Microscope and method for fluorescence imaging microscopy

• US 9,372,334 B2
• Filed: 06/13/2012
• Issued: 06/21/2016
• Est. Priority Date: 06/17/2011
• Status: Active Grant

• Current Assignee: Leica Microsystems CMS GmbH (Danaher Corporation)
• Original Assignee: Leica Microsystems CMS GmbH (Danaher Corporation)
• Inventors: Seyfried, Volker
• Primary Examiner(s): Czekaj, Dave
• Assistant Examiner(s): Rahman, Mohammad J
• Application Number:
  US13/495,538
• Publication Number:
  US 20120320184A1
• Time in Patent Office: 1,469 Days
• Field of Search: 348/79
• US Class Current: 1/1
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence

Abstract

A microscope for fluorescence imaging microscopy, in particular for wide-field fluorescence microscopy, including a pulsed light source (1) and an imaging detector (11), is characterized in that means for gating are provided, and in that the gating causes the light source (1) and the detector (11) to be synchronized in order to suppress reflected/scattered light such that suitable fluorescence components are used for evaluation and unsuitable components are rejected. Furthermore, a method is used to perform fluorescence imaging microscopy using the microscope according to the present invention.

Claims

1. A microscope for fluorescence imaging microscopy, in particular for wide-field fluorescence microscopy, comprising:

a pulsed light source sending excitation light via an objective to a sample;

an imaging detector receiving fluorescent light and scattered or reflected light from the sample via the objective; and

a synchronization unit, controllable by a computer, providing gating of the imaging detector in combination with the pulsed light source to synchronize the pulsed light source and the imaging detector to (1) suppress components of the scattered or fluorescent light, (2) allow evaluation of suitable fluorescence components of the fluorescent light, and (3) reject unsuitable fluorescence components of the fluorescent light,

wherein the fluorescence components comprise photons and wherein a timestamp representing a time of arrival of each photon, or a record of a time of arrival of each signal waveform of an image pixel, is assigned to each photon or image pixel so as to decide in a downstream data processing unit whether each photon or image pixel is suitable for evaluation.

2. The microscope as recited in claim 1,

wherein the synchronization unit gating allows the suitable fluorescence components belonging to a fluorescent light of interest to be fed to the imaging detector and components of the scattered or reflected light are not used for imaging.

3. The microscope as recited in claim 1,

wherein fluorescent light of a definable time window after illumination of the sample is used for imaging, and the fluorescent light outside of the time window is not used for imaging and can be delivered to another detection channel.

4. The microscope as recited in claim 1,

wherein a same or a simultaneous gate is provided for all image pixels.

5. The microscope as recited in claim 1,

wherein different temporal gates or gate positions are implemented for different image pixels.

6. The microscope as recited in claim 1,

wherein the synchronization unit comprises a central trigger unit which defines a master on which the pulsed light source and the detector can be triggered.

7. The microscope as recited in claim 6,

wherein the pulsed light source comprises mode-locked lasers which generate a master trigger on which the detector can be triggered.

8. The microscope as recited in claim 6,

wherein the detector generates a trigger to which the pulsed light source can be synchronized from an intrinsic or externally controlled clock.

9. The microscope as recited in claim 1,

wherein the detector comprises a gatable camera.

10. The microscope as recited in claim 9,

wherein the camera includes, or is functionally associated with, a triggerable/gatable fast optical shutter.

11. The microscope as recited in claim 10,

wherein the shutter comprises a rapidly switchable optical image intensifier which amplifies light when in the ON state.

12. The microscope as recited in claim 10,

wherein the shutter comprises an optical absorber which is saturated by short pulses of light and, therefore, becomes transparent when a gate signal is applied.

13. The microscope as recited in claim 1,

wherein the detector is gatable to be activated or deactivated.

14. The microscope as recited in claim 1,

wherein while gating is OFF, an output of the detector is short-circuited or disconnected from a signal lead-out line.

15. The microscope as recited in claim 1,

wherein the detector includes arrays of avalanche photodiodes (APD) preferably operable in Geiger mode, each individual image pixel of the imaging detector being composed of a single APD or a plurality of APDs as a subarray.

16. The microscope as recited in claim 1,

wherein the detector, after producing a detection signal, provides information qualifying a respective signal component as usable or not usable.

17. A method for fluorescence imaging microscopy, in particular for wide-field fluorescence microscopy, comprising:

sending excitation light from a pulsed light source via an objective to a sample;

receiving fluorescent light and scattered or reflected light at an imaging detector from the sample via the objective; and

gating the imaging detector in combination with the pulsed light source to synchronize the pulsed light source and the imaging detector to (1) suppress components of the scattered or reflected light, (2) allow evaluation of suitable fluorescence components of the fluorescent light, and (3) reject unsuitable fluorescence components of the fluorescent light,

wherein the fluorescence components comprise photons and wherein a timestamp representing a time of arrival of each photon, or a record of a time of arrival of each signal waveform of an image pixel, is assigned to each photon or image pixel so as to decide in a downstream data processing unit whether each photon or image pixel is suitable for evaluation.