Method and apparatus for background correction in photoluminescent analysis

• US 4,150,295 A
• Filed: 01/05/1978
• Issued: 04/17/1979
• Est. Priority Date: 01/05/1978
• Status: Expired due to Term

• Current Assignee: Irwin Wieder
• Original Assignee: Analytical Radiation Corporation
• Inventors: Wieder, Irwin
• Primary Examiner(s): Willis, Davis L.
• Application Number:
  US05/867,004
• Time in Patent Office: 467 Days
• Field of Search: 250/458, 250/459, 250/461 R, 250/461 B, 250/214 C, 250/354
• US Class Current: 250/458.1
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters

Abstract

A method and apparatus for background correction in photoluminescent analysis of a selected photoluminescent target substance with a relatively narrow emission spectrum in the presence of ambient substances with a combined photoluminescent spectrum which is relatively broad. The photoluminescence of the target substance is sampled and measured in a narrow wavelength band corresponding to the principal emission spectrum of the target. A second sample of photoluminescence is measured in one or more wavelength bands adjacent to the first wavelength band, but excluding substantially all of the photoluminescence of the target substance, and transmitting only ambient photoluminescence. The second measurement is converted into an amount which would occur if ambient photoluminescence had been sampled in the first wavelength band and then the converted amount is subtracted from the first measured amount, thereby correcting the first measured amount for photoluminescence derived from ambient substances.

Claims

1. A method of photoluminescent background correction in the detection of a photoluminescent target substance admidst photoluminescent ambient substances comprising,

selecting a photoluminescent target substance from the group consisting of fluorophores and substances labelled with fluorophores, said fluorophores having a sharp photoluminescent emission spectrum in comparison with the combined broad emission spectrum of ambient substances,

sampling and measuring an amount of photoluminescence in a first selected narrow wavelength band centered at the peak of the principal emission wavelength of the selected target substance, said amount of photoluminescence composed of both target and ambient photoluminescence, said photoluminescence derived from an amount of excitation energy,

sampling and measuring an amount of ambient photoluminescence in at least one other narrow wavelength band proximate to but substantially outside of said first selected band, said photoluminescence derived from said amount of excitation energy,

converting said measured amount of ambient photoluminescence in the other selected wavelength bands into an amount which would occur from ambient photoluminescence measured in said first selected wavelength band, and

subtracting said converted amount from the measured amount due to both target and ambient substances.

2. The method of claim 1 wherein ambient photoluminescence is sampled and measured in two selected wavelength bands, one on each side of the principal target emission band, but substantially outside of said band.

3. The method of claim 1 further defined by selecting said photoluminescent target to have a photoluminescent decay lifetime long compared with the longest of the fluorescent decay lifetimes of ambient surfaces for time domain discrimination against ambient fluorescence.

4. The method of claim 1 wherein said fluorophore is a fluorescent rate earth chelate.

5. The method of claim 1 further defined by stabilizing the amount of excitation energy used in each measuring process.

6. The method of claim 5 further defined by measuring the photoluminescence of an internal standard excited with a fixed amount of excitation energy at defined time intervals and compensating measured amounts of target and ambient photoluminescence for deviations due to drift.

7. A spectrofluorometer for detecting targets having a sharp photoluminescent emission spectrum in comparison with the combined broad emission spectrum of ambient substances comprising,

an excitation beam source,

means for supporting a photoluminescent target in said excitation beam,

means for sampling and measuring an amount of photoluminescence from said target and ambient substances present in the target area in preselected bandwidths, said sampling and measuring means having a photodetector and a wavelength band selector capable of selecting a narrow first wavelength band at the peak of the principal emission wavelength of the target substance and in at least one other narrow wavelength band proximate to, but substantially outside of said first wavelength band, and

signal processing means for converting said measured amount of ambient photoluminescence in the proximate selected wavelength bands into an amount which would occur from the ambient photoluminescence measured in said first selected wavelength band and subtracting said converted amount from the measured amount due to both target and ambient substances.