Method of fluorescent measurement of samples, and devices therefrom
First Claim
1. A method for measuring fluorescence comprising:
- providing a liquid sample having a volume contained in a sample carrier, wherein the sample volume contains at least one fluorophore amidst bulk fluorescence of the sample;
providing a laser light source to generate a scanning spot of a single wavelength having an excitation wavelength and a focus diameter to impinge on the sample volume for measuring fluorescence; and
displacing the laser spot of said single wavelength, relative to the sample volume, with a two dimensional preview scan, by moving the sample carrier and the incident beam relative to each other in an R-theta scan at a first resolution to detect in a three-dimensional space, defined by the sample volume, at least one individual volume of interest where fluorescence exceeds bulk fluorescence in other regions of the sample carrier wherein the at least one volume of interest and the laser spot have a defined relationship, thenmeasuring a depth profile of fluorescence in the at least one individual volume of interest at a second resolution higher than the first resolution as a profile of the at least one fluorophore therein amidst the bulk fluorescence,wherein a two dimensional R-theta scan of the sample at a single wavelength establishs three dimensional fluorescence profiles of the at least one fluorophore in the sample amidst the bulk fluorescence.
1 Assignment
0 Petitions
Accused Products
Abstract
The invention provides a method and device for fluorescent measurement of a volume of liquid sample in a sample carrier. The method includes impinging an incident beam having an excitation wavelength and a focus diameter on the sample in a two dimensional scan, such as an R-theta scan or a spiral scan, with a laser spot that identifies a volume of interest where higher levels of fluorescence exist. The laser spot is displaced relative to the sample volume in three dimensional space. The laser spot is translated in the depth direction of the volume of interest to detect one or more emitted fluorescence signals. A depth profile and a thickness of the sample carrier obtained from the one or more emitted fluorescence signals, are used for measuring normalized bulk fluorescence. Further, at least one microvolume of interest is obtained from the depth profile, and the incident beam is focused on the microvolume of interest to obtain at least one concentrated emitted fluorescence signal that is representative of the bulk concentration of fluorescence of a fluorphore.
53 Citations
37 Claims
-
1. A method for measuring fluorescence comprising:
-
providing a liquid sample having a volume contained in a sample carrier, wherein the sample volume contains at least one fluorophore amidst bulk fluorescence of the sample; providing a laser light source to generate a scanning spot of a single wavelength having an excitation wavelength and a focus diameter to impinge on the sample volume for measuring fluorescence; and displacing the laser spot of said single wavelength, relative to the sample volume, with a two dimensional preview scan, by moving the sample carrier and the incident beam relative to each other in an R-theta scan at a first resolution to detect in a three-dimensional space, defined by the sample volume, at least one individual volume of interest where fluorescence exceeds bulk fluorescence in other regions of the sample carrier wherein the at least one volume of interest and the laser spot have a defined relationship, then measuring a depth profile of fluorescence in the at least one individual volume of interest at a second resolution higher than the first resolution as a profile of the at least one fluorophore therein amidst the bulk fluorescence, wherein a two dimensional R-theta scan of the sample at a single wavelength establishs three dimensional fluorescence profiles of the at least one fluorophore in the sample amidst the bulk fluorescence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
-
-
17. A fluorescence measuring device comprising:
-
a sample assembly for receiving a sample carrier that comprises a sample, wherein the sample comprises at least one fluorophore amidst background bulk fluorescence; a laser light source for generating an incident beam having a single excitation wavelength and a focus diameter to impinge on the sample to yield a spot for measuring fluorescence; a means for displacing the spot in a two dimensional low resolution scan relative to the sample volume in a three dimensional space by moving the sample carrier and the incident beam relative to each other in an R-theta scan, the three dimensional space including mostly bulk fluorescence; and a means for detecting at least one individual volume of interest in the two-dimensional low resolution scan that has the at least one fluorophore amidst background bulk fluorescence therin, wherein the at least one individual volume of interest and the laser spot have a defined relationship, then displacing the spot in a high resolution scan to measure the depth profile of fluorescence in the volume of interest as a profile of the least one fluorophore in background bulk fluorescence, wherein a two-dimensional scan of the sample establishes three dimensional fluorescence profiles of the at least one fluorophore amidst the background bulk fluorescence. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
-
-
34. A method for analyzing a sample comprising:
-
scanning a liquid sample volume with a beam spot of a single wavelength over an area of the sample volume for analysis by moving a sample carrier and the beam spot relative to each other in an R-theta scan, wherein the sample volume contains at least one fluorophore amidst bulk fluorescence of the sample defining a volume of interest, wherein the volume of interest and the beam spot have a defined relationship; acquiring simultaneous fluorescent signal from fluorophores in the liquid sample volume, representing of one or more fluorescence events for the volume of interest where a depthwise fluorescent profile is measured using the spot and signal representing a normalized bulk fluorescence for the sample volume; and using the simultaneous signals to distinguish fluorescent signals from fluorophores from bulk fluorescence signals in the liquid volume. - View Dependent Claims (35, 36, 37)
-
Specification