System and method for fluorescence microscopy with detection of light emission from multiple fluorochromes

Abstract

The present invention concerns fluorescence microscopy, and more specifically, a method for detecting fluorescence emitted from a sample labeled with at least four predetermined fluorochromes, wherein the at least four different fluorochromes comprises a first and a second fluorochrome that forms a pair of fluorochromes, the pair of fluorochromes selected as a Cy3 fluorochrome analog forming the first fluorochrome, and a 594 fluorochrome analog forming the second fluorochrome, or the pair of fluorochromes is selected as a 425 fluorochrome analog forming the first fluorochrome and a 488 fluorochrome analog forming the second fluorochrome. Detection of fluorescence from the at least four predetermined fluorochromes are achieved, in accordance with the invention, by selecting an excitation wavelength interval for the second fluorochrome such that excitation of the first fluorochrome is reduced, and by selecting an emission wavelength range of the first fluorochrome so that the perfusion of the light emission from the second fluorochrome is reduced.

1 . A method for detecting fluorescence emitted from a sample labeled with a plurality of predetermined fluorochromes using a microscopy system comprising a light source arrangement, wherein the method comprises the steps of:
 selecting at least four different fluorochromes configured to emit light within the visible light spectra, the at least four different fluorochromes including a first and a second fluorochrome forming a pair of fluorochromes;   selecting excitation wavelength intervals for the at least four different fluorochromes, wherein the excitation wavelength interval for the second fluorochrome is selected such that the excitation of the first fluorochrome is reduced;   configuring a filter arrangement of the microscopy system to selectively allow light to pass through within emission wavelength intervals matching the emission wavelength intervals of the at least four different fluorochromes, wherein the emission wavelength interval for the first fluorochrome is selected to reduce light emission bleedthrough from the second fluorochrome;   sequentially emitting light within, at least a portion of the selected excitation wavelength intervals; and   detecting light emitted from the sample transmitted through the filter arrangement,   wherein the pair of fluorochromes are selected as a Cy3 analog fluorochrome forming the first fluorochrome and a 594 analog fluorochrome forming the second fluorochrome, or the pair of fluorochromes are selected as a 425 analog fluorochrome forming the first fluorochrome and a 488 analog fluorochrome forming the second fluorochrome.   
     
     
         2 . Method according to  claim 1 , further comprising the step of separating fluorescence signals from the at least four different fluorochromes. 
     
     
         3 . Method according to  claim 1 , further comprising selecting at least five different fluorochromes, wherein excitation wavelength intervals are selected for the at least five different fluorochromes and the filter arrangement is configured for the at least five different fluorochromes, the pair of fluorochromes are selected as the Cy3 analog fluorochrome forming the first fluorochrome and the 594 analog fluorochrome forming the second fluorochrome, and a further pair of fluorochromes are selected as the 425 analog fluorochrome forming the first fluorochrome and the 488 analog fluorochrome forming the second fluorochrome. 
     
     
         4 . Method according to  claim 1 , further comprising selecting at least one further fluorochrome configured to emit light within the far red/near-infrared spectra. 
     
     
         5 . Method according to  claim 1 , further comprising selecting at least one further fluorochrome configured to emit light within the infrared spectra. 
     
     
         6 . Method according to  claim 1 , wherein the remaining fluorochromes are selected from a group comprising a DAPI analog fluorochrome or a BV421 analog fluorochrome, a 425 analog fluorochrome, a 488 analog fluorochrome, Cy3 analog fluorochrome, a 594 analog fluorochrome, a PerCP analog fluorochrome or a 647/660/680/700 analog fluorochrome, and a 725/750/790 analog fluorochrome. 
     
     
         7 . A microscopy system for separating fluorescence signals emitted from a sample labeled with a plurality of predetermined fluorochromes, the microscopy system comprising:
 a light source arrangement configured to emit light;   a light guide for guiding light from the light source arrangement to the sample for excitation of the plurality of predetermined fluorochromes;   a filter arrangement configured to allow separation of fluorescence emitted by the plurality of predetermined fluorochromes labeling the sample, and   a detection device configured for receiving light transmitted through the filter arrangement,   wherein   the microscope filter sets can separate signals from at least four different fluorochromes that emit light within the visible light spectra including a first and a second fluorochrome forming a pair of fluorochromes,   the light source arrangement is configured to sequentially emit light within at least four different excitation wavelength intervals,   the excitation wavelength interval for the second fluorochrome is selected to reduce excitation of the first fluorochrome,   the filter arrangement comprises at least four matched filters configured to allow light to pass through within at least four different emission wavelength intervals,   the emission wavelength interval for the first fluorochrome is selected to reduce light emission bleedthrough from the second fluorochrome, and   the pair of fluorochromes are selected as a Cy3 analog fluorochrome forming the first fluorochrome and a 594 analog fluorochrome forming the second fluorochrome, or the pair of fluorochromes are selected as a 425 analog fluorochrome forming the first fluorochrome and a 488 analog fluorochrome forming the second fluorochrome.   
     
     
         8 . Microscopy system according to  claim 7 , wherein the detection device comprises an image capturing device configured to catch images of fluorescence of the sample. 
     
     
         9 . Microscopy system according to  claim 7 , wherein the light source arrangement comprises a plurality of narrow-banded light-emitting diodes. 
     
     
         10 . Microscopy system according to  claim 7 , wherein the light source arrangement comprises a combination of a light source emitting light within an ultraviolet and a visible wavelength spectrum and a plurality of light-emitting diodes emitting light within at least one of the far red, near-infrared or infrared spectrum.