Method and apparatus for multi-dimensional phase fluorescence lifetime imaging

• US 5,485,530 A
• Filed: 07/23/1993
• Issued: 01/16/1996
• Est. Priority Date: 01/24/1991
• Status: Expired due to Term

• Current Assignee: Joseph R. Lakowicz
• Original Assignee: Joseph R. Lakowicz
• Inventors: Johnson, Michael L., Berndt, Klaus W., Nowaczyk, Kazimierz, Lakowicz, Joseph R., Szmacinski, Henryk
• Primary Examiner(s): COUSO, YON JUNG
• Application Number:
  US08/094,016
• Time in Patent Office: 907 Days
• Field of Search: 382/6, 382/58, 382/191, 382/128, 382/133, 382/312, 382/129, 250/461.2, 250/458.1, 250/459.1, 356/73, 356/317, 356/318, 356/417, 356/346
• US Class Current: 382/191
• CPC Class Codes: G01K 11/20 using thermoluminescent materials G01K11/32 takes precedence
• CPC Class Codes: G01N 15/1427 with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences
• CPC Class Codes: G01N 2015/144 Imaging characterised by its optical setup
• CPC Class Codes: G01N 2015/1454 using phase shift or interference, e.g. for improving contrast
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging

Abstract

A method and apparatus for detection and/or measurement of physical characteristics of a sample based on multi-dimensional phase-modulation fluorescence lifetime imaging using at least one fluorescent probe having known and/or variable fluorescent lifetimes.

Claims

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.

2. The method of claim 1 further comprising using said ratio to discriminate said first physical property from said second physical property.