US 2021/0063312 A1 · METHODS FOR ULTRAVIOLET EXCITATION MICROSCOPY OF BIOLOGICAL SURFACES

Abstract

Microscopy with Ultraviolet Surface Excitation (MUSE) for use in the classroom to enhance life sciences education and curricula, or for other applications, including without limtiation the operating room, other medical environments, research environments, and low resource environments. MUSE's suitability is based on multiple key factors including its simplicity of use, the incorporation of inexpensive hardware including LED illumination, and very basic tissue preparation. The ultraviolet excitation acts as passive optical sectioning confining the generated fluorescence signal to only a few micrometers below the tissue surface thus eliminating the out of focus signals. This facilitates image capture of tissue microstructure and organization from specimens at the intact or sliced surface arising from varying fluorophore concentration within the different cellular compartments. Although just the tissue auto fluorescence maybe used, image quality is enhanced with brief application of nontoxic fluorescent dyes to selectively highlight cellular compartments. Sample preparation is safe, efficient and familiar to students with basic chemistry or biology lab experience. Mixed-dye powders may be used to simplify translation of this method for educational, medical, research, low resource, and other settings.

Claims46

Dependent claims

A method of staining a sample for examination by an ultraviolet surface excitation microscopy system, the method comprising:

(a) selecting a mixed fluorescence dye solution made from a set of fluorescing stains in preselected proportions for generating the mixed fluorescence dye solution when dissolved in a preselected volume of a solvent, the solution comprising:
(i) at least a first fluorescing stain at a first concentration and a second fluorescing stain at a second concentration,
(ii) the first fluorescing stain characterized by a first light emission spectrum when subjected to an ultraviolet light of a first wavelength by the ultraviolet surface excitation microscopy system, the second fluorescing stain characterized by a second light emission spectrum when subjected to the first ultraviolet light source wavelength, wherein the first emission spectrum is different from the second emission spectrum;
and
(b) exposing a sample for ultraviolet surface excitation microscopy examination to the mixed fluorescence dye solution for a predetermined time period to stain features of the sample;
wherein the selected mixed fluorescent dye solution is configured to prepare the sample for imaging by the ultraviolet surface excitation microscopy system at the first ultraviolet light source wavelength such that light emissions of features associated with the first fluorescing stain are imaged by the system at a first intensity and the light emissions of features associated with the second fluorescing stain are imaged by the system at a second intensity, wherein the first and second intensities are approximately the same.

3 more elements

A microscopy method utilizing an ultraviolet surface excitation microscopy system in combination with a mixed fluorescence dye solution made from a set of fluorescing stains in preselected proportions for generating the mixed fluorescence dye solution when dissolved in a preselected volume of a solvent, the method comprising:

(a) exposing a sample for microscopic analysis to the mixed fluorescence dye solution for a predetermined time period to stain features of the sample, the solution comprising at least a first fluorescing stain at a first concentration and a second fluorescing stain at a second concentration, the first fluorescing stain characterized by a first light emission spectrum when subjected to an ultraviolet light of a first wavelength of the microscopy system, the second fluorescence dye characterized by a second light emission spectrum when subjected to the ultraviolet light of the first wavelength of the microscopy system, wherein the first emission spectrum is different from the second emission spectrum;
(b) positioning the sample for imaging by the microscope, the microscope comprising an ultraviolet light source and a microscope objective;
(c) using the ultraviolet light source to simultaneously excite the first and second fluorescing stains associated with the features of the sample such that the microscope objective collects light emissions from the first and second fluorescing stains;
(d) wherein the method is configured so that the light emissions of features associated with the first fluorescing stain are imaged by the system at a first intensity and the light emissions of features associated with the second fluorescing stain are imaged by the system at a second intensity, wherein the first and second intensities are approximately the same.

1 more element

A microscopy method utilizing an ultraviolet surface excitation microscopy system in combination with a mixed fluorescence dye solution made from a set of fluorescing stains in preselected proportions for generating the mixed fluorescence dye solution when resolved in a preselected volume of solvent, the method comprising:

(a) exposing a sample for microscopic analysis to the mixed fluorescence dye solution for a predetermined time period to stain features of the sample, the solution comprising at least a first fluorescing stain at a first concentration and a second fluorescing stain at a second concentration, the first fluorescing stain characterized by a first light emission spectrum when subjected to an ultraviolet light of a first wavelength of the microscopy system, the second fluorescence dye characterized by a second light emission spectrum when subjected to the ultraviolet light of the first wavelength of the microscopy system, wherein the first emission spectrum is different from the second emission spectrum;
(b) positioning the sample for imaging by the microscope, the microscope comprising an ultraviolet light source and a microscope objective;
(c) using the ultraviolet light source to simultaneously excite the first and second fluorescing stains associated with the features of the sample such that the microscope objective collects light emissions from the first and second fluorescing stains.

Dependencies are inferred from the claim text.

Methods for ultraviolet excitation microscopy of biological surfaces

Life of this patent

Methods for ultraviolet excitation microscopy of biological surfaces

Life of this patent

Abstract

Claims46