System and method of fluorescence polarimetry

• US 4,203,670 A
• Filed: 04/21/1977
• Issued: 05/20/1980
• Est. Priority Date: 04/21/1977
• Status: Expired due to Term

• Current Assignee: Nathan S. Bromberg
• Original Assignee: Nathan S. Bromberg
• Inventors: Bromberg, Nathan S.
• Primary Examiner(s): Corbin, John K.
• Assistant Examiner(s): Rosenberger, R. A.
• Application Number:
  US05/789,636
• Time in Patent Office: 1,125 Days
• Field of Search: 356/103, 356/104, 356/114, 356/116, 356/117, 356/364, 356/366, 356/367, 356/369, 356/338, 356/339, 324/111, 324/140 D
• US Class Current: 356/367
• CPC Class Codes: G01N 21/6445 Measuring fluorescence polarisation

Abstract

A system and method for measurement of the polarization of fluorescence. A laser source provides a collimated linearly polarized beam of high intensity and small cross-section. A relatively small number of optical components are employed to minimize spurious fluorescence and spurious polarization which usually occurs from most optical materials. A photodetector provides an electrical signal in response to and representative of fluorescence polarization and is processed to directly produce first and second signals representing the difference and sum components of the polarization equation, the ratio of these components being directly provided without need for division computation.

Claims

1. A fluorescene polarimeter comprising:

an illumination source providing a defined, narrow band light beam;

a sample cell for containing a sample solution and disposed to receive light from said illumination source and to permit fluorescent radiation from a sample solution;

an analyzer;

means for providing continuous rotation of said analyzer;

an optical filter receiving fluorescent radiation from said sample cell and having a pass band centered at the emission frequency of said fluorescent radiation;

photodetector means operative in response to radiation passed by said optical filter to provide an electrical output signal representative of radiation intensity;

signal processor means operative in response to the output signal from said photodetector means and including;

means for providing first and second signals respectively representative of the difference component and the sum component of fluorescence polarization;

means providing a time interval representing the ratio of said first signal to said second signal and including;

first integrator means receiving said second signal and providing a first integrated output signal;

second integrator means receiving a signal derived from said first signal and providing a second integrated output signal of maximum level determined by the maximum level of said first integrated output signal; and

means causing the discharge of said second integrator means at a uniform rate to provide said time interval; and

means operative in response to said time interval to provide a signal representative of polarization of fluorescence.

2. The fluorescence polarimeter of claim 1 wherein said analyzer includes:

an achromatic half-wave retardation plate disposed to receive fluorescent radiation from said sample; and

a fixed linear polarizer receiving fluorescent radiation from said sample and having its polarization axis oriented along an intended reference axis.

3. The fluorescence polarimeter of claim 1 wherein said illumination source provides a linearly polarized light beam;

and wherein said analyzer includes;

a half-wave retardation plate disposed between said illumination source and said sample cell; and

a fixed linear polarizer receiving fluorescent radiation from said sample and having its polarization axis oriented along an intended reference axis.

4. The fluorescence polarimeter of claim 1 wherein said analyzer includes a continuously rotated linear polarizer.

5. The fluorescence polarimeter of claim 1 further including a depolarizer disposed in front of said photodetector means to depolarize the light received thereby.

6. The fluorescence polarimeter of claim 1 wherein said analyzer includes:

an electro-optical rotator disposed between said illumination source and said sample cell; and

a fixed linear polarizer receiving fluorescent radiation from said sample and having its polarization axis oriented along an intended reference axis.

7. The fluorescence polarimeter of claim 1 wherein said analyzer includes:

an electro-optical rotator disposed between said sample cell and said photodetector; and

a fixed linear polarizer receiving fluorescent radiation from said sample and having its polarization axis oriented along an intended reference axis.

8. The fluorescence polarimeter of claim 1 further including at least one dispersing element disposed in the optical path between said source and said photodetector means and operative to provide a spectrum.

9. The fluorescence polarimeter of claim 1 wherein said time interval providing means includes:

integrator means charged to a predetermined level and discharged at a uniform rate, the duration of discharge defining said time interval.

10. The fluorescence polarimeter of claim 1 wherein said illumination source includes a CW laser providing a collimated, monochromatic linearly polarized light beam.

11. The fluorescence polarimeter of claim 10 wherein said illumination source further includes a second optical filter receiving a light beam from said laser and having a pass band centered about the fundamental frequency of said laser and operative to exclude light above and below an intended band.

12. The fluorescence polarimeter of claim 11 wherein said second optical filter includes:

an interference filter structure operative to pass only the excitation wavelength of said illumination source;

an interference filter structure operative to reflect wavelengths longer than said excitation wavelength; and

an interference filter structure operative to reflect wavelengths shorter than said excitation wavelength.

13. The fluorescence polarimeter of claim 1 wherein said signal processor means comprises:

means for providing an AC signal directly representative of the difference component of fluorescence polarization;

means for providing a DC signal directly representative of the sum component of fluorescence polarization;

a counter;

means operative in response to said DC signal to start operation of said counter;

means operative in response to said AC signal to stop operation of said counter;

said counter providing a count accumulated during the interval between said start and stop signals representing polarization of fluorescence.

14. The fluorescence polarimeter of claim 1 wherein said optical filter includes:

an interference filter structure operative to provide a pass band matched to the spectrum of the fluorescent emission received;

an interference filter structure operative to reflect the scattered light at the excitation wavelength;

an interference filter structure operative to reflect light at the Raman wavelength of the sample solution; and

an interference filter structure operative to reflect other spurious wavelengths of received light.

28. The fluorescence polarimeter of claim 1 wherein said phase sensitive detector includes:

switch means operative to shunt the detector output to ground except during a measurement run and to open circuit the input to said second integrator means except during a measurement run.

15. A fluorescence polarimeter comprising:

an illumination source providing a defined, narrow band light beam;

a sample cell for containing a sample solution and disposed to receive light from said illumination source and to permit fluorescent radiation from a sample solution;

an analyzer;

means for providing continuous rotation of said analyzer;

an optical filter receiving fluorescent radiation from said sample cell and having a pass band centered at the emission frequency of said fluorescent radiation;

photodetector means operative in response to radiation passed by said optical filter to provide an electrical output signal representative of radiation intensity;

signal processor means operative in response to the output signal from said photodetector means and including;

bandpass filter means providing a first signal directly representative of the difference component of fluorescence polarization;

lowpass filter means providing a second signal directly representative of the sum component of fluorescence polarization;

amplifier means coupled to said bandpass filter means and providing an amplified version of said first signal;

a phase sensitive detector receiving said amplified first signal and providing an output signal;

first integrator means operative in response to said second signal to provide a first integrated output signal

second integrator means operative in response to said output signal to provide a second integrated output signal;

first comparator means providing a first comparator output signal when said first integrated output signal exceeds a reference threshold;

second comparator means providing a second comparator output signal when said second integrated output signal exceeds a reference threshold;

means operative in response to said first comparator output signal to enable operation of said phase sensitive detector and operative in response to said second comparator output signal to disable operation of said phase sensitive detector;

counter means enabled in response to said first comparator output signal and disabled in response to said second comparator output signal;

means operative in response to said first comparator output signal to cause controlled discharge of said second integrator means to provide a time interval representing the ratio of said first signal to said second signal; and

means operative in response to said time interval to provide a signal representative of polarization of fluorescence.

16. The fluorescence polarimeter of claim 15 further including for each of said integrator means:

an autozero circuit operative to compensate for offsets.

17. The fluorescence polarimeter of claim 16 wherein said autozero circuit includes:

a comparator coupled to the output of said integrator means and providing a first output if the input signal thereto is greater than a positive reference threshold, and a second output if the input signal thereto is less than a negative reference threshold;

an up/down counter incremented by one count in response to said first output signal and decremented by one count in response to said second output signal;

a digital to analog converter operative in response to the output count from said counter to provide a compensating signal to the input of said integrator means; and

timer means triggered in response to an output from said comparator and operative to provide a latch signal to said counter at the end of its timing interval.

18. The fluorescence polarimeter of claim 15 wherein each of said integrator means includes:

switch means operative to shunt the integrator except during a measurement run to provide a predetermined stable condition.

19. The fluorescence polarimeter of claim 15 wherein said first integrator means includes:

an operational amplifier integrator circuit;

first switch means connected in shunt with said first integrator circuit and closed except during a measurement run;

and wherein said first comparator means includes;

an operational amplifier comparator circuit coupled to the output of said first integrator circuit and providing a first comparator output signal of first logic level when said first switch means is closed and of second logic level when said first switch means is open and the output signal from said integrator circuit equals the reference level of said first comparator means;

and wherein said second integrator means includes;

an operational amplifier integrator circuit;

second switch means in shunt with said second integrator circuit and closed except during a measurement run to remove residual charge on said second integrator circuit;

and wherein said second comparator means includes;

an operational amplifier comparator circuit coupled to the output of said second integrator circuit and providing a second comparator output signal of first logic level when said second switch means is closed and of second logic level when said second switch means is open and the signal from said second integrator equals the reference level of said second comparator.

20. The fluorescence polarimeter of claim 19 including:

gate means coupled to the output of said first and second comparator means and operative to disable said second comparator means when said first comparator output signal is of first logic level.

21. The fluorescence polarimeter of claim 15 wherein said controlled discharge means includes:

a current source;

means operative in response to said first comparator output signal to couple said current source to said second integrator means;

said current source providing a current of a polarity to cause discharge of said second integrator means.

22. The fluorescence polarimeter of claim 15 wherein said phase sensitive detector includes:

a calibration circuit operative to compensate for circuit offsets.

23. The fluorescence polarimeter of claim 15 including:

a comparator circuit coupled to the output of said amplifier means and providing an output indication of an overload condition in response to an input signal thereto greater than a predetermined threshold.

24. The fluorescence polarimeter of claim 15 further including:

a heterodyned lock-in amplifier having an oscillator providing a signal of predetermined frequency and providing a signal to said phase sensitive detector referenced to said oscillator signal and unaffected by speed variation of the rotating analyzer.

25. The fluorescence polarimeter of claim 15 further including:

a lowpass filter receiving a signal from said amplifier means;

a mixer receiving the output signal from said lowpass filter means and a reference signal;

a bandpass filter receiving the output signal from said mixer and providing an output signal to said phase sensitive detector; and

a phase lock loop providing said reference signal and receiving clock signals from an oscillator.

26. The fluorescence polarimeter of claim 15 wherein said phase sensitive detector includes:

first and second switch means closed before and after a measurement run to shunt the output of said detector to ground to prevent application of any spurious signal to said second integrator means;

third and fourth switch means opened before and after a measurement run to open circuit the input to said second integrator means;

during a measurement run said first and fourth switch means being operative to open and close in synchronism and said second and third means being operative to open and close in synchronism to provide phase sensitive detection.

27. The fluorescence polarimeter of claim 15 wherein said phase sensitive detector includes filter means operative to reduce power line and harmonic noise frequencies.

29. The fluorescence polarimeter comprising:

an excitation path having only

a laser source providing a collimated, monochromatic, linearly polarized light beam of small cross section and high intensity; and

a first optical filter receiving said light beam and having a pass band centered about the fundamental frequency of said laser source and operative to exclude light above and below an intended band;

a sample cell for containing a sample solution and disposed to receive light from said optical filter and to permit fluorescent radiation from a sample solution;

a linear polarizer disposed to receive fluorescent radiation emitted from a sample in said sample cell;

means for continuously rotating said linear polarizer;

a second optical filter receiving light from said rotating linear polarizer and having a pass band centered at the emission frequency of said fluorescent radiation;

photodetector means operative in response to radiation passed by said second optical filter to provide an electrical output signal representative of radiation intensity; and

signal processor means operative in response to said output signal to provide a signal representative of polarization of fluoresence;

and wherein said polarizer rotating means includes;

an air bearing having a housing and a hollow rotor disposed therein for rotation about an axis coincident with the axis of received fluorescent radiation;

said linear polarizer being mounted on said rotor symmetrical about said axis for rotation with said rotor;

pneumatic means providing an air layer between the confronting surfaces of said housing and said rotor; and

pneumatic means for directing a jet of air onto a surface of said rotor to cause rotation thereof.

30. The fluorescence polarimeter of claim 29 including:

sensor means disposed in operative association with said rotor;

a plurality of magnetic slugs disposed about a periphery of said rotor for sensing by said sensor means;

said sensor means providing control signals representative of the position and speed of rotation of said rotor.

31. The fluorescence polarimeter of claim 30 wherein said signal processor means includes means controlled by said control signals.

32. For use in a fluorescence polarimeter having an illumination source for exciting a sample solution, a continuously rotating analyzer, and a photodetector providing an output signal in response to fluorescent radiation received from the sample solution, a signal processor comprising:

means for providing first and second signals respectively representative of the difference component and the sum component of fluorescence polarization;

means providing a time interval representing a ratio of said first signal to said second signal and including;

first integrator means receiving said second signal and providing a first integrated output signal;

second integrator means receiving a signal derived from said first signal and providing a second integrated output signal of maximum level determined by the maximum level of said first integrated output signal; and

means causing the discharge of said second integrator means at a uniform rate to provide said time interval; and

means operative in response to said time interval to provide a signal representative of polarization of fluorescence.

33. The fluorescence polarimeter of claim 32 wherein said signal processor means comprises:

means providing an AC signal directly representative of the difference component of fluorescence polarization;

means for providing a DC signal directly representative of the sum component of fluorescence polarization;

a counter;

means operative in response to said DC signal to start operation of said counter;

means operative in response to said AC signal to stop operation of said counter;

said counter providing a count accumulated during the interval between said start and stop signals representing polarization of fluorescence.

34. A fluoresence polarimeter comprising:

an excitation path having only

a laser source providing a collimated, monochromatic, linearly polarized light beam of small cross section and high intensity; and

a first optical filter receiving said light beam and having a pass band centered about the fundamental frequency of said laser source and operative to exclude light above and below an intended band;

a sample cell for containing a sample solution and disposed to receive light from said optical filter and to permit fluorescent radiation from a sample solution;

an analyzer;

means for continuously rotating said analyzer;

a second optical filter receiving light from said rotating linear polarizer and having a pass band centered at the emission frequency of said fluorescent radiation;

photodetector means operative in response to radiation passed by said second optical filter to provide an electrical output signal representative of radiation intensity; and

signal processor means operative in response to said output signal to provide a signal representative of polarization of fluorescence;

and wherein said rotating means includes;

a hollow rotor disposed within a housing for rotation therein about an axis coincident with the axis of received fluorescent radiation;

said analyzer being mounted on said rotor symmetrical about said axis for rotation with said rotor;

means for providing rotation of said rotor about said axis;

a plurality of sensible elements disposed about the periphery of said rotor;

and control signal providing means including a transducer operative upon detection of said sensible elements to provide control pulses representative of the position and speed of said rotor.

35. The fluorescence polarimeter of claim 34 wherein said rotation providing means includes:

a regulator for governing the driving force applied to said rotor; and

means for providing a control signal to said regulator derived from said control pulses to provide servo control of rotor speed.

36. A fluorescence polarimeter comprising:

an illumination source providing a defined, narrow band light beam;

a sample cell for containing a sample solution and disposed to receive light from said illumination source and to permit fluorescent radiation from a sample solution;

an analyzer;

means for providing continuous rotation of said analyzer;

an optical filter receiving fluorescent radiation from said sample cell and having a pass band centered at the emission frequency of said fluorescent radiation;

photodetector means operative in response to radiation passed by said optical filter to provide an electrical output signal representative of radiation intensity;

signal processor means operative in response to the output signal from said photodetector means and including;

means for providing first and second signals respectively representative of the difference component and the sum component of fluorescence polarization;

means providing a time interval representing the ratio of said first signal to said second signal and including;

first integrator means providing a first integrated output signal in response to said second signal;

second integrator means providing a second integrated output signal in response to a signal derived from said first signal;

said second integrator means being operative for an integration time determined by the integration time of said first integrator means to cause a second integrated output signal of maximum level determined by the maximum level of said first integrated output signal; and

means causing the discharge of said second integrator means at a uniform rate to provide said time interval which represents the ratio of said difference component to said sum component and thus the polarization of fluorescence; and

means operative in response to said time interval to provide a signal representative of polarization of fluorescence.

37. For use in a fluorescence polarimeter having an illumination source for exciting a sample solution, a continuously rotating analyzer, and a photodetector providing an output signal in response to fluorescent radiation received from the sample solution, a signal processing method comprising the steps of:

deriving a first signal from said photodetector signal directly representing the difference component of fluorescence polarization;

deriving a second signal from said photodetector signal directly representing the sum component of fluorescence polarization;

integrating a signal derived from said first signal to a level determined by the level of a signal derived from said second signal;

causing controlled discharge of said integrated signal to provide a time interval representing the ratio of said first signal to said second signal; and

providing in response to said time interval a signal representative of polarization of fluorescence.

38. A fluorescence polarimeter comprising:

a laser source providing a collimated, monochromatic, linearly polarized light beam of small cross section and high intensity;

a first optical filter receiving said light beam and having a pass band centered about the fundamental frequency of said laser source and operative to exclude light above and below an intended band;

a sample cell for containing a sample solution and disposed to receive light from said optical filter and to permit fluorescent radiation from a sample solution;

an analyzer;

means for continuously rotating said analyzer;

a second optical filter receiving light from said rotating linear polarizer and having a pass band centered at the emission frequency of said fluorescent radiation;

photodetector means operative in response to radiation passed by said second optical filter to provide an electrical output signal representative of radiation intensity;

signal processor means operative in response to said output signal to provide a signal representative of polarization of fluorescence;

said signal processor means including;

means providing a first signal directly representative of the difference component of fluorescence polarization;

means for providing a second signal directly representative of the sum component of fluorescence polarization;

means operative in response to said second signal to denote the start of a time interval;

means operative in response to said first signal to denote the end of said time interval; and

means operative in response to said time interval to provide an output indication of polarization of fluorescence.

39. The fluorescence polarimeter comprising:

a laser source providing a collimated, monochromatic, linearly polarized light beam of small cross section and high intensity;

a first optical filter receiving said light beam and having a pass band centered about the fundamental frequency of said laser source and operative to exclude light above and below an intended band;

a sample cell for containing a sample solution and disposed to receive light from said optical filter and to permit fluorescent radiation from a sample solution;

a linear polarizer disposed to receive fluorescent radiation emitted from a sample in said sample cell;

means for continuously rotation said linear polarizer;

a second optical filter receiving light from said rotating linear polarizer and having a pass band centered at the emission frequency of said fluorescent radiation;

photodetector means operative in response to radiation passed by said second optical filter to provide an electrical output signal representative of radiation intensity; and

signal processor means operative in response to said output signal to provide a signal representative of polarization of fluorescence;

said signal processor means including;

bandpass filter means providing a first signal directly representative of the difference component of fluorescence polarization;

lowpass filter means providing a second signal directly representative of the sum component of fluorescence polarization;

amplifier means coupled to said bandpass filter means and providing an amplified version of said first signal;

a phase sensitive detector receiving said amplified first signal and providing an output signal;

first integrator means operative in response to said second signal to provide a first integrated output signal;

second integrator means operative in response to said output signal to provide a second integrated output signal;

first comparator means providing a first comparator output signal when said first integrated output signal exceeds a reference threshold;

means operative in response to said first comparator output signal to enable operation of said phase sensitive detector and operative in response to said second comparator output signal to disable operation of said phase sensitive detector;

counter means enabled in response to said first comparator output signal and disabled in response to said second comparator output signal;

means operative in response to said first comparator output signal to cause controlled discharge of said second integrator means;

wherein said polarizer rotating means includes;

an air bearing having a housing and a hollow rotor disposed therein for rotation about an axis coincident with the axis of received fluorescent radiation;

said linear polarizer being mounted on said rotor symmetrical about said axis for rotation with said rotor;

pneumatic means providing an air layer between the confronting surfaces of said housing and said rotor;

pneumatic means for directing a jet of air onto a surface of said rotor to cause rotation thereof;

sensor means disposed in operative association with said rotor;

a plurality of magnetic slugs disposed about a periphery of said rotor for sensing by said sensor means;

said sensor means providing control signals representative of the position and speed of rotation of said rotor; and

said signal processor means including means controlled by said control signals.