Devices, systems, and methods for fluorescence lifetime imaging microscopy

Abstract

The invention provides novel non-invasive in vitro methods for assessing the metabolic condition of oocytes and/or embryos with fluorescence lifetime imaging microscope, that can be used, for example, in assessment of oocytes and embryos in assisted reproductive technologies.

What is claimed herein is: 
     
         1 . A fluorescence lifetime imaging microscopy system comprising:
 a) an environmental chamber comprising a oocyte in a medium which does not metabolically perturb the oocyte;   b) a source of fluorescence excitation light which exposes the oocyte to a fluorescence excitation light;   c) a point detector which detects the auto-fluorescence emission of endogenous NADH or endogenous FAD of an oocyte exposed to the source of fluorescence excitation light;   d) a computer executable software on a non-human machine which:
 builds a fluorescence lifetime histogram of the NADH and/or FAD auto-fluorescence emission; 
 fits the fluorescence lifetime histogram to a sum of two exponentials to provide a function comprising the parameters:
 short lifetime; 
 long lifetime; and 
 relative fraction of short lifetime vs. long lifetime; and 
 
 determines whether the parameters obtained from the fluorescence lifetime histogram from the oocyte differ statistically from parameters obtained from an fluorescence lifetime histogram reference value from a normal healthy oocyte. 
   
     
     
         2 . The system of  claim 1 , wherein
 the fluorescence excitation light is of a wavelength of about 740 nm; and   the point detector comprises an emission bandpass filter centered around about 460 nm.   
     
     
         3 . The system of  claim 1 , wherein
 the fluorescence excitation light is of a wavelength of about 340 nm; and   the point detector comprises an emission bandpass filter centered around about 460 nm.   
     
     
         4 . The system of  claim 1 , wherein
 the fluorescence excitation light is of a wavelength of about 900 nm; and   the point detector comprises an emission bandpass filter centered around about 550 nm.   
     
     
         5 . The system of  claim 1 , wherein
 the fluorescence excitation light is of a wavelength of about 450 nm; and   the point detector comprises an emission bandpass filter centered around about 550 nm.   
     
     
         6 . A method of fluorescence lifetime imaging microscopy, the method comprising:
 (a) illuminating an unperturbed oocyte with excitation light;   (b) detecting the auto-fluorescence emission of endogenous NADH or endogenous FAD of the unperturbed oocyte using a fluorescence lifetime imaging microscope (FLIM) with an emission bandpass filter set for NADH or FAD auto-fluorescence emission;   (c) building a fluorescence lifetime histogram of the NADH and/or FAD auto-fluorescence emission;   (d) fitting the fluorescence lifetime histogram of step (c) to a sum of two exponentials to provide a function comprising the parameters:
 short lifetime; 
 long lifetime; and 
 relative fraction of short lifetime vs. long lifetime; and 
 calculating whether the parameters obtained from the fluorescence lifetime histogram from the unperturbed oocyte differ statistically from parameters obtained from an fluorescence lifetime histogram reference value from a normal healthy oocyte; 
   wherein
 (i) parameters obtained from the unperturbed oocyte which do not differ statistically from the reference values indicates that the oocyte is suitable for in vitro fertilization; and 
 (ii) parameters obtained from the unperturbed oocyte which differ statistically from the reference value indicates that the oocyte is not suitable for in vitro fertilization. 
   
     
     
         7 . A method of fluorescence lifetime imaging microscopy, the method comprising:
 (a) illuminating an unperturbed oocyte with excitation light;   (b) detecting the auto-fluorescence emission of endogenous NADH or endogenous FAD of the unperturbed oocyte using a fluorescence lifetime imaging microscope (FLIM) with an emission bandpass filter set for NADH or FAD auto-fluorescence emission;   (c) building a fluorescence lifetime histogram of the NADH and/or FAD auto-fluorescence emission;   (d) fitting the fluorescence lifetime histogram of step (c) to a sum of two exponentials to provide a function comprising the parameters:
 short lifetime; 
 long lifetime; and 
 relative fraction of short lifetime vs. long lifetime; and 
 calculating whether the parameters obtained from the fluorescence lifetime histogram from the unperturbed oocyte differ statistically from parameters obtained from an fluorescence lifetime histogram reference value from a normal healthy oocyte. 
   
     
     
         8 . A method of fluorescence lifetime imaging microscopy, the method comprising:
 (a) illuminating an unperturbed oocyte with excitation light;   (b) detecting the auto-fluorescence emission of endogenous NADH or endogenous FAD of the unperturbed oocyte using a fluorescence lifetime imaging microscope (FLIM) with an emission bandpass filter set for NADH or FAD auto-fluorescence emission;   (c) building a fluorescence lifetime histogram of the NADH and/or FAD auto-fluorescence emission;   (d) calculating whether the fluorescence lifetime histogram of NADH and/or FAD from the unperturbed oocyte differs statistically from a fluorescence lifetime histogram reference value for NADH and/or FAD from a normal healthy oocyte;
 wherein
 (i) a fluorescence lifetime histogram of NADH and/or FAD of the unperturbed oocyte which does not differ statistically from the reference value indicates that the oocyte is suitable for in vitro fertilization; and 
 (ii) a fluorescence lifetime histogram of NADH and/or FAD of the unperturbed oocyte which differs statistically from the reference value indicates that the oocyte is not suitable for in vitro fertilization. 
 
   
     
     
         9 . The method of  claim 6 , comprising sequentially detecting the auto-fluorescence emission of both endogenous NADH and endogenous FAD. 
     
     
         10 . The method of  claim 6 , wherein
 the illumination step comprises two-photon fluorescence excitation using an excitation light of a wavelength of about 740 nm;   and the auto-fluorescence of endogenous NADH is detected using an emission bandpass filter centered around about 460 nm.   
     
     
         11 . The method of  claim 6 , wherein
 the illumination step comprises one-photon fluorescence excitation using an excitation light of a wavelength of about 340 nm;   and the auto-fluorescence of endogenous NADH is detected using an emission bandpass filter centered around about 460 nm.   
     
     
         12 . The method of  claim 6 , wherein
 the illumination step comprises two-photon fluorescence excitation using an excitation light of a wavelength of about 900 nm;   and the auto-fluorescence of endogenous FAD is detected using an emission bandpass filter centered around about 550 nm.   
     
     
         13 . The method of  claim 6 , wherein
 the illumination step comprises one-photon fluorescence excitation using an excitation light of a wavelength of about 450 nm;   and the auto-fluorescence of endogenous NADH is detected using an emission bandpass filter centered around about 550 nm.   
     
     
         14 . The method of  claim 6 , wherein detecting the auto-fluorescence emission of endogenous NADH or FAD comprises performing the detection in the time domain. 
     
     
         15 . The method of  claim 6 , wherein detecting the auto-fluorescence emission of endogenous NADH or FAD comprises performing the detection in the frequency domain.