Line-scanning, sample-scanning, multimodal confocal microscope

• US 10,466,460 B2
• Filed: 08/13/2015
• Issued: 11/05/2019
• Est. Priority Date: 08/13/2014
• Status: Active Grant

• Current Assignee: Surgivance Inc.
• Original Assignee: Surgivance Inc.
• Inventors: Gareau, Daniel Summer
• Primary Examiner(s): Perungavoor, Sathyanaraya V
• Assistant Examiner(s): Dang, Philip P.
• Application Number:
  US15/503,608
• Publication Number:
  US 20170242230A1
• Time in Patent Office: 1,545 Days
• CPC Class Codes: G02B 21/0024 Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
• CPC Class Codes: G02B 21/0032 Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers G02B21/0036 - G02B21/008; means for illumination of specimens in general G02B21/06
• CPC Class Codes: G02B 21/0036 Scanning details, e.g. scanning stages
• CPC Class Codes: G02B 21/0064 multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling G02B21/0076 takes precedence
• 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
• CPC Class Codes: G02B 21/0084 time-scale detection, e.g. strobed, ultra-fast, heterodyne detection
• CPC Class Codes: G02B 21/26 Stages; Adjusting means therefor
• CPC Class Codes: G02B 21/368 details of associated display arrangements, e.g. mounting of LCD monitor
• CPC Class Codes: G02B 26/04 by periodically varying the intensity of light, e.g. using choppers shutters, diaphragms for cameras G03B9/00; devices for eliminating or reducing the effect of flicker in projection systems G03B21/40

Abstract

A confocal microscope is provided that includes one or more lasers focused by an optical system into a line on the surface of a sample mounted to a stage. The microscope further includes at least one linear array detector that is optically conjugated to the focused line. The stage permits movement of the sample with respect to all other components of the microscope, which remain stationary.

Claims

1. A confocal microscope comprising:

a light source;

a stage adapted to secure a sample thereto;

a plurality of linear array detectors; and

an optical system comprising;

a cylindrical lens positioned so as to receive a first light emitted by the light source and focus the first light in a line upon a selected plane of the sample when secured to the stage;

an objective lens positioned so as to receive a second light from the sample in response to incidence of the first light upon the sample and focus the second light upon at least one of the plurality of linear array detectors;

a first beam splitter positioned to reflect second light fluorescently emitted from the sample onto a first linear detector array of the plurality of linear detector arrays; and

a second beam splitter positioned to reflect second light reflected from the sample onto a second linear detector array of the plurality of linear detector arrays,

wherein the second light fluorescently emitted from the sample possesses a wavelength different from that of the first light and wherein the second light reflected from the sample possesses a wavelength approximately the same as that of the first light, and

wherein the stage is further adapted to position the sample at about the focal plane of the objective lens and to move the sample with respect to the focused line of the first light.

2. The confocal microscope of claim 1, wherein the light source comprises a single laser source.

3. The confocal microscope of claim 1, further comprising an optical chopper and wherein the light source comprises at least two lasers, each emitting a different laser beam, wherein the optical chopper allows each different laser beam to pass on to the sample at a time different than the other laser beams.

4. The confocal microscope of claim 3, further comprising a clocking system that measures the position of the chopper, identifies the light source for which first light is permitted by the chopper to illuminate the sample, and measures the duration of that light source'"'"'s illumination upon the sample.

5. The confocal microscope of claim 4, wherein the clocking system comprises a motion detector, a signaling light, and a clock detector positioned on both sides of the chopper, wherein the clock detector generates a clock signal in response to detection of the signaling light that corresponds to the duration of illumination of the identified light source upon the sample.

6. The confocal microscope of claim 4, wherein the clocking system comprises a motion detector and a clock detector positioned opposite the light source, wherein the clock detector generates a clock signal in response to detection of the illumination of the identified light source and wherein the clock signal corresponds to the duration of illumination of the identified light source upon the sample.

7. The confocal microscope of claim 1, wherein the stage physically translates the sample with respect to the line of first light focused upon the sample without movement of the first light.

8. The confocal microscope of claim 1, wherein the path between the second light fluorescently emitted from the sample and the first linear detector array is different from the path between the second light reflected from the sample and the second linear detector array.

9. A method of imaging a sample, comprising:

providing a confocal microscope comprising;

a light source;

a stage adapted to secure a sample thereto;

a plurality of linear array detectors; and

an optical system comprising;

a cylindrical lens positioned so as to receive a first light emitted by the light source and focus the first light in a line upon a selected plane of the sample when secured to the stage;

an objective lens positioned so as to receive a second light from the sample in response to incidence of the first light upon the sample and focus the second light upon at least one of the plurality of linear array detectors;

a first beam splitter positioned to reflect second light fluorescently emitted from the sample onto a first linear detector array of the plurality of linear detector arrays; and

a second beam splitter positioned to reflect second light reflected from the sample onto a second linear detector array of the plurality of linear detector arrays,

wherein the second light fluorescently emitted from the sample possesses a wavelength different from that of the first light and wherein the second light reflected from the sample possesses a wavelength approximately the same as that of the first light, and

wherein the stage is further adapted to position the sample at about the focal plane of the objective lens and to move the sample with respect to the focused line of the first light;

positioning the stage at a first position, wherein the first light is focused in a line upon a first selected plane of the sample;

measuring, by at least one of the plurality of linear array detectors, an intensity as a function of time for the second light focused upon the first selected focal plane of the sample;

positioning the stage at second position, different from the first position, wherein the first light is focused in a line upon a second selected plane of the sample; and

measuring, by at least one of the plurality of linear array detectors, an intensity as a function of time for the second light focused upon the second selected focal plane of the sample.

10. The method of claim 9, wherein the first selected position is translated approximately perpendicular to the direction of the focused line of first light.

11. The method of claim 9, further comprising acquiring an optical image of the sample from a digital image capture device separate from the confocal microscope, the optical image having a field of view larger than the sample.

12. The method of claim 11, further comprising display of the optical image upon a display device in communication with a targeting computing device, the targeting computing device being adapted to receive vector targeting inputs from a user, wherein the vector targets correspond to a region of interest of the sample.

13. The method of claim 12, wherein the targeting computing device is in communication with the stage and wherein the stage is further adapted to:

receive the vector targets from the targeting computing device; and

position the sample such that the first light is focused in a line within the region of interest of the sample.