Optogenetics microscope
The invention provides an open-stage near-TIRF microscope in which all of the optical components are positioned underneath the sample, allowing for physical access to, and control over the environment of, the sample. The microscope can be used to image cells expressing fluorescent voltage indicators. Since the TIRF components do not interfere with the sample, living cells can be studied using a microscope of the invention. Where a sample includes electrically active cells expressing fluorescent voltage indicators, the microscope can be used to view voltage changes in, and thus the electrical activity of, those cells.
1. A near-total internal reflection fluorescence microscope comprising:
a stage comprising an object region configured to support a sample;
an illumination subsystem comprising a prism disposed beneath the object region, the prism comprising an upper surface proximal to the object region, a lower surface distal to the object region, and at least one side surface adjoining the upper surface and the lower surface, and a light source operable to transmit light onto the at least one side surface of the prism without achieving total internal reflection when the sample is on the stage, causing the prism to illuminate the sample from beneath by near-total internal reflection; and
an imaging subsystem comprising an objective lens unit disposed beneath the prism and an image capture device operable to receive an image passed through the objective lens unit from the object region.
2. The microscope of claim 1 , wherein the object region supports the sample by means of a sample dish with a transparent bottom portion.
3. The microscope of claim 2 , wherein the prism and transparent bottom portion are coupled by a low-autofluorescence index matching fluid; and wherein the prism, the index matching fluid, and the transparent bottom portion have a common index of refraction.
4. The microscope of claim 2 , wherein the sample dish can hold an aqueous medium and provide access to the sample from above.
5. The microscope of claim 4 , further comprising an environmental control subsystem operable to control one or more of humidity, temperature, and gas of the sample region above the aqueous medium to maintain living cells in the aqueous medium.
6. The microscope of claim 1 , further comprising an activation subsystem comprising an activation light source configured to transmit activation light that is spectrally distinct from the illumination light onto the sample.
7. The microscope of claim 6 , wherein the activation light source and the light source are each selected from the group consisting of: a laser, a diode laser bar; a diode laser; and an LED.
8. The microscope of claim 6 , wherein the activation subsystem transmits the activation light upwards through the objective lens unit and onto the sample.
9. The microscope of claim 6 , further comprising an activation tube lens disposed within a path defined by the activation light, operable to focus the activation light at a back aperture of the objective lens.
10. The microscope of claim 6 , wherein the activation subsystem comprises a spatial light modulator operable to spatially pattern the activation light onto the sample, wherein the spatial light modulator comprises one selected from the group consisting of: a digital micromirror device; a digital light processor; and a liquid crystal display.
11. The microscope of claim 10 , wherein the spatial light modulator is controlled by a computer operable to define an illumination pattern.
12. The microscope of claim 11 , wherein the illumination pattern is generated by the computer based on an input from the imaging subsystem, wherein the input from the imaging subsystem comprises a pattern of one or more cells in the sample.
13. The microscope of claim 12 , wherein the computer defines an illumination pattern with the spatial light modulator that corresponds to the pattern of cells in the sample, thereby causing the activation subsystem to transmit illumination light onto the one or more cells.
14. The microscope of claim 6 , further comprising a dichroic mirror to reflect the activation light upwards onto the sample, and to allow the image to pass downward through to the image capture device.
15. The microscope of claim 6 , wherein the activation light has a wavelength between 450 and 495 nm, an intensity of about 22 mW/cm2, or both.
16. The microscope of claim 1 , wherein the illumination light has a wavelength between 580 and 650 nm, an intensity of about 100 W/cm2, or both capable of illuminating a microbial rhodopsin.
17. The microscope of claim 1 , further comprising baffles positioned to prevent unwanted reflected light from entering the objecting lens.
18. The microscope of claim 1 , further comprising other beam shaping optics disposed within a path defined by the illumination light, operable to shape the illumination light.
19. The microscope of claim 1 , wherein the objective lens has a numerical aperture between 0.4 and 1.0.
20. The microscope of claim 1 , wherein the objective lens has a numerical aperture of about 0.5.