EP0454834A1 - Optique et procede de mesure de polarisation de fluorescence - Google Patents

Optique et procede de mesure de polarisation de fluorescence

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Publication number
EP0454834A1
EP0454834A1 EP19910900498 EP91900498A EP0454834A1 EP 0454834 A1 EP0454834 A1 EP 0454834A1 EP 19910900498 EP19910900498 EP 19910900498 EP 91900498 A EP91900498 A EP 91900498A EP 0454834 A1 EP0454834 A1 EP 0454834A1
Authority
EP
European Patent Office
Prior art keywords
source
polarization
polarized
light
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19910900498
Other languages
German (de)
English (en)
Inventor
Ryszard Borucki
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SCOURCE SCIENTIFIC SYSTEMS Inc
Original Assignee
SCOURCE SCIENTIFIC SYSTEMS Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SCOURCE SCIENTIFIC SYSTEMS Inc filed Critical SCOURCE SCIENTIFIC SYSTEMS Inc
Publication of EP0454834A1 publication Critical patent/EP0454834A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6445—Measuring fluorescence polarisation

Definitions

  • the invention relates to a method and apparatus for measuring fluorescence polarization. More particularly, the invention relates to a method and apparatus for measuring a quantity called "source corrected fluorescence polarization.”
  • the fluorescent light subsequently emitted by the fluorophore may be partially polarized.
  • the polarization of the emitted light will decline significantly.
  • the life time of the excited state is particularly short or if the rotational diffusion is relatively slow due to use of a highly viscous medium or due to the large size of the fluorophore, there will be a comparatively lesser decline of the polarization of the fluorescent light.
  • the measure of fluorescence polarization has found wide variety of scientific and clinical applications in several fields of biochemistry.
  • [1(1)] is the intensity of fluorescent light which is parallel to the polarization of the source light
  • 1(2)] is the intensity of fluorescent light which is perpendicular to the polarization of the source light.
  • a variety of polarization fluorometers have been described for measuring fluorescence polarization. An automated polarization fluorometer has been described by Richard Spencer et al. [Clinical Chemistry 19(8), pp 838-844 (1973)]. Spencer's polarization fluorometer splits the source light into two beams and polarizes each beam with perpendicular directions of polarization with respect to one another.
  • the two beams are then spliced by means of a mirrored chopper so that the sample fluorophore may be excited by alternating beam pulses having perpendicular directions of polarization with respect to one another.
  • the polarization of the resultant fluorescent beam will depend upon the sample fluorophore and upon the polarization of the source beam. Accordingly, after the fluorescent beam has passed through a polarizer, its intensity will vary with the same frequency as the rotational frequency of the chopper.
  • the intensity of the polarized fluorescent beam is measured by a photomultiplier tube and analyzed so as to indicate the fluorescence polarization [P] of the particular fluorophore.
  • a polarization fluorometer with an alternative optical configuration has been described by Mituta et al.
  • Mituta employs only a single polarized source beam. However, Mituta modifies the chopper and moves it from the source beam to the fluorescent beam. Mituta discloses a novel chopper which includes at least one pair of polarizers, each element of the pair having a direction of polarization which is perpendicular to the other element of the pair. After a polarized fluorescent beam passes through Mituta's chopper, the intensity of the resultant beam will vary with a frequency related to the rotational frequency of the chopper. The output and analysis of Mituta's photomultiplier tube is then similar to the output and analysis of the Spencer's photomultiplier tube.
  • Popelka A polarization fluorometer having a constant intensity of source light has been disclosed by Popelka (U.S. Pat. No. 4,516,859). Before passing the polarized source light into the sample fluorophore, Popelka employs a static beam splitter and reference detector to monitor the intensity of the source light. The output of the reference detector is then employed to adjust the power input of the light source so as to maintain a constant output of light.
  • the use of a constant intensity light source significantly enhances the stability of the fluorecence intensity.
  • Equation (1) indicates that the fluorescence polarization is dependent upon both 1(1) and 1(2), where 1(1) and 1(2) correspond respectively to the intensities of fluorescent light polarized parallel and perpendicularly with respect to the polarization of the source light.
  • Equation (1) assumes that the intensity of the source light is constant and that the transmittance of the various optical elements remains constant during the interval which separates the measurements of 1(1) and 1(2). This assumption may fail if the intensity of the light source is not regulated by feedback as disclosed by Popelka. Alternatively, the assumption may also fail if the transmittance of the various optical elements changes from one measurement to the next.
  • the polarizer is the most likely optical element to change its transmittance during the interval between one measurement and the next.
  • polarization fluorometry requires the use of a two polarizers, i.e. a first polarizer having a constant direction of polarization and a second polarizer which is adjustable between parallel and perpendicular configurations with respect to the first polarizer.
  • the first polarizer may be positioned in either the source beam or the fluorescent beam; the second polarizer is then postioned in the remaining beam.
  • the fluorescence polarization is then determined by taking a first measurement of the fluorescence intensity with the adjustable polarizer in one configuration and a second measurement of the fluorescence intensity with the adjustable polarizer in the remaining configuration.
  • a method and apparatus for measuring source corrected fluorescence polarization i.e. [P(c)] is disclosed.
  • the invention employs a reference photodetector to monitor the intensity of the source beam and employs the output of the reference photodetector to correct the fluorescent intensity with respect to each direction of. polarization.
  • the invention also positions the adjustable polarizer within the source beam. Accordingly, if the transmittances of the two configurations of the adjustable polarizer differ from one another, the reference photodetector will detect the change of source intensity.
  • the apparatus and technique of the invention enhances the accuracy and reproducibility of the measurement and dispenses with the need for stabilizing the intensity of the light source by means of electronic feedback and/or the need for employing adjustable polarizers having well matched transmittances in each their configurations.
  • source corrected fluorescence polarization i.e. [P(c)].
  • [1(c)(2)] k[I(m)(2)]/[F(2)], (4) where the measured fluorescence intensity terms, i.e. [I(m)(1)] and [I(m)(2)], are the measured signals from the fluorescence photodetector with the adjustable polarizer set in its parallel and perpendicular configurations respectively and where the reference intensity terms, i.e. [F(1)] and [F(2)], are the corresponding signals from the reference photodetector for the source beam.
  • the term "k" is a scaling factor.
  • the use of the reference intensity terms, i.e. [F(1)] and [F(2)], in equations (3) and (4) serves to correct the measured fluorescence intensity terms, i.e. [I(m)(1)] and [I(m)(2)], so as to provide the terms for the source corrected intensities, i.e. [1(c)(1)] and [1(c)(2)].
  • the reference intensity terms reflect any change of light flux within the source beam between the measurement of the fluorescent photodetector terms, i.e. [I(m)(1)] and [I(m)(2)].
  • the fluorometer of the present invention employs a reference photodetector in the path of the source beam for monitoring its flux or intensity. Furthermore, the fluorometer of the present invention positions the adjustable polarizer within the path of the source beam. Accordingly, the variability of the intensity the source beam includes any variability caused by mismatch or drift with respect to the transmittances of the two configurations of the adjustable polarizer. In particular, the variability of the intensity the source beam is monitored as the adjustable polarizer changes from one configuration to another. . The measurement of the intensity of the source light obviates the need to precisely regulate its intensity and enables the use of an adjustable polarizer having unmatched transmittances with respect to its two configurations.
  • FIG. 1 illustrates a schematic view of an example polarization fluorometer of the invention.
  • the apparatus requires a light source (2) for exciting the fluorophore (4).
  • Preferred source lights (2) include tungsten halogen lamps of the type employed with prior art polarization fluorometers. However, other conventional source lights
  • a heat glass (6) separates the source light from the remainder of the apparatus.
  • the heat glass (6) serves to block a major portion of the infra-red radiation eminating from the source light.
  • the source light is then be collimated to form a source beam (8).
  • the source light (8) may be collimated by means of a first lens (10).
  • the collimated source beam (8) may then be filtered by a first filter (12) so as to pass a source beam of monochromatic light.
  • the wavelength of the monochromatic light should correspond to the excitation energy of the particular fluorophore (4) which is targeted for excitation.
  • Preferred filters may be obtained from Corion, Inc. (Holliston, Massachusetts), e.g. P/N CFS-001564.
  • a monochromator may be employed in lieu of the filter.
  • the collimated source beam (8) is also passed through an adjustable polarizer (14).
  • the adjustable polarizer (14) has two configurations, i.e. a parallel configuration and a perpendicular configuration.
  • a parallel configuration When the adjustable polarizer (14) is set in its parallel configuration, it passes polarized light having a direction of polarization which is parallel with respect to the direction of polarization of a second polarizer (16), described below, which serves to polarize the emitted fluorescent light.
  • the adjustable polarizer (14) When the adjustable polarizer (14) is set in its perpendicular configuration, it passes polarized light having a direction of polarization which is perpendicular with respect to the parallel polarized light.
  • Preferred adjustable polarizers may be obtained from Melles Griot, Inc. (Irvine, California), e.g. P/N 03 FPG-001.
  • the polarization configuration of adjustable polarizers (14) may be changed mechanically or electronically.
  • the collimated source beam (8) is focused by a second lens (18) upon the sample fluorophore (4).
  • the sequence and/or relative positions of the first (10) and second lens (18), the first filter (12), and the adjustable polarizer (14) may be altered.
  • a source beam (8) of appropriately polarized light having a wavelength which substantially corresponds to the excitation energy of the sample fluorophore (4), will impinge upon a cuvette or other appropriate sample holder for containing such sample fluorophore (4) or blank.
  • Silicon photodiodes may serve as perferred reference photodectors. Appropriate silicone photodiodes may be obtained from Hamamatsu (Bridgewater, New Jersey).
  • the reference photodector serves to monitor the intensity of the light flux which passes through the sample holder.
  • the reference photodetector (20) is energized so as to produce a signal which is proportional to the light flux which impinges it.
  • the signal from the reference photodetector (20) is conditioned and amplified.
  • the conditioned signal from the reference photodetector (20) is then fed to an analog to digital converter (30) or to an equivalent device for reading electronic signals.
  • the sample fluorophore (4) After the source beam (8) impinges the sample fluorophore (4), the sample fluorophore (4) becomes electronically excited. The excited fluorophore (4) may then relax by emitting fluorescent light. A portion of the emitted fluorescent light is collimated by a third lens (22) to form a fluorescent beam (24). It is preferred that the third lens (22) not be aligned with the source beam (8). In the preferred embodiment, the fluorescent beam (24) forms an angle which is substantially normal to the source beam (8).
  • the fluorescent beam (24) is then passed through a second polarizer (16) having a fixed direction of polarization.
  • the direction of polarization of the second polarizer (16) serves as the standard by which the parallel direction of the adjustable polarizer (14) is estabished.
  • Preferred polarizers may be obtained from Melles Griot, Inc. (Irvine, California).
  • the fluorescent beam (24) should also preferrably pass through a second filter (26) which blocks light having an energy corresponding to the monochromatic source beam (8).
  • the second filter (26) serves to block out scattered source light while passing fluorescent light emitted by the sample fluorophore (4).
  • Preferred second filters (26) may be obtained from Corion. Inc. (Holliston, Massachusetts), e.g. P/N CFS-001565.
  • the sequence and relative positions of the third lens (22), the second polarizer (16), and the second filter (26) may be altered.
  • the fluorescent beam (24) should include only polarized light having a wavelength corresponding to the fluorescent light only.
  • the resultant fluorescent light is then allowed to impinge upon a second photodetector (28).
  • the second photodetector (28) is a high gain photomultiplier tube. Appropriate high gain photomultiplier tubes may be obtain from Hamamatsu (Bridgewater, New Jersey). However, other photodetectors conventionally employed with polarization fluorometers of the prior art may also be employed.
  • the output of the second photodetector (28) is amplified and conditions per manufacturer's suggestions and connected to an analog to digital converter (30) or an equivalent device for reading eletctronic signals.
  • an analog to digital converter (30) is employed.
  • An appropriate 16 bit precision analog to digital converter (30) may be obtained from Burr-Brown (Tucson, Arizona), e.g. P/N ACD 700.
  • a common analog to digital converter (30) may be employed for reading the signals of both the reference photodetector (20) and the second photodetector (28).
  • a microprocessor (34) connected to the analog to digital converter (30) may serve to control the signal conditioning of the second photodetector (28).
  • Example of the Method Each determination of fluorescence polarization requires four measurements, viz. I(m)(1), I(m)(2), F(1) and F(2). Measurements of I(m)(1) and F(1) are performed with the adjustable polarizer (14) set in its parallel configuration. Measurements of I(m)(2) and F(2) are performed with the adjustable polarizer (14) set in its perpendicular configuration. Measurements of I(m)(1) and F(1) are made in conjunction with one another; while measurements of I(m)(2) and F(2) are made in conjunction with one another. The measurements of I(m)(1) and F(1) may be performed in either sequence, i.e. I(m)(1) before F(1) or F(1 ) before I(m)(1); similarly measurements of I(m)(2) and F(2) may be performed in either sequence. However, it is preferred that each pair of measurements me performed as close to one another in time as possible.
  • Measurment of F(1) and F(2) may be performed with a blank cuvette or with no cuvette.
  • the blank may be empty or may contain solvent without fluorophore (4).
  • Measurment of I(m)(1) and I(m)(2) are made with a cuvette or other container loaded with a sample fluorophore (4).
  • F(1) is first measured with the adjustable polarizer (14) in its parallel configuration and without any cuvette in the sample holder.
  • a cuvette containing a sample fluorophore (4) is loaded into the sample holder and the measurement of I(m)(1) is taken.
  • the configuration of the adjustable polarizer (14) is then reset to its perpendicular configuration without disturbing the cuvette.
  • the measurement of I(m)(2) is then taken.
  • the cuvette is removed from the sample holder without disturbing the adjustable polarizer (14) and the measurement of F(2) is taken.
  • the signal from the fluorescence photodetector is fed to the analog to digital converter (30) where it is amplified and read.
  • the input to the analog to digital converter (30) is switched to output of the reference photodetector (20).
  • F(1) and F(2) may be read from the same analog to digital converter (30) as is employed to read I(m)(1) and I(m)(2).
  • a microprocessor (34) may be employed for controlling the fluorescence photodetector and the analog to digital converter (30).

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  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Une polarisation de fluorescence corrigée à la source est définie et un fluoromètre de polarisation est décrit, afin de mesurer ladite polarisation de fluorescence corrigée à la source. Le fluoromètre de polarisation se base sur un photodétecteur de référence afin de contrôler l'intensité de la source de lumière, et afin de corriger les intensités de fluorescence mesurées. Le fluoromètre de polarisation positionne le polariseur réglable dans le faisceau de la source. Une mauvaise adaptation entre les facteurs de transmission du polariseur réglable dans ces configurations parallèles et perpendiculaires est corrigée par le signal de sortie du photodétecteur de référence.
EP19910900498 1989-11-21 1990-11-21 Optique et procede de mesure de polarisation de fluorescence Withdrawn EP0454834A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US43979589A 1989-11-21 1989-11-21
US439795 1999-11-12

Publications (1)

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EP0454834A1 true EP0454834A1 (fr) 1991-11-06

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EP19910900498 Withdrawn EP0454834A1 (fr) 1989-11-21 1990-11-21 Optique et procede de mesure de polarisation de fluorescence

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EP (1) EP0454834A1 (fr)
JP (1) JPH04503118A (fr)
WO (1) WO1991007652A1 (fr)

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Publication number Priority date Publication date Assignee Title
FI20031294A0 (fi) * 2003-09-10 2003-09-10 Thermo Labsystems Oy Fluorometrin kalibrointi

Family Cites Families (2)

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Publication number Priority date Publication date Assignee Title
JPS55109949A (en) * 1979-02-16 1980-08-23 Hitachi Ltd Spectro-fluorophotometer
CA1165585A (fr) * 1981-01-09 1984-04-17 Susan R. Popelka Appareil optique pour instrument de polarisation par fluorescence

Non-Patent Citations (1)

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Title
See references of WO9107652A1 *

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Publication number Publication date
JPH04503118A (ja) 1992-06-04
WO1991007652A1 (fr) 1991-05-30

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