Method for improving fluorescence image contrast

Abstract

There is provided an improved method for enhancing fluorescence images of an object, such as a biological tissue, by selectively eliminating or reducing unwanted fluorescence from fluorophores other than the fluorophore of interest. The method is based on the measurement of the lifetime of fluorophores while preserving information related to the fluorescence intensity of the fluorophore of interest.

1 . A method for optical imaging of an object containing two or more fluorophore species, said method comprising: 
 acquiring a fluorescence signal comprising time and amplitude information for one or more pixel of a region of interest (ROI) of said object using an excitation and an emission wavelength compatible with detection of at least one of said two or more fluorophore species;    calculating a fluorescence intensity and a fluorescence lifetime from said fluorescence signal for each of said one or more pixel;    multiplying said fluorescence intensity by a weighting factor, said weighting factor being a function of said calculated fluorescence lifetime and at least one predetermined fluorescence lifetime, to generate a weighted fluorescence intensity for said one or more pixel;    generating an image of said ROI based on said weighted fluorescence intensity of said one or more pixel.    
     
     
         2 . The method as claimed in  claim 1  wherein said fluorescence signal comprises contribution from two or more fluorophore species, the method further comprising steps of: 
 deriving a contribution fraction for at least one of said fluorophore species; and    multiplying said weighted fluorescence intensity by said contribution fraction.    
     
     
         3 . The method as claimed in  claim 2  wherein said contribution fraction is determined by modeling said fluorescence signal with a multi-exponential function.  
     
     
         4 . The method as claimed in  claim 3  wherein said modeling is applied to a temporal point spread function (TPSF) of said fluorescence signal.  
     
     
         5 . The method as claimed in  claim 1  wherein said weighting factor is determined by an Indicator function.  
     
     
         6 . The method as claimed in  claim 5  wherein said Indicator function is defined by boundaries which are function of said predetermined fluorescence lifetime.  
     
     
         7 . The method as claimed in  claim 6  wherein said boundaries are also function of an error associated with said measured lifetime.  
     
     
         8 . The method as claimed in  claim 7  wherein said weighting factor is 1 when said measured lifetime is within said boundaries and 0 otherwise.  
     
     
         9 . The method as claimed in  claim 2  further comprising a step of: 
 multiplying said fluorescence intensity by a preliminary weighting factor prior to said step of multiplying said fluorescence intensity by said weighting factor or said contribution fraction, said preliminary weighting factor being a function of said calculated fluorescence lifetime and two predetermined fluorescence lifetimes corresponding to expected lifetimes of said two or more fluorophore species.    
     
     
         10 . The method as claimed in any one of claim  1 - 9  wherein said fluorescence signal acquisition is selected from frequency domain and time domain modality.  
     
     
         11 . The method as claimed in any one of claim  1 - 9  wherein said fluorescence species comprises a fluorophore that is distributed between a free state and a bound state.  
     
     
         12 . The method as claimed in any one of claim  1 - 9  wherein said weighted intensity is further processed to yield concentration of at least one of said two or more fluorophore species.  
     
     
         13 . A method for optical imaging of an object containing two or more fluorophore species, said method comprising: 
 acquiring a fluorescence signal, said signal comprising fluorescence from said two or more fluorophore species and comprising lifetime information for one or more pixel of a region of interest (ROI) of said object;    calculating a fluorescence intensity and a fluorescence lifetime from said fluorescence signal for each of said one or more pixel;    deriving a contribution fraction for at least one of said fluorophore species;    multiplying said calculated fluorescence intensity by said contribution fraction to generate a species weighted fluorescence intensity;    multiplying said species weighted fluorescence intensity by a weighting factor, said weighting factor being a function of said calculated fluorescence lifetime and at least one predetermined fluorescence lifetime, to generate a weighted fluorescence intensity for said one or more pixel;    generating an image of said ROI based on said weighted fluorescence intensity of said one or more pixel.    
     
     
         14 . The method as claimed in  claim 13  wherein said contribution fraction is determined by modeling said fluorescence signal with a multi-exponential function.  
     
     
         15 . The method as claimed in  claim 14  wherein said modeling is applied to a temporal point spread function (TPSF) of said fluorescence signal.  
     
     
         16 . The method as claimed in  claim 13  wherein said weighting factor is determined by an Indicator function.  
     
     
         17 . The method as claimed in  claim 16  wherein said Indicator function is defined by boundaries which are function of said predetermined fluorescence lifetime.  
     
     
         18 . The method as claimed in  claim 17  wherein said boundaries are also function of an error associated with said measured lifetime.  
     
     
         19 . The method as claimed in  claim 18  wherein said weighting factor is 1 when said measured lifetime is within said boundaries and 0 otherwise.  
     
     
         20 . The method as claimed in  claim 13  further comprising a step of: 
 multiplying said fluorescence intensity by a preliminary weighting factor prior to said step of multiplying said fluorescence intensity by said contribution fraction, said preliminary weighting factor being a function of said calculated fluorescence lifetime and two predetermined fluorescence lifetimes corresponding to expected lifetimes of said two or more fluorophore species.    
     
     
         21 . The method as claimed in any one of claim  13 - 20  wherein said fluorescence signal acquisition is selected from frequency domain and time domain modality.  
     
     
         22 . The method as claimed in claim  13 - 21  wherein said fluorescence species comprises a fluorophore that is distributed between a free state and a bound state.  
     
     
         23 . The method as claimed in claim  13 - 21  wherein said weighted intensity is further processed to yield concentration of at least one of said two or more fluorophore species.