Pump probe cross correlation fluorescence frequency domain microscope and microscopy

• US 5,814,820 A
• Filed: 02/09/1996
• Issued: 09/29/1998
• Est. Priority Date: 02/09/1996
• Status: Expired due to Term

• Current Assignee: Board of Trustees of The University of Illinois
• Original Assignee: Board of Trustees of The University of Illinois
• Inventors: Dong, Chen-Yuan, So, Peter, Gratton, Enrico
• Primary Examiner(s): Hannaher, Constantine
• Application Number:
  US08/599,256
• Time in Patent Office: 963 Days
• Field of Search: 250/461.2, 250/461.1, 250/458.1
• US Class Current: 250/458.1
• CPC Class Codes: G01N 21/636 using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties; non-linear optics per se G02F1/35
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence

Abstract

A scanning fluorescence lifetime microscope measures modulation and phase in fluorescence emission stimulated by spatially overlapped pump and probe beams operating at different frequencies. A pump laser modulated at a first frequency is focused onto a diffraction limited spot to excite a fluorescent sample under study. Simultaneously, a probe laser modulated at a second frequency is focused onto the same spot to induce a stimulated fluorescence emission in response to the optically combined output of the pump and probe laser light. Fluorescence emitted from the sample produces a cross-correlation signal which is dependent upon the spatial overlapping of the pump and probe beams at the focal point thereby producing a beneficial axial sectioning effect. Choosing a small differency frequency between the modulation of the first and second laser sources produces a low frequency cross correlation signal even when the modulation frequencies of the pump and probe lasers are very high. A signal processor obtains modulation and phase information from the low frequency cross correlation fluorescence signal allowing monitoring of even ultrafast fluorescence phenomena induced by high frequency modulation of the pump and probe lasers.

Claims

1. A pump-probe fluorescence microscope comprising:

a first amplitude modulated laser modulating at a first amplitude modulation frequency;

a second amplitude modulated laser modulating at a second amplitude modulation frequency;

optical transmission means for combining light output from the first and second amplitude modulated lasers, and for focusing combined light output from the first and second lasers upon a common set of molecules in a sample to stimulate fluorescence emissions from said common set of molecules;

optical collection means for collecting a fluorescence signal emitted from the common set of molecules in response to the focusing of combined light output from the first and second amplitude modulated lasers;

a filter for isolating a cross-correlation signal within said fluorescence signal, said cross-correlation signal having an amplitude modulation frequency approximately equal to the difference between said first and second frequencies; and

signal processing means for obtaining an image based upon said cross-correlation signal.

2. The microscope according to claim 1, wherein said signal processing means further comprises sampling means for obtaining a predetermined number of sampling points per cross correlation signal waveform.

3. The microscope according to claim 2, wherein said predetermined number of sampling points is four.

4. The microscope according to claim 2, wherein said signal processing means further comprises fast Fourier transform means for conducting a fast Fourier transform on a signal output from said sampling means.

5. The microscope according to claim 4, wherein said signal processing means obtains said image from phase characteristics of a signal output from said fast Fourier transform means.

6. The microscope according to claim 4, wherein said signal processing means obtains said image from modulation characteristics of a signal output from said fast Fourier transform means.

7. The microscope according to claim 4, further comprising:

an optical scanner within said optical transmission means, for raster scanning the combined light output focused upon said sample.

8. The microscope according to claim 7, further comprising:

translation means for varying the distance between the sample and said optical transmission means so that depth of focus of the combined light output upon the sample is correspondingly varied.

9. The microscope according to claim 1, wherein said first and second amplitude modulated lasers are sinusoidally modulated at said respective first and second amplitude modulation frequencies.

10. The microscope according to claim 1, wherein the strength of the cross correlation signal is proportional to the spatial integral of the intensity product of pump and probe beams emitted respectively from the first and second amplitude modulated lasers, according to a relationship:

space="preserve" listing-type="equation">∫

I(u,v)I'"'"'(u'"'"',v'"'"')d.sup.3 t,

where pump and probe beam intensity distribution is represented by I(u, v) and I'"'"'(u'"'"',v'"'"'), respectively.

11. The microscope according to claim 1, wherein the wavelength of one of the pump and probe lasers is varied so that said image includes time-resolved spectral image data.

12. A pump-probe fluorescence microscope comprising:

a first amplitude modulated laser modulating at a first amplitude modulation frequency;

a second amplitude modulated laser modulating at a second amplitude modulation frequency;

optical transmission means for combining light output from the first and second amplitude modulated lasers, and for focusing combined light output from the first and second lasers upon a sample;

optical collection means for collecting a fluorescence signal emitted from the sample in response to the focusing of combined light output from the first and second lasers;

signal processing means for obtaining an image based upon said fluorescence signal; and

respective separate pump and probe laser polarizers for said first and second amplitude modulated lasers, wherein one of said polarizers varies the polarization of its respective laser so that said image includes time-resolved polarization image data.

13. A pump-probe fluorescence microscope comprising:

a first laser emitting amplitude modulated light at a first amplitude modulation frequency;

a second laser emitting amplitude modulated light at a second amplitude modulation frequency;

a combiner which receives and combines the light from the first and second lasers;

an X-Y scanner receiving light output from said combiner and raster scanning the light output;

an objective lens receiving scanning light from said X-Y scanner, said objective lens focusing said scanning light upon a common set of molecules in a sample to induce fluorescence emissions from said common set of molecules in the sample;

a collection lens disposed to collect said fluorescence emissions;

an optical sensor disposed to receive fluorescence emissions collected by said collection lens, said optical sensor producing a fluorescence signal in response to said fluorescence emission;

an isolation filter, said filter isolating a cross correlation signal between said first and second amplitude modulation frequencies emitted from said common set of molecules;

a digitizer which receives and digitizes the cross correlation signal; and

sampling means for obtaining a predetermined number of sampling points per cross correlation signal waveform.

14. The microscope according to claim 13, wherein image data is present in the sampled cross correlation signal and said predetermined number is small enough to permit frame acquisition rates of approximately 3 to 50 seconds for a 256×

256 pixel frame.

15. The microscope according to claim 14 further comprising:

fast Fourier transform means for conducting a fast Fourier transform on a signal output from said sampling means.

16. The microscope according to claim 15, further comprising:

a display for displaying said image data, said image data being obtained from a signal output from said fast Fourier transform means; and

storage means for storing said image data.

17. A method for microscopic imaging, comprising steps of:

subjecting a common set of molecules in a sample to combined scanned laser light including amplitude modulated pump laser light of a first amplitude modulation frequency, and amplitude modulated probe laser light of a second amplitude modulation frequency;

monitoring cross correlation fluorescence emissions induced in said sample from said common set of molecules in response to said combined laser light at a difference amplitude modulation frequency approximately equal to the difference between the first and second frequencies;

sampling said cross correlation fluorescence emissions to produce a sampled signal;

Fourier transforming the sampled signal; and

obtaining image data from the Fourier transformed sampled signal, based upon phase or modulation of the Fourier transformed sampled signal.

18. The method according to claim 17, wherein said difference frequency is smaller than either said first or second frequencies.