Systems and methods for patterned illumination and imaging

An imaging system, comprising:

• a first light source module configured to emit a first beam of light at a first wavelength;
• a second light source module configured to emit a second beam of light at a second wavelength;
• a first optical system configured to project at least a portion of the first beam of light and/or the second beam of light onto a spatial light modulator configured to produce a spatial pattern of illumination;
• a second optical system configured to project the spatial pattern of illumination onto a sample being imaged;
• a back-illuminated CMOS camera having an optical acceptance angle of at least 16°, positioned to receive light from the sample being imaged;
• and
• a controller configured to modulate current through the first light source module and/or the second light source module, control the spatial light modulator to project a sequence of spatial patterns of illumination onto the sample over time, and/or control exposures of the camera.

A method of projecting two or more spatial patterns of illumination at two or more wavelengths concurrently, comprising:

• initiating an exposure of an imaging device for an exposure interval, wherein the exposure interval comprises a plurality of sub-intervals;
• controlling a first light source to emit light at a first wavelength during a first sub-interval of the exposure interval;
• controlling a spatial light modulator to project a first spatial pattern of light from the first light source onto a sample during the first sub-interval;
• controlling a second light source to emit light at a second wavelength during a second sub-interval of the exposure interval;
• controlling the spatial light modulator to project a second spatial pattern of light from the second light source onto the sample during the second sub-interval;
• and
• controlling the first light source and the second light source such that the sample is not illuminated during a third sub-interval between the first sub-interval and the second sub-interval.

A method of imaging a sample, comprising:

• illuminating a biological sample with a first sequence of spatial patterns using light of a first wavelength, wherein the biological sample comprises a plurality of cells expressing one or more fluorescent reporters and one or more light-activated molecules;
• capturing image data based on light of a second wavelength emitted by the sample using an imaging device;
• constructing a second spatial pattern configured to provide targeted illumination of cells of the plurality of cells expressing the one or more fluorescent reporters;
• constructing a third sequence of spatial patterns, wherein each spatial pattern of the third sequence of spatial patterns is configured to provide targeted illumination of a subset of cells expressing the one or more fluorescent reporters;
• and
• illuminating the biological sample with the second spatial pattern at the first wavelength and one or more spatial patterns of the third sequence of spatial patterns at a third wavelength during a stimulus period, wherein illumination with the second spatial patterns alternates with illumination with a spatial pattern of the third sequence of spatial patterns at a rate higher than a frame rate of the imaging device.

A method for registering a light-patterning device to an image capturing device, the method comprising:

• loading a sample target onto an imaging device;
• projecting, with the light-patterning device, a pattern of quick response (QR) codes onto the sample target, the pattern of QR codes encoding a plurality of digits corresponding to a number of QR codes in the pattern;
• acquiring, with the image capturing device, an image of the pattern of QR codes projected onto the sample target;
• determining a pattern of fiducial marks in the pattern of QR codes based on the acquired image of the pattern of QR codes;
• calculating, using the determined pattern of fiducial marks in the pattern of QR codes, a transformation from pixel coordinates of the light-patterning device to pixel coordinates of the image capturing device to obtain a transformed pattern of QR codes.