Microscope for routing optogenetic stimulation patterns into a sample and monitoring the resulting optical neuronal activity mapping

Abstract

When light-sensitive protein has been introduced into neurons, brain tissue (or an in vitro cluster of neurons) can be optogenetically activated using light. And when a fluorescent protein that reveals the presence of specific molecules (e.g., calcium) has been introduced into the neurons, the responses of the tissue / cluster to the activation can be monitored by capturing images of the fluorescent light. The optogenetic activation is implemented by projecting first-wavelength light onto specific portions of the tissue / cluster. The tissue / cluster is illuminated by second-wavelength light, and third-wavelength light that is emitted by the fluorescent protein within the tissue / cluster is captured by a camera. The captured images show the location of the specific molecules within the tissue / cluster, which reveals how the neurons respond to the activation.

Description

PatentAtty. Docket No. 1448-0259wo01MICROSCOPE FOR ROUTING OPTOGENETIC STIMULATION PATTERNS INTO A SAMPLE AND MONITORING THE RESULTING OPTICAL NEURONAL ACTIVITY MAPPINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of US Provisional Application 63 / 707,815, filed October 16, 2024, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with government support under MH119423 and TR002151 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Optogenetics uses light-sensitive proteins to selectively activate or inhibit neurons. And genetically encoded fluorescent calcium indicators can be used to monitor neural activity.SUMMARY OF THE INVENTION

[0004] One aspect of this application is directed to a first apparatus for activating at least one of a plurality of cells within a sample and monitoring a response of the cells to the activation. The cells can be optogenetically activated by light at a first wavelength, and the cells include a fluorescent protein that fluoresces at a third wavelength when illuminated by light at a second wavelength. The first apparatus comprises a projector, a second light source, and an optical system. The projector has a control input and is configured to project an image that includes light at the first wavelength. The nature of the image projected by the projector depends on a state of the control input. The second light source emits light at the second wavelength. The optical system is configured to (a) route the image projected by the projector towards the sample, (b) route second- wavelength light from the second light source towards the sample so the sample is illuminated with light at the second wavelength, and (c) route third- wavelength light emitted from the sample towards a camera.

[0005] In some embodiments of the first apparatus, the projector comprises a MEMS laser projector. Optionally, in these embodiments, the control input can be an HDMI port.

[0006] Some embodiments of the first apparatus further comprise the camera, which is sensitive to light at the third wavelength. Optionally, these embodiments further comprise an incubator, with the projector, the second light source, the camera, and the optical system all being positioned inside the incubator.

[0007] In some embodiments of the first apparatus, the optical system has an objective having a proximal end and a distal end, wherein the distal end of the objective is configured to be positioned adjacent to the sample, a first beam splitter positioned to route first- wavelength light from the projector into the proximal end of the objective, and a second beam splitter positioned to route second-wavelength light from the second light source into the proximal end of the objective. In these embodiments, the objective is positioned with respect to the sample so that third-wavelength light emitted from the sample enters the distal end of the objective, exits the proximal end of the objective, and subsequently travels towards the camera.

[0008] Optionally, the embodiments described in the previous paragraph further comprise the camera, which is sensitive to light at the third wavelength.

[0009] Optionally, the embodiments described in the previous paragraph further comprise a filter positioned between the proximal end of the objective and the camera that passes third- wavelength light and blocks second- wavelength light. Optionally, the embodiments described in the previous paragraph further comprise a filter positioned between the proximal end of the objective and the camera that passes third- wavelength light, blocks first-wavelength light, and blocks second-wavelength light.

[0010] In some embodiments of the first apparatus, the light at the first wavelength is red light, the light at the second wavelength is blue light, and the third- wavelength light is green light. In some embodiments of the first apparatus, the light at the first wavelength is blue light and the second wavelength is 540-580 nm.

[0011] Another aspect of this application is directed to a second apparatus for activating at least one of a plurality of cells within a sample and monitoring a response of the cells to the activation. The cells can be optogenetically activated by light at a first wavelength, and the cells include a fluorescent protein that fluoresces at a third wavelength when illuminated by light at a second wavelength. The second apparatus comprises an objective, a projector, a second light source, the first beam splitter, and a second beamsplitter. The objective has a proximal end and a distal end, and the distal end of the objective is configured to be positioned adjacent to the sample. The projector has a control input and is configured to project an image that includes light at the first wavelength. The nature of the image projected by the projector depends on a state of the control input. The first beam splitter is configured and positioned to route first- wavelength light from the projector into the proximal end of the objective. And the projector, the first beam splitter, and the objective are aligned with respect to each other so that the first- wavelength light that enters the proximal end of the objective will exit the distal end of the objective. The second light source emits light at the second wavelength. The second beam splitter is configured and positioned to route second-wavelength light from the second light source into the proximal end of the objective. And the second light source, the second beam splitter, and the objective are aligned with respect to each other so that the second-wavelength light that enters the proximal end of the objective will exit the distal end of the objective. The objective is positioned so that third- wavelength light emitted from the sample will enter the distal end of the objective and exit the proximal end of the objective. The first beam splitter and the second beam splitter are configured and positioned to route third- wavelength light that exits the proximal end of the objective towards a camera. And the objective, the first beam splitter, and the second beam splitter are aligned with respect to each other so that the third-wavelength light that exits the proximal end of the objective will arrive at the camera.

[0012] In some embodiments of the second apparatus, the projector comprises a MEMS laser projector. Optionally, in these embodiments, the control input comprises an HDMI port.

[0013] Some embodiments of the second apparatus further comprise the camera, which is sensitive to light at the third wavelength.

[0014] Some embodiments of the second apparatus further comprise the camera (which is sensitive to light at the third wavelength) and an incubator. In these embodiments, the objective, the projector, the second light source, the first beam splitter, the second beam splitter, and the camera are all positioned inside the incubator.

[0015] Some embodiments of the second apparatus further comprise the camera (which is sensitive to light at the third wavelength) and a filter positioned between the proximal end of the objective and the camera. In these embodiments, the filter can either (a)pass third- wavelength light and block second- wavelength light or (b) pass third- wavelength light, block first-wavelength light, and block second-wavelength light.

[0016] In some embodiments of the second apparatus, the light at the first wavelength is red light, the light at the second wavelength is blue light, and the third- wavelength light is green light. In some embodiments of the second apparatus, the light at the first wavelength is blue light and the second wavelength is 540-580 nm.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 depicts the optical design of one apparatus for achieving simultaneous imaging of neuronal activity and manipulation / activation of neurons.

[0018] FIG. 2 depicts one example of a suitable set of optical distances for implementing the FIG. 1 apparatus.

[0019] FIG. 3A shows the characterization of uniform resolution across a large field of view for the FIG. 1 apparatus.

[0020] FIG. 3B shows a demonstration example of capturing activity over a full field of view using the FIG. 1 apparatus.

[0021] FIG. 3C shows a characterization of uniform resolution using the FIG. 1 apparatus.

[0022] FIG. 4A shows an increase in activity rate after a patterned red light activation of ChrimsonR is turned on using the FIG. 1 apparatus.

[0023] FIG. 4B shows a preliminary example of training an ensemble in MoNNet by repeated co-activation of the whole MoNNet using the FIG. 1 apparatus.

[0024] FIG. 4C shows an example of activation of a portion of a MoNNet cell culture network using the FIG. 1 apparatus.

[0025] FIG. 5A shows that repeated co-activation of a group of neurons using the FIG. 1 apparatus leads to the emergence of synchrony in their activity.

[0026] FIG. 5B shows that training a recurrent neural network (RNN) with the data from FIG. 5A reproduces ensemble-like effects in silico.

[0027] FIG. 5C depicts an example showing how MoNNet activity changes in response to optogenetic stimulation using the FIG. 1 apparatus.

[0028] FIG. 6 shows a set of wavelengths that can be used for stimulation and excitation in the FIG. 1 apparatus.

[0029] FIG. 7 depicts a variant of the FIG. 1 apparatus in which yellow-green light is used for activity imaging and blue light from the projector is used for activation.

[0030] FIG. 8 shows a set of proteins and wavelengths that can be used for stimulation and excitation in the FIG. 7 embodiment.

[0031] Various embodiments are described in detail below with reference to the accompanying drawings, wherein like reference numerals represent like elements.DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The embodiments described herein can be used to route optogenetic stimulation patterns into a sample and monitor the optical neuronal activity mapping that is the result of the stimulation. It is particularly useful for analyzing how a slice of brain tissue or a collection of neurons (e.g., Modular Neuronal Network, i.e., MoNNet) respond to stimulation. For the Optogenetic stimulation to work, the light-sensitive protein must be introduced into the neurons (e.g., using a viral vector) before the experiment is performed. And to monitor the neuronal activity, a fluorescent protein that reveals the presence of certain molecules must also be introduced into the neurons (e.g., using a viral vector) before the experiment is performed.

[0033] In a first example, described below in connection with FIGS. 1-6, GCaMP protein is used to monitor the neuronal activity, and ChrimsonR is used to stimulate the neurons. These proteins were selected because GCaMP emits fluorescent green light when calcium ions enter a neuron, and ChrimsonR will optogenetically activate a neuron in response to red light.

[0034] FIG. 1 depicts one example of an optical design for an apparatus (referred to herein as “IncStim”) that achieves simultaneous activity imaging of neuronal activity and manipulation / activation of the neurons. In the FIG. 1 example, blue light (e.g., from an LED 20) is used to illuminate the sample for activity imaging, and red light from a projector 50 is used to optogenetically activate neurons within the sample. When the components listedbelow in Table 1 were arranged as depicted in FIG. 1 using the optical spacings depicted in FIG. 2, the resulting system had a large field of view with cellular resolution.TABLE 1

[0035] The apparatus depicted in FIG. 1 includes two optical modules: a detection I module with a wide field of view, and a patterned light stimulation module for spatiotemporally precise manipulation of neuronal activity.

[0036] The detection module includes a wide-field imaging arm with a detection objective lens 40, a tube lens 88, and a camera 90 (e.g., a consumer-grade camera such as the Teledyne Flir BFS-U3-244S8M-C). Light from a blue LED module 20 (e.g., with a wavelength of 455 nm) is collimated by collimator 22 and focused by focusing lens 24 andsubsequently directed by a short pass beam splitter (e.g., a dichroic mirror) 30 down so that it travels through objective 40 and towards the sample. The blue LED module 20, the beam splitter 30, and the objective 40 are aligned with respect to each other so that the blue light that enters the proximal end of the objective 40 will exit the distal end of the objective. This light is used for achieving Kohler illumination for calcium imaging. When this wavelength of light is used to illuminate the GCaMP protein, the GCaMP protein will emit green fluorescent light when calcium ions are present. The two modules (i.e., the detection module and the patterned light stimulation module) are combined with a long pass beam splitter (e.g., a dichroic mirror) 60. Advantageously, this detection arm provides a large field of view, and an inexpensive consumer-grade camera provides sufficient imaging capabilities.

[0037] The patterned stimulation arm includes a projector 50 e.g., a low-cost portable LASER RGB projector. The projector can be, for example, a MEMS LASER projector like the Nebra Anybeam projector, which incorporates TO-8 LASER diodes that provide blue (440-460nm), green (515-530nm), and red (632-642nm) colors. In this FIG. 1-6 example, only the red light output of the projector 50 can activate optogenetic system (red-shifted ChrimsonR). But in addition, the green light from the projector 50 can be used for calibrating the pattern illumination with the detection field of view.

[0038] Note that the system design is agnostic to specific projector used because the patterns of light that are generated by the projector 50 are formed by controlling the images that are output by the projector 50 through universal display interfaces. For example, in projectors that are controlled via an HDMI port, the HDMI port is driven with signals that cause the projector to generate images such that when the generated images are routed through the optical system and arrive at the sample, the red light from the images will optogenetically stimulate the sample according to the requirements of whatever experiment is running.

[0039] Subsequent to the projector 50, the stimulation light passes through an optical path that includes scan lenses 52 (e.g., Plossl scan lenses), a tube lens 54, and a fold mirror 56. The stimulation light then reaches the dichroic mirror 60 that reflects the long wavelength stimulation light down into the proximal end of the objective 40. The projector 50, the dichroic mirror 60, and the objective 40 are aligned with respect to each other so that the red light that enters the proximal end of the objective 40 will exit the distal end of the objective and proceed towards the sample. The dichroic mirror 60 also lets the blue light that originatesfrom the blue LED 20 to travel down towards the sample. In the presence of calcium ions, the GCaMP protein will emit fluorescent green light, and this green light will enter the distal end of the objective 40 and travel up towards the camera 90. The dichroic mirror 60 also lets the shorter wavelength green fluorescent signal pass up to the camera 90. The objective 40, the dichroic mirror 60, and the beam splitter 30 are aligned with respect to each other so that the green light that exits the proximal end of the objective 40 will arrive at the camera 90.

[0040] Optionally, the vertical height of the system can be reduced by (a) positioning a folding mirror (not shown) in the path to the camera 90 and shifting the camera 90 to a horizontal orientation and / or (b) omitting the folding mirror 56 and shifting the prior components 50-54 to a horizontal orientation.

[0041] Appropriate excitation filters 26, 58 and emission filters 85 positioned as depicted in FIG. 1 can significantly improve the performance of the system. Note that filter 58 is removable to permit the use of green light from the projector - reflected or scattered by the sample - to reach the camera. This allows visualization of the illumination pattern that would otherwise be produced by first Wavelength light, which is not directly visible in camera due to emission filter.

[0042] The red light that emanates from the projector 50 is sufficient to cause activation of the neurons (due to the presence of ChrimsonR that has been previously introduced to the neurons). And because the pattern of light that is generated by the projector 50 can be easily controlled e.g., via the projector’s HDMI input, we can easily generate whatever pattern is necessary to achieve the desired spatio-temporally precise activation.

[0043] Notably, the stimulation (using the red light that originates in the projector 50) and the activity mapping (using the blue light that originates in the blue LED 20) can occur simultaneously. The use of blue light in the detection module allows GCaMP imaging (because the GCaMP protein emits fluorescent green light), whereas the red light of the projector 50 produces ChrimsonR activation. Any patterned image of red light can be projected as required in a closed-loop manner (for example, in response to a certain activity even in the culture), including but not limited to global stimulation for large samples, selected multiple stimulations with different sizes and distances, and free-form patterns depending on the input mask. The system should be very useful for developing better in vitro models ofcomplex brain disorders as well as with potential to allow development of hybrid computing devices (for example, combining MoNNets with Al systems).

[0044] In addition, when a low-cost portable laser RGB projector is used as the projector 50, the cost of the system is dramatically reduced, as well as the system’s mechanical and optical size, thus enabling the entire system to fit in a standard lab incubator. The inventors have determined that the components listed in Table 1 remain stable in the environment of an incubator, thus making it possible to do long term experiments.

[0045] Real-time control software is used to perform activity mapping and manipulation over the duration of the experiment, which can last for a few seconds, minutes, hours, or days.

[0046] FIG. 3A shows the characterization of uniform resolution across a large field of view for the FIG. 1 apparatus. FIG. 3B shows a stimulation pattern example for the FIG. 1 apparatus in which two spots of red stimulation light are projected onto two regions of interest (ROI #1 and ROI #2) of a sample, and fluorescent return light from GCaMP within the sample (i.e., the light spots in FIG. 3B, which represent neuronal activity) is captured using the camera 90. This shows that different size and shapes of light can be precisely activated by using the projector 50 to project different light patterns into the sample. FIG. 3C shows a characterization of uniform resolution for the FIG. 1 apparatus across a large field of view in an example image of a large mouse brain section at cellular resolution. The scale bars in the left and right halves of FIG. 3C are 1 mm and 100 um, respectively.

[0047] FIGS. 4A-C show the simultaneous activity imaging and manipulation in MoNNets 3D neuronal cell cultures using the FIG. 1 apparatus. More specifically, FIG. 4A shows an increase in activity rate after a patterned red light activation of ChrimsonR is turned on. FIG. 4B shows a preliminary example of training an ensemble in MoNNet by repeated co-activation of the whole MoNNet. The region 400 in FIG. 4B identifies a potential ensemble. And FIG. 4C shows an example of activation of a portion of a MoNNet cell culture network. More specifically, the lower half of the culture (i.e., region 410) was activated by illuminating it with red light. The rasterspots in region 420 visualize the changed activity pattern (i.e., the increased activity) of the neurons in region 410 when the red light was on. The x-axis on the right half of FIG. 4C is frame numbers, and the recording rate in this experiment was 15 frames per second.

[0048] FIGS. 5A and 5B depict an example of using the FIG. 1 apparatus to achieve formation of neuronal ensembles in vitro by repeated co-activation. More specifically, FIG. 5A shows pairwise activity correlation and network state dynamics, and FIG. 5B shows output of a trained RNN.

[0049] Memories in the brain are thought to be represented by neuronal ensembles, defined as groups of neurons that become functionally connected and activate together. This organization results in interesting properties such as memory recall by pattern completion, where partial activation of an ensemble can trigger full activation. The FIG. 1 apparatus enables us to investigate the properties of neuronal ensembles by facilitating their manipulation, pattern completion, and other dynamic behaviors. The top row of FIG. 5A demonstrates that repeated co-activation of a group of neurons using this device (indicated by the ovals) leads to the emergence of synchrony in their activity, suggesting the formation of an ensemble. This synchrony persists even after stimulation ceases. The data are represented as a graph, where each node corresponds to a neuron and each edge indicates the strength of synchrony between neuron pairs. The second row of FIG. 5A shows the overall system activity projected into a reduced-dimensional space (principal components). The third row of FIG. 5A depicts pairwise correlations in neuronal activity across different conditions, showing that optogenetic stimulation with this device increases network-wide correlations. FIG. 5B shows that training a recurrent neural network (RNN) with the data from FIG. 5A reproduces these ensemble-like effects in silico.

[0050] FIG. 5C presents the corresponding activity traces under these conditions, and how MoNNet activity changes in response to optogenetic stimulation using the FIG. 1 apparatus. In each panel, the top plot shows a heatmap of neuronal activity (y-axis: individual neurons; x-axis: time), while the bottom plot shows the sum of neuronal activity over time. These plots clearly demonstrate the ensemble formation effects observed in Figure 5A.

[0051] FIG. 6 shows a set of wavelengths that can be used for stimulation and excitation in the FIG. 1 embodiment, as well as the optical responses of the various proteins contained within the sample to those stimulation and excitation wavelengths. This example advantageously has a large separation of the excitation spectrum for Calcium imaging (ex: GCaMP protein) and the activation spectrum (e.g., red-shifted Chrimson R).

[0052] The FIG. 1 embodiment advantageously provides simultaneous mapping of neuronal activity (e.g., by imaging of calcium indicators GCaMP) and its manipulation by spatiotemporally patterned optogenetics based transducer (e.g., red-shifted ChrimsonR) in a portable and compact instrument that can fit inside a standard incubator and operate inside the incubator. It has a large field of view. Moreover, using a consumer-grade pocket projector 50 to optogenetically activate the neurons and / or a consumer- grade camera 90 can yield a dramatic reduction in the implementation cost.

[0053] Potential applications include neuronal activity mapping and perturbation of in vitro models of complex brain disorders such as brain organoids and MoNNets; organ-on- chip monitoring and manipulation application; and imaging of acute brain slices such as excised human brain tissue. In addition, this setup allows for bi-directional interfacing with 3D neuronal cultures, thus allowing the possibility of developing hybrid machine learning systems (for example, by integrating with MoNNets).

[0054] The inventor successfully used the FIG. 1 embodiment to optogenetically activate MoNNets, and noticed that the MoNNets exhibited a memory effect.

[0055] Because the same system is used for both activation and imaging, it becomes possible to implement closed-loop systems that make use of both the activation and imaging functions. For example, based on how the activity in a sample is looking, an algorithm can decide in real time to modify the activation pattern, thereby implementing closed-loop manipulation of the network dynamics.

[0056] Note that the upright configuration depicted in FIGS. 1 is not the only way to implement the system. To the contrary, inverted configurations which image the sample from the bottom may be used instead of the upright configuration described above.

[0057] Note also that the specific wavelengths discussed above in connection with FIG. 1-6 (i.e., a first wavelength 11 for optogenetic activation / stimulation of the neurons, a second wavelength 12 for illumination e.g., for calcium imaging, and a third wavelength 13 for the fluorescent light that is emitted by whatever protein is used to monitor the neuronal activity) are not the only wavelengths that can be used. To the contrary, a wide variety of three- wavelength sets can be employed, depending on the particular proteins that have been introduced into the cell in order to facilitate optogenetic stimulation and activity imaging.

[0058] For example, FIG. 7 depicts a variant of the FIG. 1 apparatus in which yellowgreen light (e.g., from an LED 20’) is used to illuminate the sample for activity imaging, and blue light from the projector 50 is used to optogenetically activate neurons within the sample. Of course, because the activation light in this example has a shorter wavelength than the illumination light, the roles of the short pass and long pass filters in this embodiment are reversed (with respect to their roles in the FIG. 1 embodiment). This change is reflected by replacing the short pass filter 30 of the FIG. 1 embodiment with a long pass filter 30’ in the FIG. 7 embodiment, and by replacing the long pass filter 60 of the FIG. 1 embodiment with a short pass filter 60’ in the FIG. 7 embodiment. The excitation filters 26, 58 and emission filters 85 of the FIG. 1 embodiment should also be replaced with excitation filters 26’, 58’ and emission filters 85’ with appropriately different spectral characteristics in the FIG. 7 embodiment. The remaining components operate similar to those described above in connection with FIG. 1, with appropriate modifications to account for the different wavelengths that are used.

[0059] FIG. 8 depicts a suitable set of proteins for use in the FIG. 7 embodiment as well as the optical responses of the various proteins contained within the sample to respective stimulation and excitation wavelengths. More specifically, blue light from the projector 50 is used to activate Channelrhodopsin (which has previously been introduced into the neurons e.g., using a viral vector), and yellow-green light (540-580 nm) from the LED 20’ is used to illuminate the sample. And because RCaMP has previously been introduced into the neurons (e.g., using a viral vector) fluorescent light will be emitted in the vicinity of calcium ions. And this fluorescent light is detected by the camera 90. Note that this example has a smaller separation of the excitation spectrum for Calcium imaging (i.e., the RCaMP protein) and the activation spectrum (i.e., the Channelrhodopsin).

[0060] Note that while the examples above are described in the context of neurons, the embodiments described herein are not limited to that context, and can also be used monitor the behavior of other types of cells in response to optogenetic stimulation.

[0061] While the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. Accordingly, it is intended that the presentinvention not be limited to the described embodiments, but that it has the full scope defined by the language of the following claims, and equivalents thereof.

Claims

WHAT IS CLAIMED IS:

1. An apparatus for activating at least one of a plurality of cells within a sample and monitoring a response of the cells to the activation, wherein the cells can be optogenetically activated by light at a first wavelength, and wherein the cells include a fluorescent protein that fluoresces at a third wavelength when illuminated by light at a second wavelength, the apparatus comprising: a projector having a control input, wherein the projector is configured to project an image that includes light at the first wavelength, wherein a nature of the image projected by the projector depends on a state of the control input; a second light source that emits light at the second wavelength; and an optical system configured to (a) route the image projected by the projector towards the sample, (b) route second-wavelength light from the second light source towards the sample so the sample is illuminated with light at the second wavelength, and (c) route third-wavelength light emitted from the sample towards a camera.

2. The apparatus of claim 1, wherein the projector comprises a MEMS laser projector.

3. The apparatus of claim 2, wherein the control input comprises an HDMI port.

4. The apparatus of claim 1, further comprising the camera, wherein the camera is sensitive to light at the third wavelength.

5. The apparatus of claim 4, further comprising an incubator, wherein the projector, the second light source, the camera, and the optical system are all positioned inside the incubator.

6. The apparatus of claim 1, wherein the optical system has an objective having a proximal end and a distal end, wherein the distal end of the objective is configured to be positioned adjacent to the sample, a first beam splitter positioned to route first- wavelength light from the projector into the proximal end of the objective, and a second beam splitter positioned to route second- wavelength light from the second light source into the proximal end of the objective,wherein the objective is positioned with respect to the sample so that third-wavelength light emitted from the sample enters the distal end of the objective, exits the proximal end of the objective, and subsequently travels towards the camera.

7. The apparatus of claim 6, further comprising the camera, wherein the camera is sensitive to light at the third wavelength.

8. The apparatus of claim 7, further comprising a filter positioned between the proximal end of the objective and the camera, wherein the filter passes third- wavelength light and blocks second-wavelength light.

9. The apparatus of claim 7, further comprising a filter positioned between the proximal end of the objective and the camera, wherein the filter passes third- wavelength light, blocks first-wavelength light, and blocks second-wavelength light.

10. The apparatus of claim 1, wherein the light at the first wavelength is red light, the light at the second wavelength is blue light, and the third-wavelength light is green light.

11. The apparatus of claim 1, wherein the light at the first wavelength is blue light and the second wavelength is 540-580 nm.

12. An apparatus for activating at least one of a plurality of cells within a sample and monitoring a response of the cells to the activation, wherein the cells can be optogenetically activated by light at a first wavelength, and wherein the cells include a fluorescent protein that fluoresces at a third wavelength when illuminated by light at a second wavelength, the apparatus comprising: an objective having a proximal end and a distal end, wherein the distal end of the objective is configured to be positioned adjacent to the sample; a projector having a control input, wherein the projector is configured to project an image that includes light at the first wavelength, wherein a nature of the image projected by the projector depends on a state of the control input; a first beam splitter configured and positioned to route first-wavelength light from the projector into the proximal end of the objective, wherein the projector, the first beamsplitter, and the objective are aligned with respect to each other so that the first- wavelength light that enters the proximal end of the objective will exit the distal end of the objective; a second light source that emits light at the second wavelength; and a second beam splitter configured and positioned to route second- wavelength light from the second light source into the proximal end of the objective, wherein the second light source, the second beam splitter, and the objective are aligned with respect to each other so that the second-wavelength light that enters the proximal end of the objective will exit the distal end of the objective, wherein the objective is positioned so that third-wavelength light emitted from the sample will enter the distal end of the objective and exit the proximal end of the objective, wherein the first beam splitter and the second beam splitter are configured and positioned to route third- wavelength light that exits the proximal end of the objective towards a camera, and wherein the objective, the first beam splitter, and the second beam splitter are aligned with respect to each other so that the third- wavelength light that exits the proximal end of the objective will arrive at the camera.

13. The apparatus of claim 12, wherein the projector comprises a MEMS laser projector.

14. The apparatus of claim 13, wherein the control input comprises an HDMI port.

15. The apparatus of claim 12, further comprising the camera, wherein the camera is sensitive to light at the third wavelength.

16. The apparatus of claim 15, further comprising an incubator, wherein the objective, the projector, the second light source, the first beam splitter, the second beam splitter, and the camera are all positioned inside the incubator.

17. The apparatus of claim 15, further comprising a filter positioned between the proximal end of the objective and the camera, wherein the filter passes third- wavelength light and blocks second- wavelength light.

18. The apparatus of claim 15, further comprising a filter positioned between the proximal end of the objective and the camera, wherein the filter passes third- wavelength light, blocks first- wavelength light, and blocks second-wavelength light.

19. The apparatus of claim 12, wherein the light at the first wavelength is red light, the light at the second wavelength is blue light, and the third-wavelength light is green light.

20. The apparatus of claim 12, wherein the light at the first wavelength is blue light and the second wavelength is 540-580 nm.

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