser power by the same amount. Neutral density filters avoid this issue, but are not always the most convenient solution. The relationship between voltage and power can vary greatly between different laser models. Don’t assume your analog modulation will be linear—measure it!
Analog modulation (sinusoidal wave type) signaled through diode lasers can be very unpredictable due to the various diode thermal states that the unit must undergo. This variation manifests in wavelength drift and TEC cooling circuit instability. Once a setting has been altered on a diode laser a subsequent warm-up (stabilization) period must ensue.
Analog modulation signals through DPSS (diode pumped solid state) lasers have a more predictable result and can be easily stabilized when allowing for a warm-up period at a given setting on a given modulation routine. Most DPSS lasers are specified as to the beam characteristics and other critical values while running in a post warm-up 100% state. These beam characteristics are not always attainable in less than 100% output, therefore analog signals should be used in relation to the mapping data for each laser (as the laser may not have a linear output reaction to various signal inputs). Beam characterization in DPSS lasers can be kept within close tolerance using analog signals unless maximum output is less than 100% of the laser’s capability and only if a suitable period of stabilization is allowed after initialization of the signal routine before usage. This can be achieved using a beam 'dump' routine for free space, or an un-coupled (fibre connector removed at one end or the other) state where the beam is wasted during this warm-up and/or stabilization period. In these less than total output cycles the laser should be 'mapped' and this data logged to create datum points of the current draw (LED on the power supply typically shows this in a relative value but not necessarily an actual current value in amperes) versus optical output in milliwatts for laser usage for a given modulation routine.
What it’s good for
For most experiments, turning your laser on and off by manually flipping the power switch is not going to cut it. Instead, you’ll want to generate pulses at precise times, such as in the rhythmic pulse trains used in Cardin et al. (2009)  and Sohal et al. (2009) .
How to do it
Electronic TTL shuttering
All laser power supplies should accept TTL inputs to control the on/off status of the laser. TTL stands for “transistor–transistor logic.” TTL originally referred to signals sent between integrated circuits built from bipolar junction transistors, but it is now used as a blanket term that refers to “TTL-compatible logic levels,” usually 0 V and 5 V. Lasers can either be in TTL+ (5 V = ON) or TTL– mode (0 V = ON).
With TTL shuttering, the laser is either all on (to the extent of the output power you have set the controller at if it is an adjustable power laser, otherwise it is 100% emission) or it is all off. In theory, you should see a rapid delivery of laser energy with an equally rapid reversal of this process. TTL modulation has the disadvantage of not being able to deliver the power increase gradually, and reciprocally does not allow gradual reduction of the power, as can be done with analog modulation signals.
How to deliver TTL pulses:
- The easy way: use an existing module for delivering pulse trains, such as a Grass Stimulator or a Master-8. These require no programming skills to operate, are easy to configure (if all you need are rhythmic stimuli), and are already present in many neuroscience labs. However, they are not very flexible (try delivering a Poisson stimulus) and are expensive to purchase.
- The flexible way: the digital or analog outputs of a DAQ card, such as those made by National Instruments or Measurement Computing, can be programmed to deliver almost any stimulus imaginable. This requires some amount of programming (usually in Matlab or LabView), but allows you to control your laser in very intricate, temporally precise patterns.
- The cheap way: microcontrollers, such an Arduino, cost about $30 and can be programmed to output TTL pulses in much the same way as DAQ cards. However, unlike DAQs, they are not built with temporal precision in mind, so be sure to measure the outputs with an oscilloscope before you trust that the timing is acceptable.
Using TTL pulses to modulate the on/off state of a laser is very convenient but can result in high variability in power levels from pulse to pulse. This problem is especially bad with yellow lasers, which are useful for activating NpHR and Arch. A workaround for the instability issues seen with TTL modulation is to leave the laser on constantly, and modulate the beam with an external controller. These can include mechanical shutters or other solutions (Pockels cell, acousto-optic deflector, optical switches). Mechanical shutters are relative cheap and effective, but take at least ~300 microseconds to open. Depending on the application this delay may not be a problem. Other methods can be quicker, but are more expensive and can attenuate the laser power.
In-line fiber optic switching is an alternative to mechanical shuttering. It was originally designed for telecommunications fibre applications that require rapid and often multi-channel fibre switching of signals. The disadvantage of inline fiber optic switches is that they are acoustically noisy, often in the ultrasonic range. Alternatives include MEMS (micro electro mechanical systems) which are virtually silent yet relatively slow in switching speeds (or when used as a modulator).
In-line switches can be purchased from www.diconfiberoptics.com
What to watch for
First of all, when using a TTL pulse generator, make sure the output levels are in the right range. If they’re too low, the laser won’t turn on, and if they’re too high, you could damage the circuitry of the power supply unit.
Secondly, be aware that—as with analog modulation—lasers do not always respond in a predictable way, even to simple ON/OFF signals. In the figure to the right, you can see the mean relative power output for two lasers (blue and yellow) in response to TTL pulses of varying duration (black lines). Both lasers clearly overshoot the duration of the input pulse, and the yellow laser is unstable. Of course, for many experiments, these minor fluctuations in laser power won't make a difference. But it's important to know that your laser isn't undergoing large power swings that could invalidate your findings.
Considerations for diode lasers: Reduced diode life expectancy has been loosely attributed to TTL modulation in that this I/O, on/off regime does not allow for stabilization of the TEC (thermo electric cooling) circuitry and current delivery to the laser diode since they both pass through the power supply/control device and this thermal variation (some call it thermal shock) reduces the life of the diode. When using TTL modulation on a diode laser (versus DPSS laser) the wavelength 'drift' that is common as a result of varied power input to these diodes can be minimized by using TTL on/off and allowing the regime to stabilize by performing an adequate warm-up period where the diode and TEC circuits can stabilize within the logic controller in the power supply.
Quicklist of DPSS lasers manufacturers/suppliers
- OEM Laser Systems
- Opto Engine LLC
- RGBLase LLC
- Shanghai laser
- Shangai Dream Laser Technology
- Sigma Photonics, LLC
- Think Lasers
Detailed information about DPSS laser products and manufacturers/suppliers
Write here your reviews on lasers (price, power, available colors and options, stability, lifetime, precision of analog modulation, etc...). Don't hesitate to document your review with illustrations (e.g. measurements).
DPSS units from Laserglow come with a great variety of colors and are usually amongst the cheapest on the market (4 times cheaper than Crystalaser for the same power). This may be reflected in their lifespan; after 2 years of consistent use, at least one Laserglow unit became wildly erratic, with output power fluctuating by tens of milliwatts within the span of a few seconds.
Power Stability at Steady State
Analog Modulation at Steady State
The laser (473 nm, 100 mW) was switched to the analog mode and decreasing voltage steps were applied while the optical power was measured with a power meter (PM100 from Thorlabs).
The analog modulation is not linear (rather it looks exponential), with a 0-to-100% modulation within the 2-4V range.
TTL Pulse Generation
The laser (473 nm, 100 mW) was switched to the TTL+ mode. Pulses were obtained at various modulation values and measured using a silicon photodiode with a response time of 45 ns (SM1PD1A from Thorlabs). The photodiode output was mapped beforehand to convert it into optical power (See Light Delivery > Light Sources > Measuring Optical Power).
The laser only reaches steady state values after 800 ms. Short pulses (1 ms) clearly give much smaller power than requested by the analog modulation setting. For longer pulses (10 to 100 ms) the laser slowly converges to steady state by starts by overshooting for modulations above 4.5.
Here are some mapping data obtained with a 473 nm 50mW DPSS laser from Crystalaser.
Power Stability at Steady State
Analog Modulation at Steady State
the analog modulation appears almost linear in the 0.02-5V range.
TTL Pulse Generation
This laser clearly overshoots during short pulses (1-10 ms) for most modulation values (up to 4). For example: for a 10% analog modulation setting, the laser reaches 40% during the pulse. Longer pulses (10 ms) reveal an attenuated oscillation, and a weird rectification after 4 ms at 100% modulation (red). The steady state value is reached after 100 ms.
Shangai Dream Laser Technology
Inexpensive but reliable blue lasers with analog modulation Model: VA-I-N-473 (473nm-Blue Laser-1~100mW) Price range: US$500-1000
Viasho lasers, like many inexpensive DPSS lasers have a slow onset and sub-maximal power output when pulsed using brief analog or TTL modulation (fig. 1). This is especially problematic if you are trying to generate short pulses at maximum light output. The following is a work-around that requires a power supply with analog modulation and the ability to generate arbitrary waveforms, either with a DAQ or some other analog output device.
First, determine the maximum subthreshold control voltage. In the case of the Viasho, I used 2V. Either step up to this voltage just prior to the pulse onset or hold at it throughout the experiment.
To trigger the light pulse, step from the holding potential (2V) to the maximum (5V) (fig. 2). This produces a very quick onset (<100 us), however, returning to 2V from 5V leaves a tail. To eliminate the tail, deliver a biphasic pulse, stepping from 2V to 5V, then briefly down to 0V (for ~1 ms) before returning to the holding potential of 2V (fig. 3). This procedure produces short reliable pulses at full power.
Stadus 473-70--this is a very fast diode laser (200 MHz) using digital modulation. Here is the response of the Stadus 473-70 laser when gated with a 5 ms TTL pulse.
We obtained similar square wave forms when a 100 microsecond pulse was used.
(previously Point Source)
Opto Engine LLC
Recommended models are ( higher power ones available as well):
- MBL-III-473, 100mW, PSU-III-LED (blue)
- MGL-III-532, 200mW, PSU-III-LED (green)
- MGL-FN-561, 50mW, PSU-H-LED (yellow green)
- MGL-III-589, 50mW, PSU-III-LED (yellow)
- MRL-III-635, 300mW, PSU-III-LED (red)
405nm, 450nm, 460nm, 520nm, 556nm, 593.5nm and 640nm lasers are also available.
All feature Analog or TTL modulation with fiber coupling option and deliver adequate power for most optogenetic applications.
Cobolt offers tailored experiment-ready solutions for channelrhodopsin activation at 470nm using our Cobolt MLD™ 473nm laserlaser and halorhodopsin inhibition at 590nm using our Cobolt Mambo™ 594nm *.
Three selected laser packages have been carefully designed for optimum results and easy integration into optogenetics research set-ups and offer a very high degree of system flexibility. The packages include a laser with intensity modulation capability, an easy adjustable fiber couplers. To this system, a MM fiber (with 50μm-200μm core diameter) of your choice can be easily attached.
All packages offer full control over the pulse generation, perfect pulse-to-pulse stability, high extinction ratio and fiber coupling efficiencies of >80%.
In addition, Cobolt can also offer solutions for coupling two laser sources into one fiber.
- Cobolt MLD™ 473nm with direct modulation: Modulatable laser (80mW output power) up to 150MHz (ns pulses) mounted on Cobolt FIC-05 with fiber coupler (FC/PC or SMA)
- Cobolt Mambo™ 594nm with AOM modulation: Modulatable laser system (80mW output power) up to 3MHz (sub-μs pulses) mounted on Cobolt FIC-01 with fiber coupler (FC/PC or SMA).
- Cobolt Mambo™ 594nm with shutter modulation: Modulatable laser system (100mW output power) up to 125Hz (down to 8ms pulses) mounted on Cobolt FIC-01 with fiber coupler (FC/PC or SMA).
Other wavelengths also available.