We recently needed to verify how a large population of neurons reacts to weak optogenetic stimulation. We found that with a relatively straightforward setup, visible light optogenetic stimulation can be integrated into existing 2p rigs without resulting in problematic imaging artifacts. Here, we slightly de/hyperpolarized cells with a ~1mm beam of light aimed at the imaging window while imaging and delivering sensory stimuli, but the same approach should work for all kinds of experiments with implanted optical fibers, scanned focused light, or even patterned light stimulation.
WARNING: Don’t direct light into photomultipliers unless you’ve taken adequate precautions to ensure that they won’t be damaged. None of the methods described here have been tested other than in our specific microscope. Specifically, this method is probably not safe for GaAsP PMTs.
Fast light pulsing outside the frame acquisition times
Our 2p setup using galvos only scans in the X direction, giving us at least 200μs of flyback time during which no data is acquired. By only stimulating during this period, visible light artifacts can be massively reduced. On systems with bidirectional scanning, there should still be some dead-time at the frame edges where the galvos stop/reverse.
We pipe the line trigger outputs from the galvo controller into an arduino and generate a 50μs long trigger for the LED on every Nth line, just after the previous line has finished scanning. Depending on the details, the resulting pulse rate should be at >200Hz, which for ChR2 stimulation should be close to functionally equivalent to constant light (Lin et al. 2009). Power can be adjusted by varying either the duty cycle or LED brightness.
The method requires a light source that can switch on and back off with no residual light within ~50-100μs. We tried a few commercial LED drivers, and the ubiquitous CNI made dpss lasers and nothing was even remotely up to the task. We had success with a fast diode laser (Power Technology), but the best solution by far was simple LEDs with a very fast and stable driver circuit, the cyclops LED driver that Jon Newman, now at the Wilson lab at MIT has developed. The high linearity and <2μs rise/fall time of the driver means that no extra light bleeds into the frame even for fast scanning, and power can easily be adjusted by modulating either the duty cycle or the drive current.
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Avoiding PMT damage
Even though the light pulsing means that the images should be relatively free of the stimulation light, the PMTs would still see the full blast of light, which can either cause damage (definitely don’t try this with GaAsPs unless you’re sure that they wont see the stimulation light by accident!) or at least desensitize them. We tested this on our multi-akali PMT with around 0.1mW total integrated power at 450nm (which isn’t filtered out from the PMTs very well in our scope) out of a bare 200um fiber shining light uniformly over the imaging window which results in moderate back scatter into the imaging optics. We didn’t measure the exact power at the objective back aperture, but after only 50 trials of 1 sec each, the sensitivity of the PMT was sufficiently reduced to make imaging in deep layers of cortex almost impossible.
To resolve this issue, we attempted to filter out as much of the LED light out of the detection path as possible. We have a NIR block filter (OD 6 NIR blocking filter, Semrock) with notches at ~560nm (halo/arch) and ~470nm (chr2) that keeps most of the power of the LED away from the PMTs, plus another step of decent filtering from the primary dichroic blocking yellow light. This arrangement means that with yellow light (up to 1mW integrated power, >20mW peak, diffuse illumination directed at imaging window) we can’t see any clear imaging artifact in the line following the stimulation, and the blue LED (similar power) just leaves a very faint streak of brighter pixels across the imaging x-line after each LED pulse. In both cases, care still needs to be taken to account for residual slight brightening of the images when the LEDs are on. We haven’t been able to detect any significant PMT desensitization over the course of an imaging session using these filters.
Removing residual image artifacts
Even when filtering the LED light out of the PMT path to a degree that avoided sensitivity losses, we still observed a visible increase in image brightness over the course of ~half a image line, and weaker, but still detectable brightening over other image lines. This artifact is likely caused by a combination of the PMT bandwidth (we are, after all still saturating the signal while the LED is on), and some slower timescale tissue fluorescence elicited by the visible light.
The simple brute force solution that we found to work well was to just pulse the LED every 4th or 8th line, and simply interpolate over these to get rid of the artifacts. With ~10Hz frame rates, and selecting this pattern so that a different set of image lines is degraded & interpolated out every frame, the resulting error in the data is minimal. If using a local pixel correlation method to detect ROIs, it is advisable to either keep track of which lines were interpolated, or detect them later, and exclude these pixels from the local cross-correlation computation to avoid skewed results. Finally, even though the interpolation does a good job at removing most of the artifact, in some cases there was still a very small predictable increase in image brightness due to the stimulation which can be accounted for fairly easily by measuring it using neuropil/background ROIs over the entire imaging session and then subtracting it out for it. Additionally, when using a method like this, or any other, that could induce slight brightness changes, it is a good idea to use analysis methods that are not affected by slight changes in overall brightness.