Multispectral super resolution microscopy

Abstract

Devices, systems, reagents, and methods for the performance of multi-spectrum super resolution microscopy are disclosed.

1 . A microscope apparatus for simultaneous measurement of positional-spectral or spectral-spectral information of a sample, comprising:
 collection optics for receiving a light signal from a sample, the collection optics configured to create a first optical path having a first light signal and a second optical path having a second light signal, the collection optics includes having and right microscope image ports;   a first dispersion element disposed in the first optical path to disperse the first light signal into a first spectrum; and   a detector for receiving the spectrum from the first optical path and light from the second optical path.   
     
     
         2 . The microscope apparatus according to  claim 1 , wherein the collection optics includes an objective lens and a beam splitter disposed between the objective lens and the dispersion element, the beam splitter configured to divide light received thereby into the first and second optical paths. 
     
     
         3 . The microscope apparatus according to  claim 2 , wherein the beamsplitter is configured to divide the light into a first portion of about 70% in the first optical path and a second portion of about 30% in the second optical path. 
     
     
         4 . The microscope apparatus according to  claim 1 , wherein the collection optics includes a first objective lens disposed in the first optical path for receiving a light signal from the sample, and wherein the collection optics includes a second objective lens disposed in the second optical path at a location for receiving a light signal from the sample. 
     
     
         5 . The microscope apparatus according to  claim 1 , wherein the image ports are configured to divide the light into a first portion of about 70-80% in the first optical path and the remaining 30-20% in the second optical path. 
     
     
         6 . The microscope apparatus according to  claim 1 , wherein the position information of the sample is determined using the spectral information received by the detector. 
     
     
         7 . The microscope apparatus according to  claim 1 , wherein the first and second dispersion elements are oriented to disperse the first and second spectra in opposite directions. 
     
     
         8 . The microscope apparatus according to  claim 1 , wherein the first and second dispersion elements are oriented to disperse the first and second spectra in directions that are perpendicular to one another. 
     
     
         9 . The microscope apparatus according to  claim 1 , comprising a pair of lenses disposed between the collection optics and the first dispersion element, the pair of lenses placed in a 4f configuration with a slit placed at the intermediate image plane between the lenses. 
     
     
         10 . The microscope apparatus according to  claim 9 , wherein the slit has a longitudinal axis and the first dispersion element is oriented to disperse the first light signal along a direction parallel to the longitudinal axis. 
     
     
         11 . The microscope apparatus according to  claim 1 , wherein at least one of the first and second dispersion elements includes a prism assembly comprising an equilateral prism and at least two mirrors in optical communication with the prism. 
     
     
         12 . The microscope apparatus according to  claim 1 , wherein at least one of the first and second dispersion elements is mounted to a translation stage. 
     
     
         13 . The microscope apparatus according to  claim 1 , wherein at least one of the first and second dispersion elements includes a diffraction grating. 
     
     
         14 . The microscope apparatus according to  claim 1 , wherein at least one of the first and second dispersion elements includes a diffraction grating and at least two mirrors in optical communication with the grating. 
     
     
         15 . A method of imaging live cells, the method comprising:
 providing a microscope apparatus according to  claim 1 ; and   labeling a first biomarker with a first reagent and a second biomarker with a second reagent, where the first reagent comprises a first fluorescent label and the second reagent comprises a second fluorescent label, and where the first fluorescent label and the second fluorescent label are all excitable by light of the same wavelength and emit at a wavelength distinguishable from one another.