Method for recording and displaying fluorescence images with a high spatial resolution

• US 7,113,205 B2
• Filed: 02/21/2002
• Issued: 09/26/2006
• Est. Priority Date: 02/24/2001
• Status: Active Grant

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: Carl-Zeiss-Stiftung
• Inventors: Cappellaro, Markus
• Primary Examiner(s): Ye, Lin
• Application Number:
  US10/078,688
• Publication Number:
  US 20020176007A1
• Time in Patent Office: 1,678 Days
• Field of Search: 348/226.1, 348/229.1, 348/218.1, 348/219.1, 348/36, 382/228
• US Class Current: 348/218.1
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• CPC Class Codes: G06T 3/40 Scaling of whole images or parts thereof, e.g. expanding or contracting
• CPC Class Codes: H01L 27/14603 Special geometry or disposition of pixel-elements, address-lines or gate-electrodes
• CPC Class Codes: H04N 25/48 Increasing resolution by shifting the sensor relative to the scene

Abstract

The invention relates to a method for recording and displaying fluorescence images with high spatial resolution. Several recordations of an image of an object are made sequentially with an electronic camera and, between recordations, the image of the object and the electronic camera are shifted relative to each other. The recordations are subsequently unified to a compiled image of higher spatial resolution. Before or after the partial images are brought together, the signals, which belong to image points of the images and which are obtained at different times, are amplified with amplification factors which are dependent upon the time intervals between the image recordations and are essentially reciprocal to the time dependency of the fluorescence. Artefacts caused by decreasing fluorescence intensities (fading, bleaching) are thereby avoided. These fluorescence intensities decrease as a function of time.

Claims

1. A method for recording and displaying fluorescence images with high spatial resolution, the method comprising the steps of:

making several recordings of an image of an object sequentially at time intervals with an electronic camera to obtain partial images;

introducing lateral displacements between said electronic camera and said image between said recordings;

joining said partial images to form a compiled image of higher spatial resolution;

obtaining signals of image points of said partial images at different time points; and

,

amplifying said signals with amplification factors which are dependent upon said time intervals between said recordings.

2. The method of claim 1, wherein the dependency on time of said amplification factors is essentially a reciprocal of the time dependency of the intensity of the fluorescence.

3. The method of claim 1, wherein said signals are amplified in advance of the compilation of the sequentially made recordings.

4. The method of claim 1, wherein the sequentially made recordings are first compiled to a total image of higher resolution and then the signals belonging to the image points of said compiled image are amplified.

5. The method of claim 1, wherein said amplification factors are determined by averaging the signals in the individual sequentially made recordings.

6. The method of claim 1, wherein said amplification factors are determined by averaging the signals in the component regions of the individual sequentially made recordings.

7. The method of claim 1, wherein the amplification factors for the individual sequentially made recordings are determined by approximation of a trace of the intensity of substances excited to fluoresce;

and, said trace approximates the natural attenuation characteristic as a function of time.

8. The method of claim 1, wherein an adaptive image filtering of said compiled image is made to suppress noise.

9. The method of claim 6, comprising the further steps of:

segmenting the sequentially made recordings into image regions;

amplifying the signals corresponding to the image points of different ones of said image regions with different amplification factors; and

,

amplifying the signals, which belong to the same image region, with identical amplification factors.

10. The method of claim 1, wherein said electronic camera is a color CCD camera used for the image recordings.

11. The method of claim 10, wherein the lateral displacement between the image of the object and said electronic camera takes place so as to cause the same point of said object to be imaged sequentially onto different color channels of the same CCD pixel of said electronic camera.

12. The method of claim 11, wherein different amplification factors are used for the different color channels.

13. The method of claim 11, comprising the further steps of:

simultaneously exciting several fluorescence emissions; and

,

determining different amplification factors for the individual emissions while considering spectral sensitivity curves of said color channels.

14. The method of claim 13, comprising the further step of determining said amplification factors for individual emissions by solving a linear equation system.

15. The method of claim 13, comprising the further step of determining said amplification factors for individual emissions utilizing a fuzzy logic method.

16. The method of claim 13, comprising the further step of determining said amplification factors for individual emissions utilizing a neuro-fuzzy logic method.

17. The method of claim 13, wherein the individual emissions are displayed as false colors.

18. A fluorescence microscope comprising:

an optical illuminating system;

an optical imaging system;

an image recording system; and

,

a computer system for carrying out a method including the steps of;

making several recordings of an image of an object sequentially at time intervals with an electronic camera to obtain partial images;

introducing lateral displacements between said electronic camera and said image between said recordings;

joining said partial images to form a compiled image of higher spatial resolution;

obtaining signals of image points of said partial images at different time points; and

,

amplifying said signals with amplification factors which are dependent upon said time intervals between said recordings.

19. A computer system comprising:

means for making several recordings of an image of an object sequentially at time intervals with an electronic camera to obtain partial images;

means for introducing lateral displacements between said electronic camera and said image between said recordings;

means for joining said partial images to form a compiled image of higher spatial resolution;

means for obtaining signals of image points of said partial images at different time points; and

,

means for amplifying said signals with amplification factors which are dependent upon said time intervals between said recordings.

20. A computer program product which can be loaded into the memory of a digital computer, said computer program product comprising:

a program having a software code for executing a method for recording and displaying fluorescence images with high spatial resolution, the method including the steps of;

making several recordings of an image of an object sequentially at time intervals with an electronic camera to obtain partial images;

introducing lateral displacements between said electronic camera and said image between said recordings;

joining said partial images to form a compiled image of higher spatial resolution;

obtaining signals of image points of said partial images at different time points; and

,

amplifying said signals with amplification factors which are dependent upon said time intervals between said recordings.

21. The computer program of claim 20, wherein said program is executed when run on said digital computer.

22. A method for recording and displaying fluorescence images with high spatial resolution, the method comprising the steps of:

providing an object with fluorescing emitting portions emitting fluorescence with an intensity decaying with time according to a decay characteristic;

making several recordings of an image of said object sequentially at time intervals with an electronic camera to obtain partial images, thereby obtaining signals of image points of said partial images at different time points;

introducing lateral displacements between said electronic camera and said image of said object between said recordings;

amplifying said signals with amplification factors which are dependent upon said time intervals between said recordings and on said decay characteristic; and

,

combining said amplified signals from said several recordings to form a displayed image without artefacts of bright and dark image strips.