A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents

Abstract

Optogenetics is an excellent tool for noninvasive activation and silencing of neurons and muscles. Although they have been widely adopted, illumination techniques for optogenetic tools remain limited and relatively nonstandardized. We present a protocol for constructing an illumination system capable of dynamic multispectral optical targeting of micrometer-sized structures in both stationary and moving objects. The initial steps of the protocol describe how to modify an off-the-shelf video projector by insertion of optical filters and modification of projector optics. Subsequent steps involve altering the microscope's epifluorescence optical train as well as alignment and characterization of the system. When fully assembled, the illumination system is capable of dynamically projecting multispectral patterns with a resolution better than 10 μm at medium magnifications. Compared with other custom-assembled systems and commercially available products, this protocol allows a researcher to assemble the illumination system for a fraction of the cost and can be completed within a few days.

Figure 1.

Optical configuration of the system and components. (a) Final optical configuration for the system. Adapted from ref. 12. The epifluorescent optics are replaced by an accessory tube lens (infinity corrected) or relay lens pair (160 mm) and a modified 3-LCD projector. (b) Optical configuration of the projector in the original unmodified state. (c) Optical configuration of the constructed illumination system for an infinity corrected microscope. (d) Optical configuration of the constructed illumination system for a 160 mm microscope.

Figure 2.

Custom software for the real-time illumination of freely behaving C. elegans. (a) Three independent loops each operating at 25 Hz control image acquisition, motorized stage repositioning, and automated illumination control. (b) Acquired bright-field image of C. elegans. (c) Binary image after applied thresholding. (d) The binary image is thinned to single pixel backbone, representing the AP axis of the animal) and segmented according to user selectable parameters (number and location). The locations for segmenting are based along the relative path length of the backbone where the head is 0 and the tail is 1. (e) Resulting segmentation of the binary image. (f) Color pattern generated based on user selectable options including segment number, color (RGB), intensity (0–255) for each color, as well as illumination duration. (g) Resulting multi-color illumination pattern projected onto the moving C. elegans. Image is falsely colored based on the intended illumination pattern. Scale bar is 250 μm.

Figure 3.

Modifications of the 3-LCD projector to limit the spectral width of the RGB colors. (a) Internal filters are added to the 3-LCD projector thus narrowing the bandpass for each RGB color. (b) Action spectra for the optogenetic reagents channelrhodopsin-2 (ChR2) and MAC. (c) Measured spectra for the red, green, and blue color planes before and after addition of the internal filters. Panels a and c are adapted from ref. 12.

Figure 4.

Disassembly and insertion of custom optics into the 3-LCD projector. (a) Removal of the projection/zoom lens system. (b) Removal of the screws connecting the top of the projector case to the main body. (c) Disconnecting the top control panel to remove projector case cover. (d) Removal of the LAN board. (e) Disconnecting wires and screws connecting main board. (f) Disconnecting LCD panel cables. (g) Removal of the dynamic iris. (h) Removal of the cover of the main optical RGB path. (i) Cover showing the polarizing filters. (j) RGB optical paths. (k) Optical path after insertion of optical filters; colored boxes show location for red, green, and blue filter insertion. (l) Removal of the diverging projection lens from the zoom lens system.

Figure 5.

Characterization of the completed illumination system. Adapted from ref 12. (a) Relative intensity as a function of color pixel value (0–255) for each RBG color plane. (b) Ideal (59.6 um) and measured (68.5 um) width of a defined projection pattern using a 4× objective. This demonstrates spatial spread in illumination due to contrast transfer function of optical components. Width was measured at the point where intensity drops to 10% of the maximum value. (c) Measured spot-size using a 4× objective. This demonstrates a resolution limit of ~14 μm at 4×. (d) Measured spot-size using a 25× objective. This demonstrates a resolution limit of ~5 μm at 25×.

Figure 6.

Example application: selected area illumination of C. elegans. Adapted from ref. 12. (a) Frames from Supplementary Video 2 of ref. 12 demonstrating direct muscle control of a paralyzed animal using patterned light. (b) Sequential frames from Supplementary Video 3 of ref. 12 showing a bar of light passing over the animal from posterior to anterior as the animal is freely crawling. Initially the animal is traveling forward, but when the light reaches the anterior mechanosensory neurons expressing ChR2 (middle frame), the animal quickly reverses direction. (c) Sequential frames from Supplementary Video 8 of ref. 12 demonstrating the multi-spectral dynamic capacity of the illumination system. The animal is illuminated with blue light in the region of the anterior mechanosensory neuron which express ChR2 thus eliciting a reversal. The animal is subsequently illuminated with green light in the region of the command interneurons which express the hyperpolarizing MAC thus halting the reversal. Scale bar is 250 μm.