Methods for microscopy with ultraviolet surface excitation (MUSE) imaging
Described herein are methods for imaging a fluorescent bioassay (including a substrate, such as dispersed cellular sample, exposed to one or more exogenous fluorophore and/or fluorescent probe that accumulate in a structure of interest). The bioassay can be excited with Type-C ultraviolet (UVC) light produced by one or more light emitting diode (LED). The UVC can have a center wavelength that causes emission by the fluorescent bioassay. A digital optical device can collect a signal emitted from the fluorescent bioassay in response to the excitation. The methods relate in particular to Microscopy with Ultraviolet Surface Excitation (MUSE) imaging.
1. A method comprising:
exposing a dispersed cellular sample to one or more exogenous fluorophore and/or fluorescent probe that accumulate in a structure of interest;
positioning the dispersed cellular sample in contact with a top face of an optical window of an external accessory, wherein the external accessory comprises the optical window and one or more light emitting diodes (LED) configured to provide Type-C ultraviolet (UVC) light;
positioning the external accessory in focus with a microscope having one or more digital image sensor;
exciting the one or more exogenous fluorophore and/or fluorescent probe within the sample with the UVC light, produced by the one or more LED, having a center emission wavelength that causes emission by the one or more exogenous fluorophore and/or fluorescent probe, wherein the UVC light transmits through one or more side faces of the optical window and the optical window delivers the UVC light uniformly to a surface of the dispersed cellular sample in contact with the top face of the optical window via frustrated total internal reflection; and
collecting, with the microscope having the one or more digital image sensor, emitted light from excited ones of the one or more exogenous fluorophores and/or fluorescent probe that creates an image comprising the structure of interest in the sample, wherein the emitted light emits out a bottom face of the optical window to the microscope having the one or more digital image sensor.
2. The method of claim 1 , wherein the dispersed cellular sample is a cell suspension, a cytology smear, or a bacteria colloidal sample.
3. The method of claim 1 , wherein the one or more exogenous fluorophore and/or fluorescent probe comprise one or more of chemical dyes, quantum dots, proteins, peptides, and oligonucleotides.
4. The method of claim 1 , wherein the Type-C ultraviolet light has a center emission wavelength of between 230 nm and 290 nm.
5. The method of claim 1 , wherein the one or more exogenous fluorophore and/or fluorescent probe is excitable in a range of 235 nm to 285 nm.
6. The method of claim 1 , wherein the one or more exogenous fluorophore and/or fluorescent probe has a useful emission band from 350 nm to 700 nm.
7. The method of claim 1 , wherein one or more diffractive optical element is attached to the microscope.
8. The method of claim 1 , wherein the microscope comprises one or more cameras, each configured to detect at one or more wavelength ranges from 350 nm to 700 nm.
9. The method of claim 1 , wherein the image is projected into red, green, blue, and opacity channels of a standard display device.
10. A method comprising:
obtaining a fluorescent bioassay;
positioning the bioassay in contact with a top face of an optical window of an external accessory, wherein the external accessory comprises the optical window and one or more light emitting diodes (LED) configured to provide Type-C ultraviolet (UVC) light;
positioning the external accessory in focus with a digital optical detecting device;
exciting the bioassay with the UVC light, produced by the one or more LED, having a center emission wavelength that causes emission by the fluorescent bioassay, wherein the UVC light transmits through one or more side faces of the optical window and the optical window delivers the UVC light uniformly to a surface of the bioassay in contact with the top face of the optical window via frustrated total internal reflection; and
collecting, with the digital optical detecting device, a signal emitted from the fluorescent bioassay through a bottom face of the optical window towards the digital optical detecting device.
11. The method of claim 10 , wherein the bioassay comprises a substrate and one or more fluorescent probe.
12. The method of claim 11 , wherein the one or more fluorescent probe comprises one or more chemical dyes, quantum dots, proteins, peptides, and/or oligonucleotides.
13. The method of claim 11 , wherein the Type-C ultraviolet light has a center emission wavelength of between 230 nm and 290 nm.
14. The method of claim 11 , wherein the one or more fluorescent probes is excitable in a range of 235 nm to 285 nm.
15. The method of claim 11 , wherein the one or more fluorescent probes has a useful emission band from 350 nm to 700 nm.
16. The method of claim 10 , wherein the digital optical detecting device comprises one or more diffractive optical element and one or more electro-optical sensor.
17. The method of claim 16 , wherein the one or more diffractive optical element is a single optical lens, a compound optical lens, and/or a freeform waveguide.
18. The method of claim 16 , wherein the one or more diffractive optical element has less than 5% total optical transmittance between 230 nm and 290 nm.
19. The method of claim 16 , wherein the one or more electro-optical sensor is a camera sensor, a photodiode, or a photomultiplier tube.
20. The method of claim 19 , wherein the one or more electro-optical sensor is configured to detect and separate emitted light from fluorescent probes at one or more wavelength ranges from 350 nm to 700 nm.