CONFOCAL FLUORESCENCE SLIDE SCANNER WITH PARALLEL DETECTION

• US 20130015370A1
• Filed: 07/16/2012
• Published: 01/17/2013
• Est. Priority Date: 07/15/2011
• Status: Abandoned Application

• Current Assignee: Huron Technologies International Inc. (Confocal, Inc.)
• Original Assignee: Huron Technologies International In
• Inventors: Damaskinos, Savvas, Dixon, Arthur Edward
• Application Number:
  US13/550,291
• Publication Number:
  US 20130015370A1
• Time in Patent Office: Days
• US Class Current: 250/459.1
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 2201/10 Scanning
• CPC Class Codes: G01N 2201/1042 X, Y scan, i.e. object moving in X, beam in Y

Abstract

An instrument and method for scanning a large specimen supported on a specimen holder has a plurality of illumination sources with each illumination, source being focused on a different focus spot of the specimen simultaneously. There are a plurality of spectrally resolved detectors to receive light reflected or emitted from the different focus spots simultaneously with each spectrally resolved detector receiving light from one illumination source only.

Claims

1. An instrument for scanning a large specimen supported, the instrument comprising a plurality of illumination sources, at least one lens to focus light from each illumination source on a different focus spot of the specimen simultaneously, there being a plurality of focus spots, with each illumination source being focused on a different focus spot of the plurality of focus spots, there being a plurality of spectrally resolved detectors to receive fluorescence emitted from the different focus spots simultaneously, the detectors being isolated from one another so that substantially all of the fluorescence emitted from each focus spot is received by a different detector of the plurality of detectors.

2. An instrument as claimed in claim 1 wherein the illumination sources have different wavelengths from one another.

3. An instrument as claimed in claim 1 wherein the illumination sources have substantially the same wavelength.

4. An instrument as claimed in claim 1 wherein the different focus spots on the specimen are located apart from one another by a sufficient distance to substantially eliminate crosstalk.

5. An instrument as claimed in claim 1 wherein the plurality of illumination sources are arranged to illuminate the different focus spots in a line.

6. An instrument as claimed in claim 1 wherein the plurality of illumination sources are arranged in a line and are comprised of one of a line of lasers, a line of LEO'"'"'s and a line or pinholes illuminated from behind.

7. An instrument as claimed in claim 1 wherein a spectrometer containing two dimensional array detectors is used with a line of illumination sources to provide spectrally-resolved detection of each illumination source.

8. An instrument as claimed in claim 1 wherein a spectrometer containing a two dimensional array detector is used with a line of illumination sources to provide spectrally-resolved detection of each illumination source wherein the detectors are arranged so that each row in the two dimensional array detector receives light substantially from one illumination source only.

9. An instrument as claimed in claim 1 wherein there are a plurality of quantum dots on the specimen, the multiple illumination sources being arranged in a line, a spectrum from each illuminated spot on the specimen being detected using a spectrometer with multiple spectrally-resolved detectors.

10. An instrument as claimed in claim 1 wherein the instrument has a linear array of discrete illumination sources of the plurality of illumination sources located on an illumination arm and a linear array of discrete detectors located on a detection arm.

11. A method of using an instrument for scanning a large specimen, the instrument having a plurality of illumination sources, at least one lens to focus light from each illumination source on a different focus spot of the specimen, the instrument having one detector for each focus spot of the specimen that is to be detected, there being a plurality of focus spots, the method comprising activating the instrument including the plurality of illumination sources, adjusting the at least one lens to focus the light from each illumination source onto different focus spots of the specimen, each focus spot receiving light from only one illumination source, choosing and arranging spectrally resolved detectors to receive fluorescence emitted from the different focus spots simultaneously and isolating the detectors from one another so that each detector receives light substantially from one illumination source only.

12. A method as claimed in claim 11 wherein the focused spots excite fluorescence, the method including the step of detecting at least two fluorophores simultaneously with substantially reduced cross talk between the fluorophores.

13. A method as claimed in claim 11 including the step of using an illumination sources having different wavelengths from one another.

14. A method as claimed in claim 11 including the step of using illumination sources having substantially the same wavelength.

15. A method of using an instrument for scanning a large specimen, the instrument having a plurality of illumination sources, at least one lens to focus light from each illumination source to a different focus spot of the specimen, the instrument having one detector for each focus spot of the specimen that is to be detected, there being a plurality of focus spots, the method comprising activating the instrument including the plurality of illumination sources, adjusting the at least one lens to focus the light from each illumination source onto different focus spots of the specimen, each focus spot receiving light from only one illumination source, minimizing cross talk between the detectors by ensuring that the different focus spots on which the light, from the illumination source is focused are separated by distances of at least approximately ten spot diameters from each other, arranging the detectors to receive emitted or reflected light from the different focus spots simultaneously and isolating the detectors from one another so that each detector receives light substantially from one illumination source only.

17. A method as claimed in claim 15 including the steps of predicting an exposure required for each fluorophore using a high-speed preview scan and using the information from the preview scan to adjust the gain of each of the detectors for simultaneous detection of weak and strong fluorophores and the ability to scan very large specimens.

18. A method of using an instrument as claimed in claim 15 where the instrument is a combination of two instruments sharing a scanning area, scan lens and moving specimen holder, selecting any two or more of the instruments from the group of a confocal scanning fluorescence macroscope, spectrally-resolved confocal scanning fluorescence macroscope, confocal scanning reflection macroscope, scanning laser multiphoton fluorescence macroscope or harmonic detection macroscope, scanning photo luminescence macroscope, scanning brightfield macroscope using a white light source with a transmission RGB detector placed below the specimen and a scanning Raman macroscope.

20. A method as claimed in claim 15 where there is at least a first illumination source and a second illumination source, the method including the steps of passing the light from the first illumination source through a small-diameter fibre optic cable attached to a holder for the cable that holds an end of a fibre on a focal plane of the at least one lens.

21. A method as claimed in claim 15 including the steps of using the spectrally resolved detectors in a macroscope, reducing a scan speed significantly because a light intensity falling on each detector element is considerably reduced by spreading a beam of the light entering the detector over a range of wavelengths, allowing for longer exposure times at slower scan speed.

22. A method as claimed in claim 15 including the steps of using a confocal scanning-beam/scanning-stage optical macroscope with a single illumination arm transmitting light from an array of the illumination sources, arranging the illumination sources along a line parallel to a Y direction and a plurality of detectors being located in a single detection arm and being arranged along a line parallel to the Y direction.

23. A method as claimed in claim 15 where the instrument is a macroscope and the method includes the steps of using a linear array of light sources coupled to an area array detector and a spectrometer, for spectrally resolved detection.

16. A method of using an instrument for scanning a large specimen, the instrument having a plurality of illumination sources, at least one lens to focus light from each illumination source on a different focus spot of the specimen, the instrument having one detector for each focus spot of the specimen, that is to be detected, there being a plurality of focus spots and a plurality of detectors, the method comprising positioning the illumination sources in an XY plane such that the focus spots to be detected are along a line in the Y direction and scanning spots move across the specimen in the X direction so that the focus spots to be detected are located a distance apart, from one another to avoid any long lifetime fluorophores that are excited by a first focus spot and are detected by a second detector mixed with a fluorescence signal excited by a second illumination source, there being at least two focus spots, at least two detectors and at least two illumination sources.

19. A method as claimed in claim 16 including the steps of applying the method to confocal scanning light microscopes and microscopy.