Abstract:
A method and an arrangement for beam control in a scanning microscope are disclosed. The scanning microscope comprises means for acquiring and displaying ( 3 ) a preview image ( 7 ) and a microscope optical system ( 51 ). Means for marking ( 5 ) at least one region of interest ( 27, 29 ) in the preview image ( 7 ) are provided. A first beam deflection device ( 43, 67, 68 ) displaces the scan field ( 31, 33 ) onto the region of interest ( 27, 29 ); and a second beam deflection device ( 49, 72, 94 ) serves for meander-shaped scanning within the scan field ( 31, 33 ).

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This invention claims priority of the German patent application 100 50 529.5 which is incorporated by reference herein. 
    
    
     FIELD OF THE INVENTION 
     The invention concerns a method for scanning individual regions with a scanning microscope, the regions of interest being distributed over the entire image field. It is possible to switch rapidly between the individual regions of interest while maintaining the scanning motion. The scanning motion can be accomplished by way of a suitable motion of a scanning mirror. 
     The invention further concerns an arrangement for beam control in scanning microscopy. 
     In addition, the invention concerns a scanning microscope that comprises an arrangement for beam control which makes it possible to switch rapidly between the individual regions of interest while maintaining the scanning motion. The scanning microscope can also be configured as a confocal scanning microscope. In particular, in the scanning microscope a light beam produced by an illumination system is guided over a specimen with the interposition of several optical means, and it contains at least one detector that, by way of the several optical means, detects a light proceeding from the specimen. 
     BACKGROUND OF THE INVENTION 
     In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of the illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being at right angles to one another so that one mirror deflects in the X and the other in the Y direction. The tilting of the mirrors is brought about, for example, using galvanometer positioning elements; both fast resonant galvanometer positioning elements and slower (more accurate) non-resonant ones are used. In order to scan a sample in the specimen plane, it is important that the rotation axes of the mirror lie in or at least near a plane, also referred to as the Fourier plane, conjugated with the focal plane. One possible beam deflection device that meets the requirements for telecentric imaging is known, for example, from DE 196 54 210. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. Usually the positioning elements are equipped with sensors for ascertaining the present mirror position. 
     In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam. 
     A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the excitation stop), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection stop, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen arrives via the beam deflection device back at the beam splitter, passes through it, and is then focused on the detection stop behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop, thus yielding a point datum that results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually obtained by acquiring image data in layers. 
     Ideally, the track of the scanning light beam on or in the specimen describes a meander that fills the entire image field (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). At high beam deflection speeds, deviations from the ideal track occur because of the inertia of the deflecting moving parts, for example the galvanometer shaft and the mirrors. At usable scanning rates (&gt;100 Hz) the scanning track of the light beam actually describes a sine curve, which in fact a necessitates a correction of the deviations from the ideal situation resulting therefrom. 
     The power level of the light coming from the specimen is measured at fixed time intervals during the scanning operation, and thus scanned one grid point at a time. The reading must be unequivocally associated with the pertinent scan position so that an image can be generated from the measured data. Advantageously this is done by also continuously measuring the status data of the adjusting elements of the beam deflection device, or (although this is less accurate) by directly using the reference control data of the beam deflection device. 
     In some microscopy applications the user is interested only in information about individual regions within the image field, while the surrounding sample regions are not of interest. The regions of interest should moreover be scanned as quickly as possible in succession. 
     Known arrangements offer only a limited capability for scanning individual sample regions of interest. Scanning the entire image field and subsequently selecting the data of the regions of interest is feasible, if at all, only to a limited extent given the required rapid sequential acquisition of information about the regions of interest. 
     The approach of sequentially scanning the individual regions of interest is better. It is possible in principle to activate the beam deflection device in such a way that each of the regions of interest is separately scanned, for example, in meander fashion, and the surrounding regions that are not of interest are not scanned. This procedure is possible, however, only if the beam deflection device allows the scanning light beam to be specifically controlled and specifically directed onto individual points in the image field. 
     This is not possible when using resonantly operating beam deflection devices which are based, for example, on the use of resonant galvanometers or micromirrors, because these beam deflection devices operate exclusively at the particular resonant frequency dictated by their design. It is not possible to “park” the light beam in one region of the image field. Difficulties also occur with rapidly deflecting non-resonantly operating beam deflection devices in terms of the positionability that can be achieved, since the positioning elements react to an activation signal in delayed fashion because of their inertia. 
     SUMMARY OF THE INVENTION 
     It is therefore the object of the invention to describe a method for scanning microscopic preparations with a light beam that solves the problem described above. 
     This object is achieved by way of a method that comprises the following steps: 
     acquiring a preview image; 
     marking at least one region of interest in the preview image; 
     displacing a scan field onto the region of interest by means of a first beam deflection device; and 
     acquiring an image by meander-shaped scanning of the region of interest with a second beam deflection device. 
     What has been recognized according to the present invention is that it is not necessary to forgo the use of fast or resonant beam deflection devices if the scan field swept by the first beam deflection device is displaced within the image field onto the regions of interest with the aid of a further suitable beam deflection device that allows exact positioning. 
     A further object of the invention is to create an arrangement for beam control which makes it possible to switch rapidly among several regions of interest and, in that context, to collect information from regions of interest based on a consistent pattern. 
     This object is achieved by an arrangement for beam control in a scanning microscope. The arrangement comprises: 
     a scanning microscope defining a scan field; 
     means for acquiring and displaying a preview image 
     a microscope optical system; 
     means for marking at least one region of interest in the preview image; 
     a first beam deflection device for displacing the scan field onto the region of interest; and 
     a second beam deflection device for meander-shaped scanning within the scan field. 
     In a particular embodiment, according to the present invention an imaging optical system is provided between the beam deflection devices in order to guarantee the principle of telecentric scanning. 
     A further object of the invention is to create a scanning microscope that makes possible rapid sequential scanning of sample regions of interest. 
     This object is achieved by a scanning microscope which comprises: 
     an arrangement for beam control, 
     means for acquiring and displaying a preview image 
     a microscope optical system, 
     means for marking at least one region of interest in the preview image, 
     a first beam deflection device for displacing the scan field onto the region of interest; and 
     a second beam deflection device for meander-shaped scanning within the scan field. 
     The invention has the advantage that it is not necessary to forgo the use of fast or resonant beam deflection devices if the scan field swept by the first beam deflection device is displaced within the image field onto the regions of interest with the aid of a second suitable beam deflection device that allows exact positioning. Ideally, the track of the scanning light beam on or in the specimen describes a rectangular curve (scan one line in the X direction at a constant Y position, then stop the X scan and slew by Y displacement to the next line to be scanned, then scan that line in the negative X direction at constant Y position, etc.). At increasingly high deflection speeds, the scanning track deviates more and more from the rectangular shape. This phenomenon is attributable substantially to the inertia of the moving elements. With rapid scanning, the scanning track is more similar to a sine curve. In this context, the scan field is swept by the light beam in such a way that the reversing points of the sinusoidal track lie outside the region of interest. The scanning light beam thus describes approximately straight tracks on the region of interest. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The subject matter of the invention is depicted schematically in the drawings and will be described below with reference to the Figures, in which: 
     FIG. 1 schematically depicts selection of the regions of interest; 
     FIG. 2 schematically depicts the shape of the scanning track; 
     FIG. 3 shows a scanning microscope having the arrangement according to the present invention; 
     FIG. 4 shows a scanning microscope according to the present invention; 
     FIG. 5 shows a further embodiment of the scanning microscope according to the present invention; and 
     FIG. 6 shows a further embodiment of the scanning microscope having an arrangement according to the present invention. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     FIG. 1 shows a PC  1  having a monitor  3  and a cursor controller  5 . In one embodiment, cursor controller  5  is a computer mouse. Further possibilities for a cursor controller  5  are, for example, a joystick, a trackball, or any other conceivable cursor control device. A preview image  7  of the entire image field  19  is displayed on the monitor. Using the computer mouse, image areas  11 ,  13  of regions of interest  27 ,  29  are marked with cursor  9 . The marking is displayed in the form of bordering lines  15 ,  17 . 
     FIG. 2 schematically depicts the shape of the scanning track, which is made up of partial scanning tracks  21 ,  23 , and  25 . Partial scanning tracks  21  and  25  are caused by a fast, resonant beam deflection device, whereas partial scanning track  23  is caused by a beam deflection device for specific positioning. Region of interest  27  is traversed by partial scanning track  21 , whereas region of interest  29  is traversed by partial scanning track  25 . Scan fields  31  and  33  are consequently scanned sequentially. Partial scanning track  23  is swept because of the displacement of scan fields  31 ,  33  using the second beam deflection device for specific positioning. 
     FIG. 3 schematically shows a confocal scanning microscope. Light beam  37  coming from an illumination system  35  is reflected from a beam splitter  39  via deflection mirror  41  to first beam deflection device  43  for specific positioning, which contains a gimbal-suspended scanning mirror (not shown) and serves to displace the scan field in a plane perpendicular to the illumination direction. By way of an intermediate image using an imaging optical system  47  that generates an intermediate focal plane  45 , light beam  37  arrives at second beam deflection device  49  which is embodied as a K-mirror (see DE 196 54 210) and contains two resonant galvanometers (not shown). Second beam deflection device  49  provides meander-shaped scanning within the scan field. The size of a scan field can be varied by defining the deflection angle of the galvanometers. Light beam  37  is consequently guided over or through sample  53  via scanning lens  55 , optical system  57  and through microscope optical system  51 . In the case of nontransparent specimens  15 , light beam  37  is guided over the specimen surface. In the case of biological samples (preparations) or transparent samples, light beam  37  can also be guided through sample  53 . This means that different focal planes of sample  53  are successively scanned by the focus of light beam  37 . Detected light  59  proceeding from sample  53  arrives, on the reverse light path via first and second beam deflection devices  43 ,  49 , back at beam splitter  39 , passes through it, and is then detected with detector  61 . Detector  61  comprises a photomultiplier that converts the detected light data into electrical signals. Subsequent assembly of the signals and allocation to the respective scan positions then yields a three-dimensional image of region of interest  27 ,  29  of sample  53 . Illumination pinhole  63  and detection pinhole  65  that are usually provided in a confocal scanning microscope are shown schematically for the sake of completeness. Certain optical elements for guiding and shaping the light beams are omitted, however, for better clarity; these are sufficiently known to those skilled in this art. 
     FIG. 4 shows a confocal scanning microscope having a first beam deflection device  67  for specific position. Second beam deflection device  72 , which is constituted by a first and a second beam deflection module  71  and  75 , is provided for rapid scanning of the scan field. The two beam deflection modules  71  and  75  can, for example, each contain a resonantly oscillating micromirror. Imaging optical systems  69 ,  73  are provided between first and second beam deflection devices  67  and first and second beam deflection modules  71  and  75  to maintain the telecentric principle. 
     FIG. 5 shows a confocal scanning microscope having a total of four beam deflection modules  77 ,  79 ,  81 ,  83  that are distributed respectively among two beam deflection devices  78  and  82 . Beam deflection modules  77  and  81  deflect in the X direction, whereas beam deflection modules  79  and  83  deflect in the Y direction. Beam deflection device  78  serves to position scan field  31 ,  33 . It contains, for example, galvanometers that operate non-resonantly. Beam deflection device  82  operates resonantly, and serves to scan scan field  31 ,  33  in meander-shaped fashion. Beam deflection modules  77 ,  79  and  81 ,  83  are arranged close to planes  89  and  87 , respectively, that are conjugated with the focal plane of microscope optical system  51 . Imaging optical system  85  is provided to maintain the telecentric principle. 
     FIG. 6 shows a further exemplary embodiment of a confocal scanning microscope. Here it is the gimbal-mounted mirror  91 , driven in non-resonant fashion, that serves to position scan field  31 ,  33 . In order to maintain the telecentric scanning principle, mirror  93  that deflects in the Y direction and is driven by a resonant galvanometer, and mirror  95  that deflects in the X direction and is driven by a resonant galvanometer, are arranged close to plane  99  conjugated with the focal plane of microscope optical system  51 . Imaging optical system  98 , which is made up of optical systems  97  and  101  and generates at the rotation point of mirror  91  a further plane  99  conjugated with the focal plane of microscope optical system  51 , is located between mirrors  93  and  91 . In this exemplary embodiment, second beam deflection device  94  comprises mirrors  93  and  95 . 
     First beam deflection device  43 ,  67 ,  78  and/or second beam deflection device  49 ,  72 ,  82 ,  94  contain, inter alia, a gimbal-mounted mirror or a micromirror or an acoustooptical deflector or a galvanometer mirror or a resonantly oscillating mirror system. 
     The present invention was described with reference to a particular exemplary embodiment. It is, however, self-evident that changes and modifications can be made without thereby leaving the range of protection of the claims recited hereinafter. 
     Parts List 
     1 PC 
     3 Monitor 
     5 Cursor controller 
     7 Preview image 
     9 Cursor 
     11 Image area 
     13 Image area 
     15 Bordering line 
     17 Bordering line 
     19 Image field 
     21 Partial scanning track 
     23 Partial scanning track 
     25 Partial scanning track 
     27 Region of interest 
     29 Region of interest 
     31 Scan field 
     33 Scan field 
     35 Illumination system 
     37 Light beam 
     39 Beam splitter 
     41 Deflection mirror 
     43 First beam deflection device 
     45 Intermediate focal plane 
     47 Imaging optical system 
     49 Second beam deflection device 
     51 Microscope optical system 
     53 Sample 
     55 Scanning lens 
     57 Optical system 
     59 Detected light 
     61 Detector 
     63 Illumination pinhole 
     65 Detection pinhole 
     67 First beam deflection device 
     69 Imaging optical system 
     71 First beam deflection module 
     72 Second beam deflection device 
     73 Imaging optical system 
     75 Second beam deflection module 
     77 First beam deflection module 
     78 First beam deflection device 
     79 Second beam deflection module 
     81 First beam deflection module 
     82 Second beam deflection device 
     83 Second beam deflection module 
     85 Imaging optical system 
     87 Conjugated plane 
     89 Conjugated plane 
     91 Mirror 
     93 Mirror 
     94 Second beam deflection device 
     95 Mirror 
     97 Optical system 
     98 Imaging optical system 
     99 Conjugated plane 
     101 Optical system