Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2000-11-03
2003-10-28
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C200S458000, C200S459000, C200S461000, C200S461000
Reexamination Certificate
active
06639668
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to devices useful for the acquisition of fluorescence data from a sample, such as biological tissue, in order to analyze the status or composition of the sample, and, in particular, to devices and methods that utilize asynchronous fluorescence scanning to assess the presence or absence of pathology in skin or other tissue.
2. Description of Background
Fluorescence, by definition, involves the excitation of a fluorophore by light of a given wavelength, &lgr;
ex
. The fluorophore then re-emits light at a lower energy, and hence a longer wavelength, &lgr;
em
. Thus, &lgr;
em
is always greater than &lgr;
ex
for fluorescence. In addition, the amount of emitted fluorescent light is typically much less than the excitation light, and therefore instrumental limitations (e.g., band pass width, straylight rejection) force a minimum separation &Dgr;&lgr;
min
between the more intense excitation wavelength &lgr;
ex
and the less intense emission wavelength &lgr;
em
, so that the less intense signal does not get swamped. &Dgr;&lgr;
min
may be on the order of 15-30 nm in realistic instruments.
Traditionally, fluorescence spectra have been taken by either (a) fixing the excitation wavelength and varying the emission wavelength (an “emission scan”), or (b) fixing the emission wavelength and varying the excitation wavelength (an “excitation scan”). By taking a series of either excitation or emission scans and piecing them together, a complete excitation/emission fluorescence map of a sample may be constructed.
In addition to the emission and excitation scans, a “synchronous scan” has also been described. In a synchronous scan, both excitation and emission wavelengths are incremented in synchrony by the same amount, so that the difference between them remains constant. That is, &lgr;
em
=&lgr;
ex
+&Dgr;&lgr;, where &Dgr;&lgr; is a fixed value. For example, a “50 nm synchronous scan” might increment the excitation wavelength from 250 to 350 nm in 2 nm increments, while simultaneously incrementing the emission wavelength from 300 to 400 mn in 2 nm steps. Synchronous scans using a fixed value for &Dgr;&lgr;
min
are discussed by Tuan Vo-Dinh in Chapter 5 of Modern Fluorescence Spectroscopy, edited by E. L. Wehry (Plenum, 1981), and are further described in PCT publication WO 96/07889, incorporated herein by reference. Commercial spectrofluorometer products such as those made by Instruments S. A. of Edison, N.J. are programmed to make synchronous scans. However, fluorescent data which can be collected from such instruments are limited to spectra whose optimal collection wavelengths vary by the set constant with respect to the wavelength of the excitation radiation.
SUMMARY OF THE INVENTION
The invention overcomes the problems and disadvantages associated with current strategies and designs and provides new instruments and methods for acquiring fluorescence data from skin and other tissue, to facilitate detection of the presence or absence of disease or other abnormality in a sample. The present invention is useful for biomedical diagnostics, chemical analysis or other evaluation of a target sample.
The present invention differs from the conventional synchronous scan in that it allows &Dgr;&lgr; to vary during the scan according to the equation &lgr;
em
=&lgr;
ex
+&Dgr;&lgr;, subject to the minimum separation condition that &Dgr;&lgr;>&Dgr;&lgr;
min
.
Accordingly, one embodiment of the invention is directed to a method of analyzing a sample comprising the steps of exposing the sample to an excitation radiation having a wavelength, &lgr;
ex
, thereby generating an emission radiation having a wavelength, &lgr;
em
, scanning the wavelength of the excitation radiation and the wavelength of the emission radiation to collect a spectrum, and correlating the spectrum to a condition of the sample. The excitation and emission wavelengths are varied according to a formula selected from the group consisting of:
&lgr;
em
&lgr;
ex
+&Dgr;&lgr;, wherein &Dgr;&lgr; varies during scanning and &Dgr;&lgr;>&Dgr;&lgr;
min
;
More specifically, emission may depend on excitation according to either:
(a) &lgr;
em
=m&lgr;
ex
+b, where m≠1, and &Dgr;&lgr;=&lgr;
em
−&lgr;
ex
>&Dgr;&lgr;
min
; or
(b) &lgr;
em
=f(&lgr;
ex
), where f(&lgr;
ex
) represents any simple curved arc, and &Dgr;&lgr;=&lgr;
em
−&lgr;
ex
>&Dgr;&lgr;
min
.
Another embodiment is directed to an apparatus for testing a sample comprising means for exposing the sample to an excitation radiation, &lgr;
ex
, thereby generating an emission radiation, &lgr;
em
; means for scanning the wavelength of the excitation radiation and the wavelength of the emission radiation to produce a spectrum; and means for correlating the spectrum to a condition of the sample. The excitation and emission wavelengths are varied according to a formula:
&lgr;
em
=&lgr;
ex
+&Dgr;&lgr;, and &Dgr;&lgr; varies during scanning and &Dgr;&lgr;>&Dgr;&lgr;
min
.
More specifically, emission may depend on excitation according to either:
(a) &lgr;
em
=m&lgr;
ex
+b, where m≠1, and &Dgr;&lgr;
min
=&lgr;
em
−&lgr;
ex
>&Dgr;&lgr;
min
; or
(b) &lgr;
em
=f(&lgr;
ex
), where f(&lgr;
ex
) represents any simple curved arc, and &Dgr;&lgr;=&lgr;
em
−&lgr;
ex
>&Dgr;&lgr;
min
.
Another embodiment is directed to a fluorescence spectral acquisition system comprising means for exposing a sample to an excitation radiation having an excitation wavelength, &lgr;
ex
, and means for scanning the excitation wavelength and radiation re-emitted by the sample, the radiation having an emission wavelength, &lgr;
em
. Both the excitation wavelength, &lgr;
ex
, and emission wavelength, &lgr;
em
, are varied, the variation having the mathematical formula &lgr;
em
=m&lgr;
ex
+b, wherein m≠1 and the variation is subject to the constraint that &Dgr;&lgr;=&lgr;
em
−&lgr;
ex
>&Dgr;&lgr;
min
.
Another embodiment is directed to a fluorescence spectral acquisition system comprising means for exposing a sample to an excitation radiation having an excitation wavelength, &lgr;
ex
, and means for scanning the excitation wavelength and radiation re-emitted by the sample, the radiation having an emission wavelength, &lgr;
em
. Both the excitation wavelength, &lgr;
ex
, and emission wavelength, &lgr;
em
, are varied, the variation having the mathematical formula &lgr;
em=f(&lgr;
ex
), where f(&lgr;
ex
) represents any simple curved arc and the variation is subject to the constraint that &Dgr;&lgr;=&lgr;
em
−&lgr;
ex
>&Dgr;&lgr;
min
.
Other embodiments and advantages of the invention are set forth in part in the description which follows, and in part will be obvious from this description, or may be learned from the practice of the invention.
REFERENCES:
patent: 4037960 (1977-07-01), Macemon
patent: 5599717 (1997-02-01), Vo-Dinh
patent: 5612540 (1997-03-01), Richards-Kortum et al.
patent: WO9951142 (1999-10-01), None
Hueber, Dennis M., et al., “Fast Scanning Synchronous Luminescence Spectrometer Based on Acousto-Optic Tunable Filters,”Applied Spectroscopy, 49:1624-1631 (1995).
Alarie, Jean Pierre, et al., “Development of a Battery-Operated Portable Synchronous Luminescnece Spectrofluorometer,”Review of Scientific Instruments, 64:2541-2546 (1993).
Vo-Dinh, T., “Synchronous Excitation Spectroscopy,”Modern Fluorescence Spectroscopy 4, pp. 167-192 (1981).
Argose, Inc.
Elrifi, Esq. Ivor R.
Evans F. L.
Geisel Kara
Karnakis, Esq. Christina V.
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