Systems and methods for optogenetic imaging
Provided herein are systems and methods for simultaneous imaging and stimulation using a microscope system. The microscope system can have a relatively small size compared to an average microscope system. The microscope can comprise in part an imaging light source and a stimulation light source. Light from the imaging light source and the stimulation light source can be spectrally separated to reduce cross talk between the stimulation light and the imaging light.
1. A microscope system for simultaneous imaging and stimulation of tissue within a subject, the microscope system comprising:
a) an optical assembly that includes:
i. an illumination optical path comprising one or more light-emitting elements and configured to direct imaging light to the tissue within a specified field-of-view;
ii. a stimulation optical path comprising one or more light-emitting elements and configured to direct stimulation light to the tissue within said field-of-view, to the tissue outside of said field-of-view, or to the tissue partially within said field-of-view;
iii. an imaging optical path comprising one or more image sensors and configured to receive light reflected, scattered, or emitted by the tissue to generate an image of the tissue within said field-of-view;
b) a corrective optical element optically cooperated with an element of the optical assembly to achieve a spatial resolution of better than 2 μm over the entirety of the specified field-of-view; and
a baseplate is configured to be mounted on the subject in a fixed position and to receive the optical assembly, thereby aligning the optical assembly relative to the tissue within the subject upon attachment to the baseplate, and wherein the optical assembly is removable from the baseplate.
2. The microscope system of claim 1 , wherein the specified field-of-view is about 1 mm×1 mm.
3. The microscope system of claim 1 , wherein the specified field-of-view is about 2 mm×2 mm.
4. The microscope system of claim 1 , wherein the specified field-of-view is about 4 mm×4 mm.
5. The microscope system of claim 1 , wherein the imaging light is fluorescence excitation light.
6. The microscope system of claim 5 , wherein the fluorescence excitation light has a wavelength within a wavelength range of 400 nm to 500 nm, and the stimulation light has a wavelength within a wavelength range of 500 nm and 800 nm.
7. The microscope system of claim 5 , wherein the fluorescence excitation light has a wavelength within a wavelength range of 500 nm to 650 nm, and the stimulation light has a wavelength within a wavelength range of 350 nm and 560 nm.
8. The microscope system of claim 1 , wherein the optical assembly is configured to acquire images with a spatial resolution of better than 2 μm at a center of the field-of-view.
9. The microscope system of claim 1 , wherein the optical assembly further comprises one or more deformable lenses configured to adjust a corresponding focal depth of the imaging optical path, the stimulation optical path, or both.
10. The microscope system of claim 9 , wherein the illumination optical path, stimulation optical path, and imaging optical path share at least one deformable lens of said one or more deformable lenses.
11. The microscope system of claim 9 , wherein the one or more deformable lenses are configured to adjust the corresponding focal depth of the imaging optical path, the stimulation optical path, or both over a range of about 300 μm without loss of spatial resolution.
12. The microscope system of claim 9 , wherein the one or more deformable lenses are selected from the group consisting of liquid lenses, liquid crystal lenses, piezo-actuated tunable lenses, and any combination thereof.
13. The microscope system of claim 9 , wherein the one or more deformable lenses are configured so that the imaging optical path and the stimulation optical path direct light to the same focal plane.
14. The microscope system of claim 9 , wherein the optical assembly is configured to acquire images over a tissue volume of about 1 mm×1 mm×300 μm.
15. The microscope system of claim 1 , wherein the stimulation optical path is further configured to direct the stimulation light to the tissue in a user-defined spatial pattern.
16. The microscope system of claim 15 , wherein the user-defined spatial pattern has a spatial resolution of better than 5 μm at a corresponding focal plane.
17. The microscope system of claim 1 , further comprising a processor and a memory device, wherein the processor is configured to execute a series of software-encoded instructions stored in the memory device.
18. The microscope system of claim 17 , wherein the software-encoded instructions include instructions for: (i) modulating intensity of the imaging light in a time-dependent manner or a spatially-dependent manner, (ii) modulating intensity of the stimulation light in a time-dependent manner or a spatially-dependent manner, (iii) varying a corresponding focal depth of the imaging optical path, the stimulation optical path, or both in a time-dependent manner, and (iv) capturing one or more images of the tissue within the specified field-of-view at specified times, or any combination thereof.
19. A microscope system for simultaneous imaging and stimulation of tissue within a subject, the microscope system comprising:
a) an optical assembly that includes
i. an illumination optical path comprising one or more light-emitting elements and configured to direct imaging light to the tissue within a specified field-of-view;
ii. a stimulation optical path comprising one or more light-emitting elements and configured to direct stimulation light to the tissue within said field-of-view, to tissue outside of said field-of-view, or to the tissue partially within said field-of-view;
iii. an imaging optical path comprising one or more image sensors and configured to receive light reflected, scattered, or emitted by the tissue to generate an image of the tissue within said field-of-view; and
iv. one or more deformable lenses configured to adjust a corresponding focal depth of the imaging optical path,
the stimulation optical path, or both over a range of about 300 μm without loss of spatial resolution; and
b). a baseplate configured to be mounted on the subject in a fixed position and to receive the optical assembly, thereby aligning the optical assembly relative to the tissue within the subject upon attachment to the baseplate, and wherein the optical assembly is removable from the baseplate.
20. The microscope system of claim 9 , wherein the one or more deformable lenses are configured so that the imaging optical path and the stimulation optical path direct light to the same focal plane.