Method and apparatus for ROI-scan with high temporal resolution

#The present invention relates to a method and an apparatus for ROI-scan with a high temporal resolution, preferably the method is used in confocal scanning microscopy.

#In confocal scanning microscopy, a specimen is scanned with a focused light beam; this is generally achieved by tilting two mirrors arranged in the beam path of the confocal scanning microscope. The focus of the light beam is thereby moved in the focal plane, the deflection directions of the light beam most often being arranged perpendicular to one another so that, for example, one mirror deflects the beam in the X direction and another mirror deflects the beam in the Y direction. The motion or tilting of the mirrors is usually brought about with the aid of galvanometer actuating elements. Special control devices connected to the galvanometer actuating elements provide a position signal of the light beam on the specimen.

#The German patent Application DE 198 29 981 discloses a method which involves coupling laser light of different spectral ranges in an, in at least two coordinates diverted microscope beam path, and directing the laser light successively on places of a test. The test is supplied in at least one plane, place for place and line for line with the laser light, and an image of the sampled plane is generated from reflected and/or emitted light. The spectral composition and/or the intensity of the laser light is changed while the scanning is continued, and at least two adjacent points of the test are supplied with light of different spectral characteristics and/or different intensity. An Independent claim is provided for a laser-scanning microscope implementing the method. The above mentioned method applies the same intensity in the scan direction and in the opposite scan direction. In other words the region of interest is illuminated with the same wavelength and/or illumination condition in the scan direction and in the opposite scan direction. A detection of the influence of the illumination condition on the region of interest is detectable only in the successive frame. For some biological processes the delay between illumination and detection is to long.

#There are various ways of advantageously embodying and developing the teaching of the present invention. Reference is made to the drawings. In the drawings:

#FIG. 1 schematically depicts an apparatus according to the present invention for carrying out a method according to the present invention,

#FIG. 2 is a schematic representation of a frame to be scanned wherein the frame encompasses two regions of interest,

#FIG. 1 shows an apparatus for scanning a specimen 11 with a light beam 4. The light beam 4 may be generated by combining the light of at least a first and an second light source 1 and 2. The first and the second light source differ in wavelength and are configured as lasers. The second light source is a multi line laser. A beam combiner 3 is arranged with respect to the first and second light source 1 and 2 so that the light beam 4 is generated form the two individual light sources 1 and 2. In this embodiment, the light beam travels though an acoustooptical tunable filter (AOTF) 5. The AOTF allows the user of a confocal scanning microscope, which is the apparatus for scanning a specimen, to select various intensities per each wavelength of the light beam 4. The AOTF 5 is connected to a high frequency driving device 6 which itself is connected to a control device 13. A beam dump 7 is arranged to eliminate deflected light 4a of the light beam 4 which is not needed for further investigation of the specimen 11. Passing light 4b of the light beam 4 reaches a beam splitter 8 and is reflected from there to a scanning device 9 which leads light beam 4b through a microscope optical system or an objective 10 via the specimen 11. Fluorescent light produced in the specimen 11 passes through the objective 10 and reaches the scanning device 9. From the scanning device 9 the fluorescent light passes through the beam splitter 8 and reaches at least one detector 12. The control device 13 is connected to the scanning device 9 as well. The control device 13 is connected to a computer 14 which itself has a display 15 for displaying image signals and/or user interfaces. Furthermore, the electric signals generated by the detector 12 are delivered to the computer 14 as well. Image formation on the display 15 is accomplished in consideration of the signals from the scanning device 9, the detector 12 and the control device 13.

#FIG. 2 is a schematic representation of a scan frame 20 to be scanned wherein the scan frame 20 encompasses a first and second region of interest 24 and 25. The scan pattern 23 of the scan frame 20 of the specimen 11 is started in the x-direction (represented by arrow x). The scanning device 9 is configured to reverse the scan pattern 23 (opposite to the x-direction). The scan pattern 23 defines a plurality of lines 23a across the frame 20 so that every other line 23a points in the same direction. The switching from one line 23a to the next is done in y-direction (represented by arrow y) outside the scan frame 20. According to one embodiment of the invention, the scan pattern 23 intersecting the first region of interest 24 defines a first scan line 21, in the x-direction, to which light with a first wavelength λ1 is applied and the intersection defines a second scan line 22, opposite to the x-direction, to which light with a second wavelength λ2 is applied. The same applies to the intersection of the scan pattern 23 with the second region of interest 25. The coordination of the appliance of the various wavelengths is carried out by the control device 13. The control device 13 is connected to an AOTF (not shown) and with the information about the beam position of the according to the scanning device 9 a switching between wavelength λ1 and wavelength λ2 can be easily done. The switching is synchronized with the intersection of the scan pattern 23 and the user selected region of interest.

#FIG. 3a shows an embodiment of the invention wherein a first representation 20a and a second representation 20b are shown simultaneously on the display 15. The first representation 20a is generated by first illumination conditions, which are constituted by a first parameter setting 26. The second representation 20a is generated by second illumination conditions which are constituted by a second parameter setting 27. The arrows across the scan frame 20 of the first representation 20a represent the substantial parallel part of the scan pattern 23 applied to the scan frame 20. The same is valid for the second embodiment as shown in FIG. 3b. The first representation 20a on the display is gained by scanning the sample line by line as shown by the arrows. The second representation 20b is obtained in that the scanning spot is move back substantially on the scan pattern 23 of the first representation 20a. In other words, the scan spot is substantially moved back along the line represented by the arrows in FIG. 3a. During the move back of the scan spot the light intensity impinging onto the specimen 11 is substantially reduced, the laser is switched to a standby mode or the laser light is blocked completely. Then the line (defined by one arrow) is scanned once again and the data gained are the second representation 20b on the display 15. The scan to gain the first representation 20a is carried out with different illumination conditions than the scan for the second representation 20b. The parameter settings comprise wavelength, intensity level or the like. In the first representation 20a the intersection of the arrows with the first and second region of interest 24 and 25 result in a plurality of first scan lines 21. In the second representation 20b the intersection of the arrows with the first and second region of interest 24 and 25 result in a plurality of second scan lines 22. The illumination conditions along the first scan line 21 are different from the illumination conditions of the second scan line 22. In a more complicated embodiment of the invention the illumination conditions of the first scan line 21 in the first region of interest 24 are different from the illumination conditions in the second region of interest 25.