Method and apparatus for multi-dimensional phase fluorescence lifetime imaging

Claim 1

A method for performing multi-dimensional optical measurements on a subject in a sample containing at least one fluorescent probe, comprising the following steps: (a) illuminating said fluorescent probe with intensity modulated light at a first wavelength of said probe, said light being intensity modulated at a first frequency and a first phase, to produce secondary light as an excitation emission from said probe, said secondary light being intensity modulated at the first frequency and having a second phase; (b) generating a gating signal modulated at said first frequency and having a third phase, differing by a first detector phase angle from said first phase; (c) receiving said secondary light and intensifying said secondary light in response to said gating signal; (d) generating at least a two dimensional image in response to said intensified secondary light; and (e) detecting said at least two dimensional image; (f) repeating, at least once, steps (b), (c), (d) and (e) with a second detector phase angle to produce at least two detected images each of which corresponds to a change between the phase and/or modulation of said secondary light and said intensity modulated light; and (g) arithmetically processing the detected images to produce a final lifetime image, wherein said final lifetime image is (a) indicative of a value of the fluorescence lifetime of said at least one fluorescent probe and (b) is independent of the amplitude of the emission signal; wherein said generating step (d) comprises: (a) generating a first stored picture by setting said detector phase angle to π/2 relative to said first phase, detecting said resultant image and storing said detected image as a first picture; (b) generating a second stored picture without modulation on said grating signal, detecting said resultant image and storing said detected image as a second picture; (c) generating a third stored picture by setting said detector phase angle to 0° relative to said first phase, detecting said resultant image and storing said detected image as a third picture; (d) subtracting said second stored picture from said first stored picture to generate a first intermediate image; (e) subtracting said second stored picture from said third stored picture to generate a second intermediate image; and (f) generating the ratio of said first intermediate image and said second intermediate image, said ratio identifying a pixel intensity that is proportional to said first physical property with respect to said second physical property.

Claim 2

A method for performing multi-dimensional optical measurements on a subject in a sample containing at least one fluorescent probe, comprising the following steps: (a) illuminating said fluorescent probe with intensity modulated light at a first wavelength of said probe, said light being intensity modulated at a first frequency and a first phase, to produce secondary light as an excitation emission from said probe, said secondary light being intensity modulated at the first frequency and having a second phase; (b) generating a gating signal modulated at said first frequency and having a third phase, differing by a first detector phase angle from said first phase; (c) receiving said secondary light and intensifying said secondary light in response to said gating signal; (d) generating at least a two dimensional image in response to said intensified secondary light; and (e) detecting said at least two dimensional image; (f) repeating, at least once, steps (b), (c), (d) and (e) with a second detector phase angle to produce at least two detected images each of which corresponds to a change between the phase and/or modulation of said secondary light and said intensity modulated light; and (g) arithmetically processing the detected images to produce a final lifetime image, wherein said final lifetime image is (a) indicative of a value of the fluorescence lifetime of said at least one fluorescent probe and (b) is independent of the amplitude of the emission signal; wherein said generating step (d) comprises: (a) generating a first stored picture by setting said detector phase angle to π/2 relative to said first phase, detecting said resultant image and storing said detected image as a first picture; (b) generating a second stored picture without modulation on said grating signal, detecting said resultant image and storing said detected image as a second picture; (c) generating a third stored picture by setting said detector phase angle to 0° relative to said first phase, detecting said resultant image and storing said detected image as a third picture; (d) subtracting said second stored picture from said first stored picture to generate a first intermediate image; (e) subtracting said second stored picture from said third stored picture to generate a second intermediate image; and (f) generating the ratio of said first intermediate image and said second intermediate image, said ratio identifying a pixel intensity that is proportional to said first physical property with respect to said second physical property.