Luminescence system and method for determining the nature of substances by measuring fluorescence and phosphorescence properties

• US 5,039,219 A
• Filed: 05/26/1989
• Issued: 08/13/1991
• Est. Priority Date: 05/26/1989
• Status: Expired due to Term

• Current Assignee: Photop Technologies Incorporated (Coherent Corp.)
• Original Assignee: Photop Technologies Incorporated (Coherent Corp.)
• Inventors: James, Douglas R., Ware, William R.
• Primary Examiner(s): McGraw, Vincent P.
• Application Number:
  US07/357,118
• Time in Patent Office: 809 Days
• Field of Search: 356/317, 356/318, 250/458.1, 250/459.1, 250/461.1
• US Class Current: 356/318
• CPC Class Codes: G01J 3/4406 Fluorescence spectrometry
• 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
• CPC Class Codes: G01N 2201/024 Modular construction

Abstract

Disclosed is an integrated, multi-module luminescence measuring system. The system can selectively conduct lifetime decay or steady-state measurements of light emitted by a sample which receives exitation light. The system includes: a sample chamber which directs monochromatized, focused excitation light from a selected source at a selected sample and directs any resultant emitted light through a monochromator to either a first gated photodetector or a second regular photodetector. Also present is a reference module with a regular photodetector which may selectively receive a portion of the excitation light. A lifetime decay detector module pulses a selected light source and the gated photodetector so that a converter produces digital signals related to output signals from the photodetector, the digital signals representing the integral of the intensity of the emitted light over a selected time. A steady-state detector module pulses a selected light source and includes a resettable integrator-A/D converter combination which receives the output from the regular photodetectors and produces digital signals representing the integrals over varying time intervals of such outputs. The integrator is reset and the A/D convertor is turned on whenever the light source is pulsed. A data processing module controls sample selection, the wavelengths of the excitation and emitted lights, light source selection, the number and time-width of the various integrations, and the storage and processing of the resulting digital signals.

Claims

1. An integrated luminescence-measuring system for selectively conducting lifetime decay or steady-state measurements of fluorescent or phosphorescent light emitted by a sample when the sample receives excitation light, wherein the system comprises:

(A) chamber means containing a plurality of samples for directing excitation light received therein at a selected sample, and for directing fluorescent or phosphorescent light emitted by the selected sample to the exterior thereof;

(B) first monochromator means for for transmitting excitation light which comprises selected wavelengths and which is derived from the input light into the chamber means;

(C) second monochromator means for for producing output light which comprises selected wavelengths and which is derived from the emitted light;

(D) illumination module means which includes a plurality of sources of focused light for transmitting such light to the first monochromator means;

(E) a gated photodetector having a gating input and an output, signals directly related to the intensity of received light appearing on the output if a signal is also present on the gating input;

(F) a first photodetector having an output on which signals appear which are directly related to the intensity of received light;

(G) means for selectively directing the output light from the second monochromator means to the gated photodetector or the first photodetector;

(H) reference module means, which includes

(i) a second photodetector having an output on which signals appear which are directly related to the intensity of received light, and

(ii) means for directing the excitation light to the chamber means or for directing some of the excitation light to both the second photoconductor and the chamber means;

(I) a first detector subsystem, which includes

(i) a first normally deenergized source of first serial pulses,

(ii) means for delaying the first pulses by a fixed amount and for pulsatingly energizing a selected light source in response thereto,

(iii) means for selectively delaying the first pulses by a variable amount and for applying the variably delayed pulses to the gating input of the gated photodetector, and

(iv) first convertor means which receives signals on the output of the gated photoconductor and for producing digital signals related thereto, the digital signals produced by the first convertor means occurring a selected time after light pulses from the selected light source, being directly related to the intensity of the output light at such selected time, and representing the integral of such intensity taken over the time-width of the variably delayed pulses;

(J) a second detector subsystem which includes

(i) a second normally deenergized source of second serial pulses,

(ii) means for pulsatingly energizing a selected light source in response to the second pulses,

(iii) resetable integrator means connected to the outputs of the first and second photodetectors,

(iv) means for resetting the integrators in response to second pulses,

(v) normally "off" analog-to-digital convertor means for receiving from the integrator means analog signals representing the time integrals of the signals on the outputs of the first and second photodetectors and for producing digital signals, respectively representative thereof,

(vi) means for gating "on" the analog-to-digital convertor means in response to second pulses, and

(vii) means for adjusting the phase relationship among the second pulses received by the selected light source, the integrator resetting means, and the convertor gating "on" means, so that respective outputs of the second and analog-to-digital convertor means carry signals representative of the respective time integrals, taken during an integration window of selected widths, of signals on the respective outputs of the first and second photodetectors; and

(K) digital data processing means

(i) for selecting a sample for receipt of excitation light, for selecting the wavelengths of the excitation light and the output light, for selectively energizing a selected pulse source, selectively directing the output light to the gated photodetector or to the first photoconductor and, if the latter, to direct excitation light to the second photodetector,

(ii) if the gated photoconductor receives output light and if the first pulse source is energized, for varying the delay of the first pulses by differing amounts so that a selected number of integrals of the intensity of the output light are taken over varying time-widths, and for storing digital signals from the analog-to-digital convertor means representing a selected number of integrals over each such time-width, and

(iii) if the first and second photoconductors receive light and if the second pulse source is energized, for storing a selected number of digital signals from the second and third analog-to-digital convertor means representative of the time integrations performed by the integrator means and using the integration of the second photodetector output as a reference for the integration of the first photodetector output.

2. An integrated luminescence measuring system for selectively conducting lifetime decay or steady state measurement of fluorescent or phosphorescent light emitted by a sample at a test site when the sample receives excitation light, wherein the system comprises:

(A) A chamber surrounding the test site which holds a plurality of samples and which includes means for directing excitation light received in the chamber to the test site and for directing fluorescent or phosphorescent light emitted by a sample at the test site to the exterior of the chamber;

(B) a first adjustable monochromator for receiving input light and for transmitting excitation light to the chamber, the excitation light being derived from and including selected wavelengths of the input light;

(C) a second adjustable monochromator for receiving emitted light from the chamber and for producing output light, the output light being derived from and including selected wavelengths of the emitted light;

(D) an illumination module which includes

(i) a plurality of selectively energizable sources of pulsed, focused light, and

(ii) means for permitting the focused, pulsed light from a selected source to be received as input light by the first monochromator;

(E) a gated photodetector having a gating input for receiving output light from the second monochromator, the gated photodetector producing an output directly related to the intensity of received output light when a signal is present on the gating input;

(F) a first photodetector for receiving output light from the second monochromator, the photodetector producing an output directly related to the intensity of the received output light;

(G) adjustable means for selectively directing the output light from the second monochromator to the gated photodetector or to the first photodetector;

(H) a reference module, which includes

(i) a second photodetector which produces an output directly related to the intensity of excitation light incident on the second photodetector, and

(ii) adjustable means for selectively

(a) permitting all of the excitation light to reach the chamber and for permitting none of the excitation light to reach the second photodetector, or

(b) directing excitation light to the second photoconductor and to the chamber;

(I) a first detector subsystem, which includes

(i) a first selectively energizable source of serial pulses,

(ii) means for delaying pulses from the first source by a fixed amount and for energizing and pulsing one of the light sources in response thereto,

(iii) means for selectively delaying pulses from the first source by a variable amount and for applying the variably delayed pulses to the gating input of the gated photodetector,

(iv) first convertor means for receiving the output of the gated photoconductor and for producing digital signals representative thereof, whereby the digital signals produced by the first convertor means occur a selected time after the occurrence of the light pulses from the one light source, are directly related to the intensity of the output light at such selected time, and represent the integral of such intensity taken over the time-width of the variably delayed pulses on the gating input;

(J) a second detector subsystem which includes

(i) a second selectively energizable source of pulses,

(ii) means for energizing and pulsing another of the light sources in response to pulses from the second pulse source,

(iii) a first resetable integrator which receives the output of the first photodetector,

(iv) a second resetable integrator which receives the output of the second photodetector,

(v) means for resetting the integrators in response to pulses from the second pulse source,

(vi) normally "off" analog-to-digital convertors for respectively receiving from the integrators analog signals representing the time integral of the signals on the outputs of the first and second photodetectors and for producing digital signals representative thereof,

(vii) means for gating "on" the convertors in response to pulses from the second pulse source, and

(viii) means for adjusting the phase relationship among the pulses received from the second pulse source by the other light source, the integrator resetting means, and the convertor gating "on" means, so that the convertors produce signals representative of the respective time integrals, taken during integration windows of selected widths, of the respective outputs of the first and second photodetectors; and

(K) digital data processing means

(i) for presenting a selected sample at the test site,

(ii) for adjusting the monochromators so that the excitation light and the output light, respectively, comprise selected wavelengths,

(iii) for selectively energizing one of the pulse sources to energize one of the light sources, to permit light from the selected light source to be received by the first monochromator, to adjust the output-light-directing means, and to selectively adjust the excitation-light-permitting means,

(iv) if the gated photoconductor receives output light and if the first clock is energized, for affecting the variable delaying means to selectively vary the delay of the pulses from the first clock by differing amounts so that a selected number of integrals of the intensity of the output light are taken over varying the time widths, and for storing digital signals from the first convertor representing a selected number of integrals over each such time-width, and

(v) if the first photoconductor receives output light, if the second clock is energized, and if the second photoconductor receives excitation light, for storing a selected number of digital signals from the second and third analog-to-digital convertors representative of the time integrations performed by the integrators and using the integration of the second photodetector output as a reference for the integration of the first photodetector output.

3. An integrated luminescence-measuring system for selectively conducting lifetime decay or steady-state measurement of fluorescent or phosphorescent light emitted by a sample at a test site when the sample receives excitation light, wherein the system comprises:

(A) A sample chamber, which includes

(i) a rotatable turret,

(ii) a plurality of sample holders on the turret, which holders are selectively locatable at the test site upon rotation of the turret,

(iii) means for rotating the turret,

(iv) first means for directing excitation light received in the chamber to the test site, and

(v) second means for directing fluorescent or phosphorescent light emitted by a sample at the test site to the exterior of the chamber;

(B) a first monochromator for receiving input light and for transmitting excitation light, which is derived from the input light, to the first directing means, the first monochromator including means for selectively permitting the output excitation light to comprise selected wavelengths of the input light;

(C) a second monochromator for receiving emitted light from the second directing means and for producing output light which is derived from the emitted light, the second monochromator including means for selectively permitting the output light to comprise selected wavelengths of the emitted light;

(D) an illumination module, which module includes

(i) first and second pulsable, selectively energizable sources of light,

(ii) means for focusing light issuing from each source, and

(iii) multipositionable means for, depending on the position thereof, permitting the focused light from the first or the second light source to be received as input light by the first monochromator;

(E) a gated photodetector for receiving output light from the second monochromator, the gated photodetector including a gating input and an electrical output, there appearing on such output, when a signal is present on the gating input, an electrical signal directly related to the intensity of the received output light;

(F) a first photodetector for receiving output light from the second monochromator, the first photodetector including an electrical output on which there appear electrical signals directly related to the intensity of the received output light;

(G) means for selectively directing the output light from the second monochromator to the gated photodetector or to the first photodetector;

(H) a lifetime measurement reference module, which includes

(i) a second photodetector having an electrical output on which there appear electrical signals directly related to the intensity of light incident on the second photodetector, and

(ii) selectively positionable means for

(a) in a first position, permitting all of the excitation light to reach the first directing means and permitting none of the excitation light to reach the second photodetector, and

(b) in a second position, directing some of the excitation light to the second photoconductor and to the first directing means;

(I) a first detector subsystem, which includes

(i) a first, selectively energizable clock for producing serial pulses,

(ii) means for delaying pulses from the first clock by a fixed amount and for energizing and pulsing the first light source in response thereto,

(iii) means for selectively delaying pulses from the first clock by a variable amount and for applying the variably delayed pulses to the gating input of the gated photodetector,

(iv) a first analog-to-digital convertor for receiving the electrical signals on the output of the gated photoconductor and for producing digital signals related thereto, whereby the digital signals produced by the first convertor occur a selected time after the occurrence of the light pulses from the first light source, are directly related to the intensity of the output light at such selected time, and represent the integral of such intensity taken over the time-width of he delayed pulses on the gating input;

(J) a second detector subsystem which includes

(i) a second selectively energizable clock for producing serial pulses,

(ii) means for energizing and pulsing the second light source in response to pulses from the second clock

(iii) a first resetable integrator connected to the output of the first photodetector,

(iv) a second resetable integrator connected to the output of the second photodetector,

(v) means for resetting the integrators in response to pulses form the second clock,

(vi) a normally "off" second analog-to-digital convertor for receiving from the first integratory analog signals representing the time integral of the signals on the output of the first photodetector and for producing digital signals representative thereof on an output,

(vii) a normally "off" third analog-to-digital convertor for receiving from the second integrator analog signals representing the time integral of the signals on the output of the second photodetector and for producing digital signals representative thereof on an output,

(viii) means for gating "on" the second and third analog-to-digital convertors in response to pulses from the second clock, and

(ix) means for adjusting the phase relationship among the pulses received from the second clock by the second light source, the integrator resetting means, and the convertor gating "on" means, so that respective outputs of the second and third analog-to-digital convertors carry signals representative of the respective time integrals, taken during an integration window of selected widths, of signals on the respective outputs of the first and second photodetectors; and

(K) digital data processing means

(i) for selectively rotating the turret to locate a selected sample holder at the test site,

(ii) for selectively affecting the monochromators so that the output excitation light and output light, respectively, comprise selected wavelengths,

(iii) for selectively energizing one of the clocks to energize one of the light sources, to selectively position the multipositionable permitting means, to selectively adjust the selective output-light-directing means, and to selectively position the excitation-light-related selectively positionable means,

(iv) if the gated photoconductor receives output light and if the first clock is energized, for affecting the variable delaying means to selectively vary the delay of the pulses from the first clock by differing amounts so that a selected number of integrals of the intensity of the output light are taken over varying time-widths, and for storing digital signals from the first analog-to-digital convertor representing a selected number of integrals over each such time-width, and

(v) if the first photoconductor receives output light, if the second clock is energized, and if the second photoconductor receives excitation light, for storing a selected number of digital signals from the second and third analog-to-digital convertors representative of the time integrations performed by the integrators and using the integration of the second photodetector output as a reference for the integration of the first photodetector output.