Scanning confocal microscope providing a continuous display

• US 5,283,433 A
• Filed: 10/05/1992
• Issued: 02/01/1994
• Est. Priority Date: 10/05/1992
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Tsien, Roger Y.
• Primary Examiner(s): Nelms, David C.
• Assistant Examiner(s): SHAMI, KHALED
• Application Number:
  US07/958,038
• Time in Patent Office: 484 Days
• Field of Search: 250/216, 250/234, 250/235, 250/236, 250/459.1, 250/486.1, 250/327.2, 359/368
• US Class Current: 250/234
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/008 Details of detection or image processing, including general computer control

Abstract

A scanning confocal microscope scans a sample with an incident beam of radiation, in a raster scan pattern, causing the sample to fluoresce and emit visible radiation in at least two wavelengths. A portion of the fluorescent light retraces a portion of the path optical of incident beam, to a dichroic mirror that separates it from the incident beam for detection by a pair of photomultiplier tubes. A data processor accumulates digital data from the photomultiplier tubes to form a succession of image data frames of the sample being scanned, in the two wavelengths. Image data for a selected number of frames in each wavelength is averaged and then recorded on a single track of a recording system, in an alternating fashion with averaged data for the other wavelength. In addition, a ratio of the averaged data for the two wavelengths is delivered to a video display.

Claims

1. A confocal microscope for scanning a sample doped with a predetermined fluorescent indicator, comprising:

a scanner for repeatedly scanning a laser beam in two dimensions across the sample, whereupon the sample fluoresces in first and second predetermined wavelengths;

first and second detectors for detecting light emitted by the sample in the first and second wavelengths and for generating a succession of frames of image data representing a sequence of related pairs of simultaneous two-dimensional images of the sample, in the first and second wavelengths;

first and second frame memories for temporarily storing individual frames of image data for the respective first and second wavelengths;

a permanent data storage device;

transfer means for transferring image data derived from the data generated by the first and second detectors to the permanent data storage device in an alternating fashion;

ratio means for repeatedly computing and temporarily storing the ratio of image data for the respective first and second wavelengths, to produce a sequence of image data ratios; and

video means for receiving the sequence of image data ratios produced by the ratio means and providing a signal suitable for coupling to a video display, to provide a repeatedly updated display of the scanned sample that is substantially unaffected by any variations in the intensity of the scanning laser beam or in the local concentration in the sample of the fluorescent indicator.

2. A confocal microscope as defined in claim 1, wherein:

the microscope further includes first and second averaging means for averaging a selected number of successive frames of image data representing the respective first and second wavelengths, to produce a sequence of related pairs of average image data for delivery to the ratio means;

the ratio means repeatedly computes and temporarily stores the ratio of average image data received from the respective first and second averaging means; and

the video display provides a repeatedly updated display of the sequence of ratios of average image data.

3. A confocal microscope as defined in claim 2, wherein the video display provides a continuous display of the ratio of average image data currently stored by the ratio means, until the next successive ratio of average image data is received.

4. A confocal microscope as defined in claim 3, wherein:

the scanner scans the laser beam across the sample continuously;

the first and second detectors detect light emitted by the sample continuously and generate the succession of frames of image data continuously; and

the frames of image data transferred by the transfer means to the permanent data storage device reflect all of the data generated by the first and second detectors.

5. A confocal microscope as defined in claim 4, wherein the transfer means is adapted to transfer frames of image data to the permanent data storage device at a frame rate that is the same as the frame rate at which the first and second detectors generate frames of image data.

6. A confocal microscope as defined in claim 2, wherein the first and second averaging means each include:

an adder for adding the current image data frame to a previous sum of image data frames to produce a current sum of image data frames;

means for delivering the current sum of image data frames for storage in the associated first or second frame memory;

means for selectively supplying the image data frame currently stored in the associated frame memory to the adder; and

means operable during the time of the last frame in each sequence of image frames being averaged, for dividing by a predetermined integer the current sum of image data frames produced by the adder, to produce the average image data for delivery to the ratio means.

7. A confocal microscope as defined in claim 2, wherein:

the ratio means includes a single, bifurcated frame memory;

the transfer means transfers frames of average image data for the first and second wavelengths to the permanent data storage device in an alternating

the successive average image data frames for the first wavelength are each stored for one frame duration in the first frame memory; and

the successive average image data frames for the second wavelength are each stored for two frame durations in one half of the bifurcated frame memory of the ratio means.

8. A confocal microscope as defined in claim 7, wherein:

the first and second frame memories and the frame memory of the ratio means are of the same predetermined size; and

the first and second frame memories each store just a single frame of image data and each receive data for storage immediately after previously-stored data is read out.

9. A confocal microscope as defined in claim 1, wherein:

the scanner scans across the sample in a raster-scan pattern; and

the first and second detectors generate data representing video signals for each of the first and second wavelengths.

10. A method for scanning a sample doped with a predetermined fluorescent indicator and providing a video display of the sample, comprising the steps of:

repeatedly scanning a laser beam in two dimensions across the sample, whereupon the sample fluoresces in first and second predetermined wavelengths;

detecting light emitted by the sample in the first and second wavelengths and generating a succession of frames of image data representing a sequence of related pairs of simultaneous two-dimensional images of the sample, in the first and second wavelengths;

temporarily storing in first and second frame memories individual frames of image data for the respective first and second wavelengths;

transferring image data derived from the data for the first and second wavelengths, generated in the step of detecting, to a permanent data storage device in an alternating fashion;

repeatedly computing and temporarily storing the ratio of image data for the respective first and second wavelengths, to produce a sequence of image data ratios; and

receiving the sequence of image data ratios produced in step of repeatedly computing and providing a video signal suitable for coupling to a video display, to provide repeatedly updated display of the scanned sample that is substantially unaffected by any variations in the intensity of the scanning laser beam or in the local concentration in the sample of the fluorescent indicator.

11. A method as defined in claim 10, wherein:

the method further includes a step of averaging a selected number of successive frames of image data representing the respective first and second wavelengths, to produce a sequence of related pairs of average image data for use in the step of repeatedly computing;

the step of repeatedly computing repeatedly computes and temporarily stores the ratio of average image data provided in the step of averaging; and

the step of receiving provides a repeatedly updated display of the sequence of ratios of average image data.

12. A method as defined in claim 11, wherein the step of receiving provides a continuous display of the ratio of average image data currently stored in the step of repeatedly computing, until the next successive ratio of average image data is received.

13. A method as defined in claim 12, wherein:

the step of repeatedly scanning is performed continuously;

the step of detecting is performed continuously such that the succession of frames of image data are continuous; and

the frames of image data transferred to the permanent data storage device in the step of transferring reflect all of the data generated in the step of detecting.

14. A method as defined in claim 13, wherein the step of transferring includes a step of selectively transferring frames of image data to the permanent data storage device at a frame rate that is the same as the frame rate at which the step of detecting generates frames of image data.

15. A method as defined in claim 11, wherein the step of averaging includes steps of:

adding the current image data frame to a previous sum of image data frames to produce a current sum of image data frames;

delivering the current sum of image data frames for storage in the associated first or second frame memory;

selectively supplying the image data frame currently stored in the associated frame memory to the adder; and

during the time of the last frame in each sequence of image frames being averaged, dividing by a predetermined integer the current sum of image data frames produced in the step of adding, to produce the average image data for use in the step of repeatedly computing.

16. A method as defined in claim 11, wherein:

the step of repeatedly computing utilizes a single, bifurcated frame memory;

the step of transferring transfers average image data frames for the first and second wavelengths to the permanent data storage device in an alternating fashion;

the successive average image data frames for the first wavelength are each stored for one frame duration in the first frame memory; and

the successive average image data frames for the second wavelength are each stored for two frame durations in one half of the bifurcated frame memory used in the step of repeatedly computing.

17. A method as defined in claim 16, wherein:

the first and second frame memories used in the step of temporarily storing and the frame memory used in the step of repeatedly computing are of the same predetermined size; and

the step of repeatedly storing includes a step of receiving data for storage in the first and second frame memories immediately after previously-stored data is read out.

18. A method as defined in claim 10, wherein:

the step of repeatedly scanning includes a step of scanning across the sample in a raster-scan pattern; and

the step of detecting includes a step of generating data representing video signals for each of the first and second wavelengths.

19. A confocal microscope for scanning a sample doped with a predetermined fluorescent indicator, comprising:

a scanner for repeatedly scanning a laser beam in a raster-scan pattern across the sample, whereupon the sample fluoresces in first and second predetermined wavelengths;

first and second photodetectors for detecting light emitted by the sample in the first and second wavelengths and for generating video signals constituting a succession of frames of image data representing a sequence of related pairs of simultaneous two-dimensional images of the sample, in the first and second wavelengths;

first and second frame memories for temporarily storing individual frames of image data for the respective first and second wavelengths;

first and second averaging means for averaging a selected number of successive frames of image data representing the respective first wavelength and the second wavelength, to produce a sequence of related pairs of average image data, wherein the first and second averaging means each include

an adder for adding the current image data frame to a previous sum of image data frames to produce a current sum of image data frames,

means for delivering the current sum of image data frames for storage in the associated first or second frame memory,

means for selectively supplying the image data frame currently stored in the associated frame memory to the adder, and

means, operable during the time of the last frame in each sequence of image frames being averaged, for dividing by a predetermined integer the current sum of image data frames produced by the adder, to produce the average image data;

a permanent data storage device;

transfer means for transferring the average image data to the permanent data storage device alternately from the first and second averaging means;

ratio means for repeatedly computing and temporarily storing in a third frame memory the ratio of average image data received from the first and second averaging means, to produce a sequence of image data ratios; and

video means for receiving the sequence of image data ratios produced by the ratio means and providing a signal suitable for coupling to a video display, to provide a continuous, repeatedly-updated display of the scanned sample that is substantially unaffected by any variations in the intensity of the scanning laser beam or in the local concentration in the sample of the fluorescent indicator.

20. A confocal microscope as defined in claim 19, wherein:

the scanner includes

a laser for supplying the laser beam,

a microscope objective,

means for positioning the microscope objective adjacent to the sample to be scanned,

means for repeatedly scanning the laser beam in a raster-scan pattern across the sample; and

the confocal microscope further includes a video display for displaying the signal provided by the video means.