Method for detecting fluorescence phenomena in microscope

Details Method for detecting fluorescence phenomena in microscope Method for detecting fluorescence phenomena in microscope

2001-12-21

2004-05-25

Evans, F. L. (Department: 2877)

Optics: measuring and testing

By dispersed light spectroscopy

With sample excitation

C250S459100

Reexamination Certificate

active

06741346

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The process that is presented relates to confocal and 2-photon fluorescence microscopy as described by M. Göppert-Mayer, Ann. Physik 9, 273 (1931) and T. Wilson,
Theory and Practice of Scanning Optical Microscopy
(Academic Press, 1984). Both methods are assumed to be known.
Both confocal fluorescence microscopy and 2-photon microscopy are modified by the method described below to the extent that an additional contrast parameter is possible: the fluorescence lifetime.
2. Related Art
Temporal resolved fluorescence and/or the use of the lifetime as a contrast parameter in confocal/2-photon microscopy can be carried out by two different methods—by time domain detection and frequency domain detection.
In the case of time domain detection (described in Published patent application DE 41 04 014 of Wabnitz, entitled “Method for Determining the Calcium Concentration in Cells”; and in Patent DE 36 14 359 C2 of Gröbler, entitled “Device for the Analysis and Imaging of Real Time Intensity of Fluorescence Radiation resulting from Point-by-Point Excitation of a Preparation by means of Laser Light”) a fluorescent sample is excited so as to produce fluorescence by means of a pulsed light source, and the fluorescence emission is detected with time resolution either by means of time correlated single photon counting (TCSPC)(described in Patent WO 98/09154 to Müller et al., entitled “System for Differentiating Fluorescing Molecular Groups by means of Time-Resolved Fluorescence Measurement”; and in Published patent application DE 42 31 477 A1 of Han, entitled “Method for Optical Sorting of Plastics by means of Time-Resolved Laser Spectroscopy”) or by means of time gated detection (described by H. Schneckenburger et al., “Time-Gated H. Microscopic Imaging and Spectroscopy in Medical Diagnosis and Photobiology,”
Optical Engineering
33 (8) 2600 (1994); R. Cubeddu et al., “A Real Time System for Fluorescence Lifetime Imaging,”
SPIE
2976 (1997) 98; and K. Dowling et al., “Two Dimensional Fluorescence Lifetime Imaging using a 5 kHz/110 ps Gated Image Intensifier” (www.kentech.co.uk/pdf_files/K_Dowling_et_al.pdf)).
In the case of frequency domain detection, a fluorescent sample or preparation is excited with a light source that is either actively modulated or pulsed (for example, by means of passive mode coupling). Since any arbitrary modulation of the excitation by means of a Fourier analysis breaks down into sinusoidal components, the observation of a sinusoidal excitation is adequate. The frequency domain detection technique is based on the delay of the fluorescence emission by a phase f and a change in the modulation depth M compared with the excitation light as a function of the modulation frequency &ohgr;(=2 &pgr;f
mod
) and the lifetime &tgr;.
&phgr;=
a
tan(&ohgr;&tgr;)  (1)
M
=
1
1
+
ω
2
⁢
τ
2
(
2
)
However, the resulting fluorescence signal, oscillated with the modulation frequency, is out of phase and demodulated. For typical fluorescence lifetimes ranging from &tgr;=1 . . . 10 ns, modulation frequencies ranging from f
mod
=10 . . . 100 MHZ are adequate.
Since it generally does not make any sense to scan the fluorescence signal at such high modulation frequencies, a frequency mixing process is used to detect the signal. For mixing, any detection element with a modulatable amplification is suitable.
In essence two methods are distinguished—the homodyne and the heterodyne detection techniques.
To understand the principle, one observes generally two “signals” S
1
, S
2
, (where, for example S
1
is modulated excitation, and S
2
is modulated amplification).
S
1
=A
0
+A
1
cos(&ohgr;
a
t
+&agr;)
S
2
=B
0
+B
1
cos(&ohgr;
b
t
+&bgr;)  (3)
Multiplication results in:
S
1
S
2
=A
0
B
0
+A
0
B
1
cos(w
b
t+b
)+
B
0
A
1
cos(w
a
t+a
)+
A
1
B
1
{cos((
w
a
w
b
)
t
+(
a+b
))+cos((
w
a
w
b
)
t
+(
a−b
))}  (4)
Homodyne Detection
If &ohgr;
a
=&ohgr;
b
(homodyne), the second harmonic and a frequency independent component are generated by the mixing process of the “signals” S
1
and S
2
. A low pass filter results in a suppression of the components at &ohgr;
a
and 2&ohgr;
a
. Only the DC background and the phase dependent DC component are detected. The signal, filtered by means of a low pass (LP) filter, can be written as:
 
LP
(
S
1
S
2
)=
A
0
B
0
+A
1
B
1
cos(&agr;−&bgr;)  (5)
In the case of homodyne detection this frequency independent (DC) signal can be detected in multiple relative phases. To measure the fluorescence lifetime, at least 3 three different phase positions are necessary. At just two relative phase positions, the phase shift or the demodulation, induced by the fluorescence lifetime, can be used as the contrast parameter (as described by P. C. Schneider et al., “Rapid Acquisition, Analysis and Display of Fluorescence Lifetime Resolved Images for Real time Applications, ”
Rev. Sci. Instrum
. 68 (11) 4107 (1997) (hereafter, “Schneider et al.”)).
Heterodyne Detection—Cross Correlation
If &ohgr;
b
=&ohgr;
a
+Dw (heterodyne), the mixing process generates a high frequency signal at the total frequency and a signal at the cross correlation frequency &Dgr;&ohgr;. Again the high frequency components are suppressed by a low pass filter.
LP
(
S
1
S
2
)=
A
0
B
0
+A
1
B
1
cos(&Dgr;&ohgr;
t
+&agr;−&bgr;)  (6)
In the case of heterodyne detection, the differential frequency &Dgr;&ohgr; is detected. Phase position and modulation depth of the signal at the differential frequency make it possible to determine the lifetime. Typical cross correlation frequencies range a few Hz up to about 100 kHz.
A more comprehensive presentation of the heterodyne method can be found in the publication by E. Gratton et al., “A Continuously Variable Frequency Cross Correlation Phase Fluorometer with Picosecond Resolution,”
Biophys. J
. 44 (1983) 315 (hereinafter, “Gratton”), and “Multifrequency Phase and Modulation Fluorometer, ”
Ann. Rev. Biophys. Bioeng
. 13 (1984) 105 (hereinafter, “Gratton 2”).
In both the homodyne and heterodyne detection technique the high frequency change is reflected, so to speak, on the low frequency range.
Owing to the multi-exponential decay behavior of the fluorescence emission, the lifetimes, determined from the modulation depth and the phase shift, vary. Therefore, for precise measurement of the lifetime, the excitation frequency has to be varied as described by Gratton 2.
If, in contrast, the lifetime is supposed to be used, for example, as a contrast parameter in an imaging process, this is generally not necessary. Frequently it suffices, for example, to show the phase shift or the demodulation by means of the lifetime or by means of the lifetime, calculated from the phase shift and demodulation data, at a fixed modulation frequency.


REFERENCES:
patent: 4786170 (1988-11-01), Groebler
patent: 5034613 (1991-07-01), Denk et al.
patent: 5459323 (1995-10-01), Morgan
patent: 5485530 (1996-01-01), Lakowicz et al.
patent: 5504337 (1996-04-01), Lakowicz et al.
patent: 3 614 359 (1987-02-01), None
patent: 41 04 014 (1991-08-01), None
patent: 42 31 477 (1994-03-01), None
patent: WO 98/09154 (2000-10-01), None
J. Pawley (Ed.),Handbook of Biological Confocal Microscopy, Chapters 28 and 31 (New York: Plenum Press, 1995).
X. Wang and B. Herman (Eds.),Fluorescence Imagin Spectroscopy and Microscopy, vol. 137, Chapters 8-11 (New York: John Wiley & Sons, Inc., 1996).
K. Dowling et al., “Two Dimensional Flourescence Lifetime Imaging using a 5 kHz/110 ps Gated Image Intensifier,” Kentech Instruments Ltd. (www.kentech.co.uk/pdf_files/K_Dowling_et_al.pdf) (Oct. 24, 199).
P.T.C. So,Timer-resolved fluorescence microscopy using two-photon excitation, Bioimaging, 3 (1995) 49.
H. Schneckenburger, et al.,Time-gated microscopic imaging and specrtoscopy in medical diagnosis and photobiology, Optical Engineeri

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