Microscope and Method for Fluorescence Imaging Microscopy

• US 20120320184A1
• Filed: 06/13/2012
• Published: 12/20/2012
• Est. Priority Date: 06/17/2011
• Status: Active Grant

• Current Assignee: Leica Microsystems Gmbh (Danaher Corporation)
• Original Assignee: Leica Microsystems Gmbh (Danaher Corporation)
• Inventors: SEYFRIED, Volker
• Granted Patent: US 9,372,334 B2
• Time in Patent Office: Days
• US Class Current: 348/79
• 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, including a pulsed light source (1) and an imaging detector (11),

wherein means for gating are provided, and 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.

2. The microscope as recited in claim 1,

wherein by means of the gating, light components belonging to the fluorescent light of interest can be fed to the detector (11), and the light components that belong to the reflected/scattered light are not used for imaging.

3. The microscope as recited in claim 1,

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

4. The microscope as recited in claim 1,

wherein the same and/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 can be implemented for different image pixels.

6. The microscope as recited in claim 1,

wherein the means for gating include a preferably central trigger unit (14) which defines a master on which the light source are the detector can be triggered.

7. The microscope as recited in claim 6,

wherein, in particular in the case of mode-locked lasers, the light source (1) generates the master trigger on which the detector (11) can be triggered.

8. The microscope as recited in claim 6,

wherein the detector (11) generates the trigger to which the light source (1) can be synchronized from an intrinsic or externally controlled clock.

9. The microscope as recited in claim 1,

wherein the detector (11) is in the form of a gatable camera.

10. The microscope as recited in claim 9,

wherein the camera (11) includes, or is functionally associated with, a triggerable/gatable fast optical shutter (18).

11. The microscope as recited in claim 10,

wherein the shutter (18) is in the form of a rapidly switchable optical image intensifier which amplifies the light when in the ON state.

12. The microscope as recited in claim 10,

wherein the shutter (18) is in the form of an optical absorber which is saturated by short pulses of light and, therefore, becomes transparent when the gate signal is applied.

13. The microscope as recited in claim 1,

wherein the detector (11) is itself gatable, namely suitably activatable/deactivatable.

14. The microscope as recited in claim 1,

wherein while the gate is OFF, the output of the detector (11) is short-circuited or disconnected from the signal lead-out line.

15. The microscope as recited in claim 1,

wherein as [sic. the] detector (11) includes arrays of avalanche photodiodes (APD) which are preferably operable in Geiger mode, each individual image pixel of the imaging detector (11) being composed of a single APD or a plurality of APDs in the sense of a subarray.

16. The microscope as recited in claim 1,

wherein the detector (11), after producing a detection signal, provides information qualifying the respective signal component as usable or not usable.

17. The microscope as recited in claim 1,

wherein a timestamp representing the time of arrival, or a record of the signal waveform, is assigned to each photon or image pixel so as to decide in a downstream data processing unit (13) whether the photon/image pixel can be used for evaluation.

18. A method for fluorescence imaging microscopy, in particular for wide-field fluorescence microscopy, including a pulsed light source and an imaging detector, in particular using a microscope according to claim 1,

wherein with regard to the signals that are usable for imaging, gating is performed, said gating causing the light source and the detector to be synchronized in order to suppress reflected/scattered light such that suitable fluorescence components are used for evaluation and unsuitable components are rejected.