Autofocus method for a microscope, and system for adjusting the focus for a microscope

#This application claims priority of the German patent application 102 17 404.0-42 which is incorporated by reference herein.

#The invention concerns an auto focus method for a microscope.

#The invention also concerns a system for adjusting the focus for a microscope. In particular, the system concerns a microscope having: an objective arranged in a working position and a microscope stage, such that a relative motion can be generated in the Z direction between the objective and the microscope stage; a camera that is connected to the microscope in order to acquire images of a specimen; a microscope control device; and a computer that is connected via at least one connection to the microscope control device.

#Further advantages and advantageous embodiments are the subject matter of the description below of the Figures, in which, in detail:

#FIG. 1 schematically depicts the system for adjusting the focus for a microscope;

#FIG. 2 schematically depicts the method for determining the Z position having the optimum focus position;

#FIG. 1 schematically depicts a system 1 for adjusting the focus for a microscope 2. System 1 encompasses microscope 2, a microscope control device 4, a computer 6 in which a frame grabber (not depicted) is installed, and a display 8 having a keyboard 10. Microscope 2 comprises and automatic microscope (e.g. Leica DM LA having microscope control device 4. Microscope 2 and microscope control device 4 communicate via a special control cable 25. The configuration of microscope 2 will not be discussed in further detail. Only those components of microscope 2 that are of essential significance for the invention will be described further. Microscope 2 comprises a stand 12 on which a objective turret 14 is mounted. Objective turret 14 carries at least one objective 16. In its working position, objective 16 is provided opposite a microscope stage 18. Microscope stage 18 is movable in X direction X, Y direction Y, and Z direction Z. The focus position can be modified or arrived at by generating a relative motion between microscope stage 18 and objective 16 in the working position. For determination of the focus position, a video camera 20 is mounted on microscope 2 in such a way that the image of the specimen imaged through objective 16 is acquired with video camera 20. Video camera 20 is connected via a video cable 21 to computer 6. The signals acquired from video camera 20 are digitized by means of the frame grabber in computer 6. Computer 6 is connected via a first serial cable 22 to microscope control device 4. Computer 6 is additionally connected via a second serial cable 24 to microscope control device 4. Microscope control device 4 serves to control the automatic functions of microscope 2, for example Z drive motion (movement in Z direction Z), movement of the microscope stage in X and Y directions X and Y, selection of a suitable objective, and much more. Computer 6 and microscope control device 4 communicate via first serial cable 22. Video camera 20 is mounted on a photo tube 26 of microscope 2. Second serial cable 24 transfers trigger signals from computer 6 to microscope control device 4. These solutions trigger position determinations in the Z drive. The Z drive can bring about either a displacement of microscope stage 18 in the Z direction, or a displacement of objective 16 in the Z direction.

#For the description below, a displacement of the Z position is to be understood as a displacement of microscope stage 18 in Z direction Z. When the focus function is started with microscope control device 4, an arrival at starting position 26 occurs at the beginning of the focus function. The arrival at starting position 26 is performed from instantaneous position z0 to starting position z1=z0−r/2, where r is the search region (referred to as the focus capture region) in Z direction Z. As a rule, the focus capture region is a range on either side of the ideal focus position. Next a movement 28 of the Z drive (or microscope stage 18 ) of microscope 2 occurs at constant velocity. Movement 28 through the focus capture region is performed until a final position z2=z0+r/2 is reached. Acquisition of images 30, which is performed concurrently with motion of the Z drive, is accomplished by means of video camera 20. In this context, images are acquired continuously at the maximum possible frequency (e.g. 25 images per second). Digitization 32 of these images is performed by the frame grabber present in computer 6. A time scale 39 (arbitrary units) is also plotted in FIG. 2. It is evident from this that a determination 36 of the instantaneous Z position, movement 28 of Z drive of microscope 2, and acquisition of images 30 occur concurrently. It is also to be noted that during the entire time (from T=0 to T=E) until the suitable focus position has been set, microscope stage 18 is only minimally halted. Between the motion through the capture region (region within which the optimum focus position is located) and the subsequent motion to the focus position, computational determination of that focus position is performed (interpolation). An interruption of minimal duration occurs between the two motions.

#Computer 6 determines, on the basis of the image data, the contrast value of each individual image. In the determination of the contrast, it is not necessary to employ the entire individual image. As a rule, it is sufficient to limit the contrast determination to a central subregion of the individual image. A contrast calculation 34 is implemented in such a way that it is performed for an image concurrently with infeed of the image subsequent thereto. The maximum movement velocity of the Z drive is selected so that significant contrast values can still be obtained from the individual images. Trigger signals synchronized in time with the image acquisitions are sent from computer 6 via second serial cable 24 to microscope control device 4. Each trigger signal triggers, in microscope control device 4, a determination 36 of the instantaneous Z position. All the Z positions belonging to one measurement are stored temporarily in microscope control device 4.