Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0036—Scanning details, e.g. scanning stages
  • G02B21/0044—Scanning details, e.g. scanning stages moving apertures, e.g. Nipkow disks, rotating lens arrays
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0072—Optical details of the image generation details concerning resolution or correction, including general design of CSOM objectives
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
  • G02B21/365—Control or image processing arrangements for digital or video microscopes
  • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
  • G02B21/368—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements details of associated display arrangements, e.g. mounting of LCD monitor
• • H—ELECTRICITY
  • H04—ELECTRIC COMMUNICATION TECHNIQUE
  • H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
  • H04N25/00—Circuitry of solid-state image sensors [SSIS]; Control thereof
  • H04N25/40—Extracting pixel data from image sensors by controlling scanning circuits, e.g. by modifying the number of pixels sampled or to be sampled
  • H04N25/46—Extracting pixel data from image sensors by controlling scanning circuits, e.g. by modifying the number of pixels sampled or to be sampled by combining or binning pixels
• • H—ELECTRICITY
  • H04—ELECTRIC COMMUNICATION TECHNIQUE
  • H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
  • H04N25/00—Circuitry of solid-state image sensors [SSIS]; Control thereof
  • H04N25/50—Control of the SSIS exposure
  • H04N25/53—Control of the integration time

Definitions

• Microscope with several microscope objectives of different magnification factors can be used for rapid acquisition and display of a sample at different magnifications. Depending on the desired resolution or magnification, the appropriate microscope objective is automatically selected, and the sample is recorded in the desired field of view in the desired magnification and displayed on a screen.
• microscopes with such an interchangeable lens are relatively expensive. In order to use a digital microscope with only a single lens, it must be equipped with a high magnification factor, in order to allow a greatly enlarged shot of the sample in the field of view in the first place. If a lower resolution is desired by the physician, the sample must first be scanned through a variety of high-resolution images, so that they can be assembled picture by picture.
• a viewing section of the sample is selected, a microscope objective is moved in a scanning route over this viewing section, first a sequence covering the section offset from one another images are digitally taken and displayed, and then the scan route continues with a sequence of images outside the selected field of view.
• the invention is based on the consideration that a doctor - or, more generally: an operator - may take a while to examine the sample in the field of view before moving the field of view. This time can be used to record the sample also for the images outside the selected field of view. If the operator selects a different field of view, this may at least partially already be recorded, so that the field of view can be displayed on the screen without any time delay. As a result, the sample can be examined relatively quickly with only one microscope objective.
• a viewing section of the sample is selected.
• the viewing section may be an area within which an operator may see one or more images of the sample offset from each other after the images have been taken.
• the viewing section is expediently smaller than the sample.
• the sample may be a unit that is placed in a test fixture of a digital microscope.
• the sample may have an examination area and a support area.
• the examination area is expediently the area which is intended for microscopic examination and in which, for example, biological material to be examined can be arranged.
• the examination area may be the area covered by a cover glass.
• the carrier region is usually located outside the sample area and may contain a code area in which, in particular, machine-readable information about the sample is available, such as a sample name, a sample type, a type of examination, a sample origin, a tissue type and / or other information.
• the viewing section is expediently located in the sample area.
• the selection of the visible section can be automatic or manual.
• the viewing section can be automatically selected by a predetermined algorithm on the basis of, for example, a sample surface, a sample outline and / or a sample shape from which a partial region is determined according to the algorithm
• Viewing section is selected.
• the viewing section is selected in an automatic selection depending on machine-readable information on the sample, for example a barcode on the carrier area of the sample.
• abstract operator information can be information which does not directly determine the field of view, that is to say contains no coordinates or the like.
• the algorithm contains instructions that associate a visual clipping with the abstract operator information.
• the abstract operator information is, for example, an examination method, wherein the field of view is predefined directly in terms of its position or appearance, depending on the examination method. It is also possible to automatically select the viewing section based on a sample parameter. If the sample parameter determines a type of tissue, such tissue can be generated by automated web recognition are localized. The viewing section can then form a partial area of the sample surface in which the tissue lies.
• the operator is shown on a display means an overview image of the sample area or of the entire sample.
• the sample area is also referred to simply as a sample, even if it means only that area which is intended to be picked up by a microscope objective.
• a scan route can be determined which lies at least partially in the field of view.
• their beginning is in the field of view.
• the scan route is determined as a function of the position of the visible section in the sample.
• a lens in the scan route is moved over this field of view, expediently after the scan route has been determined, wherein the movement is a relative movement to the sample, regardless of which element rests in absolute terms.
• the objective is expediently a microscope objective, that is to say an objective with m> 1, in particular m> 5.
• the microscope objective expediently has a larger magnification than the first objective - if this exists at all.
• the digital microscope for carrying out the method according to the invention has only a single microscope objective, that is to say a magnifying objective with m> 1. It is cost effective if the microscope objective has a pre-set and fixed magnification.
• the images taken by the microscope objective can be displayed to the operator, who can now view the sample in the field of view at a higher resolution than would be possible using the overview image.
• the scan route can be continued outside the selected field of view with a sequence of images, even without the viewing section being moved or changed.
• the recordings can now be used in various ways.
• the images are not displayed to the operator as long as the operator does not move the clipping by continuing the scan route. Only when the view section is moved over the continued scan route, the images of the continued scan route are now displayed in the field of view.
• the operator is shown the position of the recorded images of the scanning route which lie outside the current viewing section.
• that has Display means a region in which the sample is displayed as a whole or a smaller portion of the sample, but which is greater than the field of view, for example, only as a frame of the sample.
• Each captured image can now be displayed as a point or area according to its location on the sample. The operator can recognize which area of the sample has already been picked up with the microscope objective. If he has the choice, he can now move the field of view specifically in such an area that contains already recorded images.
• Another possibility is to display the newly acquired images as images, e.g. next to the display field of the visible section.
• images e.g. next to the display field of the visible section.
• Each figure is covered by the following figure, so that the figures outside the field of view are only briefly shown. But this may be enough to give the operator the opportunity to recognize interesting images as such. He can then move the field of view targeted on such pictures.
• the representation of one or more recursive images on a screen is expediently such that it has a lower resolution than the image itself.
• the recording takes place with a higher optical resolution, as requested by the viewer and / or this is displayed on the screen.
• it is additionally possible to increase the detailing of the representation ie to zoom in on the representation of the sample until the resolution of the representation reaches the resolution of the image.
• the field of view of the sample on the screen comprises several images, these are combined to form an overall image that completely fills the viewing area.
• the operator can now be shown all or part of this composite image.
• the viewing section is expediently displayed on a display unit.
• a screen is called simplifying, without any limitation of the invention would be connected.
• the representation of the field of view on the screen may be composed of one or more images of the sample taken at the location of the field of view of the sample.
• the field of view of the microscope objective can continue to travel beyond the sample to capture further images outside the field of view, so that the field of view at the time of presentation need not coincide with the field of view of the microscope objective on the sample.
• the representation of the field of view is therefore usually not a live presentation, but uses one or more stored images back. Nevertheless, the presentation of images on a screen can be done in real time, ie immediately after capture. Thus, there is no need to wait until the entire sample is covered by images, which are then displayed - assembled into an overall image.
• the display is expediently carried out in real time, so at the moment of the first representation of a figure, a live representation of this image takes place.
• this image is kept on the screen as the field of view of the microscope objective continues to move and the subsequent ones Illustrations created.
• the previously recorded image is then no longer live presentation, for their representation is resorted to a non-volatile memory.
• the scanning route ie the movement of the microscope objective over the sample and thus the position of the sequence of the images of the sample, is expediently determined by a stored algorithm and may depend on one or more parameters which the algorithm incorporates.
• There are a variety of parameters that may affect the course of a scan route across the sample particularly sample parameters, acquisition parameters, and environmental parameters.
• the sample parameters primarily determine the initial scan route, ie the route first traveled by the microscope objective.
• the acquisition parameters and environmental parameters are primarily relevant for a change in an already partially traveled scan route.
• a sample parameter is taken into account in the calculation of the initial scan route, it is possible to achieve an efficient recording of the sample with regard to the intended examination, so that a rapid examination of the sample is made possible.
• the above object is also achieved by a method for digitally recording a sample through a microscope, in which a sample parameter is determined according to the invention, a scan route is determined as a function of the sample parameter and a microscope objective is moved along the scan route over the sample.
• Sample parameters may be determined by the nature of the sample, ie an outer shape of the sample, an arrangement of one or more sample areas on a sample carrier, for example as a micro-array or as a slide in a large area and / or a thickness of the sample through which Number of scan layers lying one above the other in the z-direction is determined. Sample parameters may also be determined by a type of tissue in the sample, by an assay method by which the sample is to be assayed, by a patient ID or patient class from which the sample is derived, by pathological information about the sample, by a color of the sample, and or a color adjustment that can be additive, ie a colored illumination, or subtractive in the form of a color filter in a imaging beam path.
• Changes in the course of the scan one or more parameters is advantageously processed immediately after the change, ie in real time, the algorithm, expediently in the way that the scan route is changed or recalculated depending on the change. This can already be done while the microscope objective is being moved along the old scan route. The old route can be canceled and continued with the new one.
• Changing the route in response to a parameter change can significantly speed up a sample examination.
• the above object is also achieved by a method for digitally recording a sample through a microscope, in which a scanning route for moving a microscope objective is determined and the microscope objective is moved along the scanning route over the sample. It is proposed that, according to the invention, a parameter is changed already during the movement of the microscope objective along the scanning route over the sample, the scanning route is at least partially redetermined and thereby changed as a function of this, the movement of the Microscope lens along the old scan route aborted and continued along the new scan route.
• the scanning process can be adapted directly to the specifications of an operator, whereby a study of the sample is possible quickly.
• Parameters which expediently lead to a change of an existing scan route are, for example, acquisition parameters. If a recording parameter is changed, the scan route is expediently aborted and transferred to a new one.
• a recording parameter can be changed by an operator input.
• a change in the acquisition parameter can be done by moving the field of view over the sample, changing the detailing of the representation of the sample on the screen, ie changing the zoom level when viewing the sample, changing the depth of focus in the sample, changing the color by filtering or lighting and / or changing the exposure time of the individual images.
• an operator inactivity leads to a change of an existing scan route. If there is no operator input for a predetermined period of time, the scan route may also be changed, e.g. in longer straight lines without changing direction to reduce a scan sound. With the same advantage scanning can be slowed down.
• the digital microscope expediently comprises an acceleration sensor, which is signal-technically connected to the control unit for controlling the scanning process.
• a temperature change above a threshold value, in particular a limit value per time, can also be critical for high-quality images since the material can expand the focus in the sample due to material expansion. Accordingly, it is advantageous if the scan route is changed with a change in temperature above a limit, for example, a new autofocus process is performed and the scan route is then moved to a new autofocus level.
• the temperature may be a temperature at or about the microscope objective or another component within or on the digital microscope.
• Also relevant to the scan route is a result of taking one or more images. For this a contrast analysis can be performed during the scan. If the contrast provides sufficient indication that the focus is too inaccurate, an autofocus process may be performed and the scan route may be relocated to a new autofocus level, depending on the result.
• a test image is taken in a deviating from the current scan plane scan plane and its contrast is evaluated. Depending on the results, a new test image is taken, or the scan route is moved to a level where the test image is located.
• an autofocus process is carried out during the scanning process, that is to say the scanning of the current scanning route.
• x and y coordinates of one or more autofocus points can be set, which are then approached by the microscope objective during the scan. Then an autofocus will be done there.
• the autofocus plane is shifted, ie the plane in which the focus is located during the scan as a result of the autofocus method. It is also advantageous to set the scan route either initially or subsequently so that the autofocus points are approached quickly, so that the calculation of the autofocus can start as early as possible.
• a recording parameter influencing the scanning route is, for example, the position of the viewing section on the sample. If the viewing section covers the sample only partially, ie beyond the edge of the sample, then the scanning route is expediently limited to the overlap area of the viewing section with the sample.
• the area of the sample is conveniently the area of a microscopy area, e.g. the area that can contain tissue.
• a sample carrier such as a glass plate containing the sample, may go beyond that. If a field of view has been selected by an operator, the recorded images partially or completely cover the field of view. Depending on the size of the selected view, it can already be completely covered by a single image.
• the entire field of view is composed of several juxtaposed images.
• the images are offset from each other and may partially overlap one another to facilitate stitching the images to a larger overall image, that is, automated composition based on image content comparison of adjacent images in their coverage area.
• a displacement of the images can also take place in the vertical direction, ie in the z-direction, if, for example, several images lie in different focal planes with one another or one above the other, ie are offset from each other in the z-direction.
• the images are formed by moving the field of view of the microscope objective relative to the sample and imaging the sample in the positions of the field of view offset by the movement.
• the microscope objective can be at rest at the time of recording or, in particular, can be continuously moved further, even at very short exposure times, without a disturbing smear occurring in the images.
• the movement of the microscope objective relative to the sample and thus also the selection of the position of the images is expediently controlled by the algorithm as a function of the size of the selected visual excision in the sample.
• the field of view is displayed on the screen so that the sample can be viewed by an operator of the digital microscope.
• the representation of the images lying outside the field of view expediently does not occur at this point in time until the visible section is moved into these already recorded images. If the viewing section is moved into a region of the sample which has not yet been completely or partially recorded, the part of the area of this visible section which has not yet been recorded will be immediately afterwards added to the shift or even during the shift.
• the course of the scan route depends on the movement of the field of view.
• an operator may specify the field of view, for example, from a previously acquired overview image of the sample, or the field of view may be determined by an algorithm, for example, depending on the type of sample.
• an overview image of the sample is initially produced, in which the entire sample area or the entire sample field of the sample is displayed. Based on this overview image, the operator can select the viewing section that he would like to see first. This is done, for example, by the operator marking the region of interest in the overview image of the sample with a marking agent, for example a mouse. The marking can be done by creating a window or by marking a point in the overview image of the sample. The field of view can now be placed in or around the marking, for example in the size of the marked window or in a preset size, in particular symmetrically, around a marked point. The size of the field of view may be conveniently selected by the operator, for example by determining a geometric size, e.g.
• the size of the view on the screen may depend on the magnification you select.
• the size of the field of view is greater than the predetermined by the microscope objective size of a single image, so that the field of view is covered with a plurality of images. The scan route is now selected so that the view section is scanned and assembled picture by picture.
• a time in which the operator finds an area of interest from an overview image and selects it by a marker can be advantageously used by already defining a scan route initially and traversing image by image before the viewing area is selected.
• the scanning route is advantageously placed in the selected viewing section in such a way that it is guided outwards from the center of the field of view, in particular in a spiral shape.
• the first image therefore covers the center of the field of view, and the following figures are around the first image.
• the viewing window of interest to the operator is filled from inside to outside with pictorial content.
• a mostly quieter and faster variant is a meandering scan route, which is particularly suitable for fast image sequences, eg for short exposure times. Also advantageous is a scan route in response to a movement of the viewing window. If the viewing window has been moved in one direction, filling the new viewing window in this direction is the most ergonomic. For example, if the viewing window was moved to the right, the scan route could fill the viewing window vertically perpendicular to the right.
• the scan route continues outside the field of view in a spiral around the viewing section expanding outward.
• the sample is scanned with a plurality of individual images, that is to say with images or route parts, which are each completely surrounded by an image-free region.
• a larger area can hereby be scanned dot-by-dot, in order, e.g. To find tissue.
• Priority area may be an area of tissue or other substance to be studied.
• those areas of the field of view which have not yet been imaged are advantageously initially, in particular exclusively, taken up by the microscope objective.
• the scan route moves in the direction from the center of the field of view to the edge of the field of view.
• the route is optimized for the fastest possible scanning of the entire field of view while still partially scanned viewing section.
• the scan route outside the field of view it is advantageous to increase the scan rate, ie to achieve a larger number of images per time than was the case with the scan route within the field of view. This is usually achieved by long straight scan lines.
• the course of the scan route can be a compromise between fast scanning of a region, e.g. around the current field of view, and a high scan rate.
• the operator may change a parameter.
• a parameter change is a change in the magnification factor.
• the selected field of view will be in the area of the image of the overview image, whereby the field of view is assigned a higher magnification than the overview image.
• the sample can now be displayed in the area of the field of view with the resolution of the overview image, this is not the resolution desired by the operator, which is why the overview image is a non-current image.
• Another parameter change occurs when the focal plane is changed. The different image planes are now in z-direction below or above each other.
• Yet another parameter change occurs when a spectral region of the images is changed, for example in fluorescence microscopy.
• An image plane then has a different spectral range of the images than another image plane.
• a parameter change such that an exposure time or a color channel selection of the images has been changed. For example, in order to save time a sequence of images with a low exposure time has been taken, for example using digital image brightening, and if an image is to be taken in the same local position with a higher exposure time, this also represents a parameter change. because the previously taken picture is not up-to-date regarding its picture quality.
• a change in the scan route is described below as an image plane change in which the images of the old, inactive scanning route lie in a different image plane, ie the new images of the current scan route.
• An image plane may here be referred to as a location or an area in a multi-dimensional parameter space, in which each parameter takes on a dimension. If a parameter or, strictly speaking, a parameter value is changed, the image plane shifts in the parameter space.
• the scan route within or outside the field of view may be guided independently of or otherwise from already recorded images of an inactive image plane. If, for example, the depth of focus in the sample is changed, it may make sense to replace blurry in-focus images with new sharp ones.
• the new image plane can then be recorded separately and independently of other image layers. In this case, however, images of another image plane can also be displayed so that current images and inaccurate images of the visible section are displayed side by side. As a result, the operator can orientate himself faster on the sample or in his field of view.
• the inactive recording was recorded in a different spectral range than the sequence of the current images, for example by a spectral filter or another recording mode, such as a bright field recording in comparison to a fluorescence recording.
• a spectral filter or another recording mode such as a bright field recording in comparison to a fluorescence recording.
• the in-focus shot was taken with a different focus position in the sample.
• the representation of the in-focus image is distinguished from the representation of the current image such that a distinction is made between the in-state image and the current image Illustration is possible. This can be done by coloring the unsuitable areas, obscuring or other marking.
• an algorithm can calculate a probability that a recording parameter will be changed next, especially during the current scan. It is calculated, for example, where the field of view in a fixed time window is moved to the corresponding probability range of the sample.
• Another possibility is to set the scan route based on constraints that are given.
• information about the type of sample is obtained from an image of the sample and the scanning route is selected outside the selected field of view as a function of the type of sample.
• different tasks of the operator or doctor for analyzing the sample can be set, from which a movement of the visual excerpt can be predicted with sufficient probability.
• Information about, for example, the nature of the sample can be obtained from a label on the sample, conveniently a machine-readable code, such as a barcode.
• a further advantageous possibility for detecting the type of sample lies in a tissue recognition in an image of the sample.
• Image recognition allows more meaningful areas of the sample to be distinguished from less meaningful areas, such as areas with more tissue from areas with little or no tissue. In this way, areas can be classified into higher classified areas and lower classified areas. Also from the type of tissue can be closed on more or less interesting areas, ie higher or lower classified areas.
• a tissue recognition can take place based on an image of the sample, for example a previous image, an in-current image and / or an overview image of the sample. Now the scan route is expediently controlled outside the field of view as a function of tissue recognition results.
• the scan route first moves to areas of higher classification and then to areas of low classification, for example first areas with recognized tissue and then tissue-free areas.
• Certain types of specimens may include analysis instructions that require an operator to perform a scan of a specimen.
• a typical example is a screening in which a doctor looks at the entire sample along a meandering screening route, for example.
• Such a screening route can be scanned in advance by always covering such areas of the screening route with pictures in which the field of view is not yet.
• the field of view moves next, and the scan route outside the field of view is guided as a function of the calculation result.
• an earlier movement of the field of view by an operator can be taken into account.
• the type of sample is also or alternatively included in the calculation.
• the type of sample may be entered by an operator or optically determined by a control unit of the digital microscope.
• a prediction of a future position of the field of view can also be improved by determining a character of the sample in the current field of view and approaching character-like areas of the sample from the scan route in front of dissimilar areas.
• the character of the sample in the current field of view may be included, and the calculation also incorporates character similarities of regions of the sample from other areas of the sample into the calculation.
• tissue recognition the sample is searched for given tissues or image contents so that regions with recognized tissue can be distinguished from tissue-free regions. It is even more accurate when different types of tissue or image content types are distinguished and preferably a certain type of tissue is approached.
• tissue type is used below to refer to a type of sample or structures / image contents in general.
• the tissue type can be determined by the choice of the position of the visual excision in the sample.
• a type of tissue can be determined in the field of view, and this type of tissue can determine preferred scanning areas insofar as they contain such a type of tissue, possibly with a predetermined edge area around the tissue type areas found.
• a field of view is selected by an operator or automatically selected by a control unit of the digital microscope, which is significantly larger than a single image of the microscope objective whose magnification is thus significantly lower than the magnification of the microscope objective, the scanning of the field of view can take a relatively long time, especially if this is repeatedly postponed.
• a pixel binning of a detector receiving the images takes place, ie merging a plurality of pixels into a common pixel for signal amplification. In this way, the exposure can be shortened and the image Fertilize can be done more quickly in a row.
• the images in this case have a reduced resolution, this is tolerable in a view section with a low magnification.
• a pixel binning is expediently carried out automatically, in particular as a function of a ratio of a resolution of the representation of the visual excerpt on a display means (zoom level) for the resolution of the recorded images. For example, if the ratio is less than a threshold, pixel binning may be performed automatically. Especially with a long exposure time, as with fluorescence images, pixel binning is advantageous. But even with a high level of user activity, pixel binning is advantageous if, for example, the user quickly shifts the field of view. However, if the field of view always remains long in several places, it may be possible to do without binning in order to achieve a better image quality.
• pixel binning can be performed automatically depending on zoom level, imaging channel and / or user activity.
• Pixel binning should generally also be understood to mean the selective readout of only a part of the total detector elements, even without the merging of detector elements.
• reduction of the exposure time without pixel binning may also be considered to increase an imaging speed.
• Subsequent image processing, for example enlargement of the contrast and / or brightening of the image makes it possible to make the otherwise dark images more analyzable. In this respect, it is advantageous if an exposure time of the images is selected as a function of a size of the field of view.
• a spectral channel selection of the images may increase the imaging speed depending on a size of the field of view. For example, it is only recorded in one spectral channel in order to first give the operator an overview of the viewed area of the sample.
• an autofocus method can be carried out, with which a suitable autofocusing of the microscope objective for the creation of the images is determined.
• a suitable autofocusing of the microscope objective for the creation of the images is determined.
• an autofocus depth or autofocus position is determined at a plurality of locations of the specimen, so that an autofocus plane can be laid through these points.
• the scan route expediently runs at least initially in the autofocus plane.
• the depth of focus can be understood as meaning a depth in the z-direction, ie perpendicular to the sample plane in which the focus of the microscope objective lies. The sample is now sharply focused in the depth of focus.
• An autofocus depth may be a depth of focus in which the focus has been adjusted by an autofocus method.
• An autofocus depth is expediently located in a material region of the sample which is to be examined.
• Several autofocus depths that are different in the x and / or y direction can form an autofocus plane, which is expediently parallel to the sample plane.
• the operator may still manually adjust the focus position at a high magnification selected by the operator to optimize for him or her. le picture of the sample to get.
• the scan route expediently initially extends in the depth direction around an autofocus depth. This is particularly expedient if the operator selects a zoom factor or an enlargement of the visible section above a limit value, for example over 10x. If, after the operator has changed the focus position of the microscope objective, the scan route is first
• the scan route runs in the plane of the adjusted focus depth.
• the operator will scan the sample at that depth of focus, so that a preliminary scan of that focal plane can speed up the examination.
• the scan route is parallel to an autofocus plane determined in an autofocus method.
• the scan route can again run horizontally, e.g. in the current focal plane of the microscope objective.
• the invention is also directed to a digital microscope, in particular for carrying out the method according to the invention.
• the digital microscope expediently has a sample receiver, a microscope objective, a drive for moving the microscope objective over the sample, a camera for taking the sample through the microscope objective, and a control unit for controlling the drive and for taking pictures of the camera.
• the control unit is also prepared to display the recordings on a display means, for example a screen. The screen can also be part of the digital microscope.
• control unit be prepared in accordance with the invention to move the microscope objective over a viewing section in a scan route, to digitally record a sequence of images offset from one another and then to display the scan route with a sequence of images outside continue the selected field of view.
• future displaced views can be displayed quickly.
• a movement of the microscope objective over the sample is a relative movement, so that, viewed in absolute terms, the microscope objective moves over the sample or the sample is moved under the absolutely stationary microscope objective.
• FIG. 1 shows a digital microscope with a microscope objective and an overview lens over a
• FIG. 2 shows the sample from FIG. 1 in a plan view with a sample field with two tissue regions, two information fields next to the sample field and a visual excision which lies above one of the two tissue regions,
• FIG. 3 shows the viewing section from FIG. 2 with scanning routes along which images of the sample are taken
• FIG. 4 shows the view detail from FIG. 2 with a coarse-dissolved outer and colored region and an inner region with higher resolution through four images
• FIG. 5 shows the sample field of the sample from FIG. 2 with a viewing section which is meandering over the sample along a screening route, FIG.
• FIG. 6 shows the sample field with a viewing section, which is guided over the sample, depending on the tissue
• FIG. 7 shows a view detail and an overview of a sample field in which already recorded images are recorded
• 8 shows a stack of images which were recorded with focal positions varying in the z-direction and a scanning route started at a desired depth of focus.
• Sample 1 shows a digital microscope 2 with a sample holder 4, in which a sample 6 is inserted.
• Sample 6 has a sample carrier 8 and a cover glass 10 and biological material arranged between sample carrier 8 and cover glass 10, as indicated in FIG. 2 in the plan view of sample 6.
• the cover glass completely covers the sample area, ie the area of the sample in which the material to be examined can be arranged.
• the sample area is also referred to below as sample field 48.
• a carrier area ie the area of the entire sample around the sample area.
• two information fields 40, 42 are arranged, which contain sample information.
• the sample holder 4 is movable within a housing 14 of the digital microscope 2, so that the sample 6 can be inserted outside the housing 14 in the sample holder and the sample holder 4 is moved by the drive 12 into the housing 14 and under a microscope 16 ,
• the microscope 16 comprises a microscope objective 1 8, which is shown only schematically in FIG. 1, and with which the sample 6 is imaged onto a matrix detector 20 of a camera 22.
• the microscope objective 18 is attached to a lens carrier 24 and can be moved two-dimensionally by means of a drive 26, as indicated by the two arrows in FIG.
• the image acquisition by the microscope objective 18 is controlled by a control unit 28 which also controls the drive 26 for moving the microscope objective 18 over the sample 6.
• the sample 6 can be moved under the fixed microscope objective 18, so that the drive 26 can be dispensed with.
• the digital microscope 2 is equipped with an overview camera 30, which has a detector 32 and an overview objective 34 for imaging the sample 6 on the detector 32.
• the overview camera 30 can likewise be fastened to the objective carrier 24 and moved into a suitable position over the sample 6, so that the sample 6 is taken in total by the overview camera 30.
• the overview camera 30 may be firmly fixed relative to the housing 14 in such a position that it can completely receive the sample 6 when the sample 6 has been brought by the drive 12 into its examination position within the housing 14.
• the images recorded by the overview camera 30 and the microscope camera 22 are displayed to an operator.
• an input unit 38 for example a keyboard and a mouse
• the operator can enter inputs and commands which are processed by the control unit 28, which controls the position of the microscope camera 22, for example.
• the information field 40 carries a machine-readable code, for example a bar code, which contains information about the sample 6, for example about the type of sample.
• the information Field 42 contains information in ASCII format, that is, with letters, numbers, and characters, from which the operator can obtain important information about Sample 6 for him / her.
• the digital microscope 2 is suitable for light and dark field analysis in reflected light and transmitted light and also for fluorescence analysis of the sample 6 and this prepared by appropriate lighting units, the representation of the sake of clarity in FIG 1 was omitted.
• the following section first describes bright field examination methods and then fluorescence examination methods.
• the sample 6 is inserted by an operator in the sample holder 4, which is located outside of the housing 14 of the digital microscope 2.
• the sample holder 4 is drawn into the housing 14 and moved by the drive 12 in the examination position.
• the operator can decide based on an input on the input unit 38, according to which method he would like to examine the sample 6.
• an overview image of the entire sample 6 including the information fields 40, 42 can optionally be recorded.
• the overview image is displayed on the screen 36 to the operator. Even with a fluorescence examination, it makes sense that an overview image is recorded in bright field or dark field.
• the sample holder 4 can accommodate a plurality of samples 6 side by side or with each other, so that multiple samples 6 can be examined in one operation with the digital microscope 2. It is also possible for a sample 6 to be divided into a plurality of sample regions arranged separately from one another, for example if it comprises a microarray with a plurality of small sample containers.
• the overview image can be an image of all samples 6 on the sample holder 4, or a separate overview image of each sample 6 is created, which is then displayed individually or together on the screen 36.
• the operator can then select in what order he wants to examine the samples 6.
• the operator selects a sample 6 for examination, for example, the sample 6 shown in FIG. 2.
• the control unit 28 determines, for example, a type of sample and an examination method, possibly patient data and / or pathological information. These sample parameters are used to calculate a scan route.
• the operator selects a region of interest by clicking on or marking it with the input unit 38.
• a viewing cutout 44 is produced.
• the viewing section 44 can also be preset, that is to say be selected automatically.
• An automatic selection is made by an algorithm which is executed in the control unit 28 on the basis of data provided for this purpose, for example the predetermined examination mode, which is explicitly or implicitly indicated on an information field 40, 42, for example.
• the viewing section 44 can be selected on an information field 40, 42 in the case of automatic selection as a function of machine-readable information. It is also possible to automatically select the viewing area based on a sample parameter.
• the algorithm includes instructions that associate a vision area 44 with such abstract operator information.
• General selection data may be the sample surface, the sample outline and / or a sample shape, ie the area, the outline and / or the shape of the sample field 48.
• the initial view section 44 always in the upper left corner of the sample field 48 or in its center.
• the viewing section 44 is displayed enlarged on the screen 36. Depending on the magnification of the visible section 44, the display section 44 is displayed with image data of the overview image or with images taken by the microscope objective 18.
• the microscope objective 18 Before taking the images through the microscope objective 18, it makes sense to set a suitable focus position of the microscope objective 18 in the sample 6. This can be done, for example, using an autofocus method.
• the autofocus method is performed autonomously, for example, after the overview image is taken and displayed on the screen 36 to the operator.
• the microscope objective 18 moves over the sample 6 and moves in the z-direction 46, ie in the depth direction of the sample 6 and perpendicular to the sample plane of the sample 6.
• the distance of the biological material to the microscope objective 18 or a focus position of the microscope 16 in the biological material are determined.
• This focus position is expediently carried out in the middle of the sample field 48, as indicated by a small cross in the center of the sample field 48 in FIG.
• the microscope objective 18 is moved in the x and y directions and the appropriate autofocus position is measured at several other locations of the sample field 48, as indicated for example by the four outer crosses in the sample field 48 of FIG. From the multiple autofocus positions an autofocus plane is calculated, which lies in the biological material.
• the autofocus plane is used as the starting plane for the first images of the sample material.
• a scan route 50 (FIG. 3) is calculated by the control unit 28.
• the selected field of view 44 of the sample 6 is displayed on the screen 36 with.
• the magnification of the visible section 44 may be predetermined, or the operator determines with the selection of the visible section 44 also the size of the visual excision or the optical magnification with which he wants to view the sample 6 through the viewing section 44.
• the resolution of the overview image it may be that the resolution of the overview image is sufficient to display the sample 6 in the preset or selected field of view 44 on the screen 36. Irrespective of this, the microscope objective 18 starts to travel over the sample 6 along the calculated scan route 50 and to record images 52 of the sample 6. If the resolution of the overview image is sufficient, the representation of the images 52 can be dispensed with. If the resolution is insufficient, the images 52 are displayed on the screen 36.
• An optional case distinction is shown in FIGS. 3 and 4.
• FIG. 3 shows the viewing detail 44 displayed on the screen 36.
• the image content comes from a previously recorded image of the sample 6, for example the overview image, and generally does not coincide with the current position of the microscope objective 18 above the sample 6.
• the overview image which, depending on the resolution, shows the sample 6 sharply resolved, as shown in FIG. 3, or shows a blurred image, as in the outer regions of the visible section 44 in FIG indicated by the thick lines.
• the control unit 28 controls the microscope objective 18 in a scanning route 50 over the sample and controls the taking of a sequence of images 52 through the microscope objective 18.
• the images 52 are shown in FIG This sequence is offset from each other within the viewing section 44, so that the sample 6 is taken within the field of view 44 Figure 52 for Figure 52 in the resolution or magnification of the microscope objective 18.
• the scan route 50 begins in the middle of the field of view 44.
• the first image symmetrically or asymmetrically covers the center of the field of view 44.
• the scan route 50 which is shown in FIG. 3 with dash-dotted lines or arrows, runs outward from the center in a spiral shape towards the edge of the viewing section 44.
• the microscope objective 18 travels over the sample 6 along the scan route 50 and picks up image 52 for illustration 52, as shown in FIG.
• the first image 52 is shown in solid lines, the second image 52 is dashed, and the third image 52 is dotted. Further illustrations are not shown for the sake of clarity. However, it can be seen that, over time, the entire field of view 44 is covered with images 52 in the order of the scan route 50, so that high-resolution image data of the sample are available in the entire field of view 44.
• the procedure is basically the same.
• the microscope objective 18 moves along the identical scan route 52 for image 52 over the sample 6.
• the in-focus image area of the field of view 44 shows blurred image details, while the sections of the images 52 of the field of view 44 are displayed sharply because they are from the image data of Figures 52. Accordingly, the sharp image area grows from the inside to the outside, as indicated in FIG.
• the inactive image sections are colored, for example with a gray tone. This is particularly advantageous if the resolution of the inactive image areas is still quite good, so that the image quality differences from the current to the inactive image sections are not immediately recognizable.
• the entire field of view 44 is covered with images 52 so that the image representation within the entire field of view 44 can be fed with actual images 52 or sharp image data.
• the scan can now be stopped and serviced until the viewing section 44 is moved by the operator to another area of the sample 6. Then also this view section 44 would be scanned image 52 for Figure 52, so that the image of the field of view 44 piece by piece composed in the current representation.
• this takes a certain amount of time in each shifting field of view 44 by the operator, it is advantageous if the current image data of a displaced field of view already exist at the time of the movement. For this purpose, an anticipatory scan is also required outside of the field of view 44.
• the scanning route 50 runs outside the field of view in a spiral around the viewing cutout 44 and expands radially outward, web-by-web, until the
• Limits of a region of interest or the sample field 48 are reached. If such a limit is reached, the scanning route 50 reverses and moves the next outer imaging row in the opposite spiral. If the viewing section 44 is now shifted a bit by the operator, current image data can be used and the viewing section 44 can be displayed currently. If the viewing cutout 44 has been displaced and placed over already taken pictures 52, these are displayed directly in the new view cutout 44. If the new field of view 44 is only partially occupied by existing images 52 and part of the field of view 44 is not yet occupied by images 52, the continuation of the new scan route depends on which part of the field of view 44 already exists with images and which does not.
• the scan route 50 continues in the middle of the visible section 44 so that it is filled outward from the center. If the center is already covered, then the new scan route 50 connects meander-shaped from the inside outwards to the existing images 52. This is true even if the center is not covered, but the existing images 52 reach the center to a predetermined distance, for example, down to less than 20% of a line-of-sight edge length. If all images 52 of the new visible section 44 are already taken after the movement, it is possible to dispense with a change in the scanning route so that it is not changed outside of the visible section 44.
• An actual display does not necessarily mean that a momentary view of the sample 6 is displayed. It is always resorted to stored images 52, even if the sample 6, for example, moved or taken out of the digital microscope 2. Currently, in this sense, it should be understood that the images 52 have been made in the way that the operator - or the control unit 28 in a default setting - has adjusted.
• the operator may change a variety of parameters such that a representation of the field of view 44 on the screen is changed. If the operator changes a parameter, this is registered by the control unit 28 and included in the calculation of the scan route 50. As a rule, the scan route 50 is influenced and changed hereby. This has the consequence that the departure of the current scan route 50 is stopped, and the microscope objective 18 now travels along the recalculated scan route 50 and picks up new images 52. If a parameter is changed, e.g. a capture parameter, the image plane changes and the previously acquired images 52 become inactive.
• the parameters may include the resolution or magnification, the depth of focus, an exposure, a pixel binning of the matrix detector 20, in the fluorescence analysis in particular the spectral range, and further parameters.
• the representation can be marked accordingly on the screen 36, for example by coloring.
• An exception may be a change in the magnification recording value when shooting at the same or greater magnification. For example, if you change from 10x to 20x, ie from 10x to 20x, and there are already 40x images, these images will remain current and they will only be displayed larger.
• the control unit 28 expediently selects one as a function of a future parameter.
• the future parameter may indicate with which probability the viewing section 44 is displaced at which location within the sample by the operator.
• the control unit 28 can carry out a calculation as to where the viewing section 44 next migrates and then control the scanning route 50 outside the visible section 44 as a function of the calculation result or the future parameter obtained therewith.
• one or more sample properties or one or more operator inputs may be used.
• Information about the type of sample 6 can be taken, for example, from an information field 40, 42, for example the barcode of the information field 40. give an examination of the type of examination with which sample 6 is to be examined.
• a screening route 54 may be connected to the sample type, as shown by way of example in FIG.
• FIG. 5 shows the sample field 48 of the sample 6 with tissue regions 56 and tissue-free regions around the tissue regions 56. If, for example, a complete screening of the sample field 48 is specified, the visual segment 44 automatically jumps to the beginning of the screening route 54 after a corresponding input by the operator systematically, for example meandering, is guided over the entire sample field 48. This is indicated in FIG 5 using the meandering dashed arrow.
• the operator moves the field of view, for example, with an operator, such as a mouse, along the screening route 54.
• the controller 28 controls the scan route 50 to extend outside the field of view 44 along the screening route 54. If the visible section 44 comprises only one image 52, the scan route 50 can run identically to the screening route 54.
• the scan route 50 may, for example, meander along the screening route 54, as indicated in FIG.
• the scan route 50 generally advantageously leads the movement of the viewing section 44 along the screening route 54, so that the viewing section 44 always moves into already existing images 52. In this way, it is possible to proceed until the viewing cutout 44 has moved over the entire sample field 48.
• the screening route 54 also passes through tissue-free areas that are not of great interest to the operator. Accordingly, it can be assumed that he will guide the viewing section 44 relatively quickly through tissue-free areas.
• tissue regions 56 there may be 6 regions of interest, such as the tissue regions 56, and non-interest or less interesting regions, such as the tissue-free regions in FIG. 5.
• a capture mode for controlling acquisition of the images 52 is dependent of the course of the screening route 54 and / or the scan route 50 through differently categorized areas such as a region of interest and a region of no interest. Illustrations 52 are performed faster in a lower categorized area than in a higher categorized area.
• the scan route 50 can remain the same here, ie be placed independently of the area categories, if appropriate, as for example in the case of a defined screening route 54. Otherwise, the scan route 50 can also be made dependent on area categories.
• the area categories can be created using image processing methods of a previously captured image of the sample 6, for example, based on an overview image. For example, tissue regions 56 are recognized as higher rated categorized regions and distinguished from low categorized regions, such as regions with no or other tissue.
• Acceleration of the acquisition of the images 52 can be achieved by one or more of the following measures.
• Channels or elements of the matrix detector 20 are combined by pixel binning so that sufficient exposure is achieved after a shorter exposure time.
• the exposure time can also be reduced without pixelbinning
• the brightness of the images 52 may be subsequently increased by image processing, such as by increasing image brightness, contrast, and / or other means.
• image processing such as by increasing image brightness, contrast, and / or other means.
• mappings 52 along the scan route 50. If, with sufficient probability, no region of interest or higher categorization lies in the corresponding image 52 or in its image field, then the corresponding image 52 can be dispensed with. In the case of a fluorescence image, the images 52 can be recorded in only one fluorescence channel, and the recording in other spectral channels is omitted.
• one or more of these imaging accelerating measures can be used even if the selected resolution or image magnification of the viewing section 44 is relatively low, or more precisely: at least to a preset level lower than the resolution or magnification of the microscope objective 18. In this case the magnification of the microscope objective 18 is not fully utilized anyway, so that one or more measures that impair the sharpness of the image can be made at the desired magnification without significant loss of quality in the representation of the images 52.
• an acceleration measure such as pixel binning, can be carried out automatically, ie triggered automatically, without the operator prescribing the measure. The measure is triggered in particular as a function of the zoom level of the current view section 44 on the screen 36. For example, if the zoom level is less than a threshold, the action can be triggered automatically.
• a user activity can also use a trigger parameter for an acceleration measure, such as pixel binning, for example the dwell time of the field of view 44 in one location.
• FIG. 1 Another possibility of an intelligent guidance of the scan route 50 via the sample 6 or its sample field 48 is shown in FIG. 1
• FIG. 6 shows the visible section 44 within the sample field 48, as selected by the operator or preset by the control unit 28.
• the control unit 28 performs a tissue recognition of this
• the tissue recognition can take place on the basis of the overview image, in which the tissue 56 is already shown.
• the scan route 50 outside the current field of view 44 is now performed prioritized within the higher-categorized areas 56.
• the scan route 50 will first pass across all higher-categorized areas before passing into a lower-categorized area.
• the scanning route 50 extends outward in a spiral around the viewing section 44 for reversing the spiral direction in the next outermost imaging path at range boundaries of the higher categorized region 56. This finally leads in a meandering path over the entire fabric area 56 until it is completely scanned.
• the scan route 50 jumps to the next tissue region 56 and rasterizes it in a meandering manner. If the field of view 44 is displaced by the operator, then it may be that it comes to rest over an area that has not yet been scanned.
• a scan route 50 within the viewing section 44 in dependence on a movement of the viewing section 44 is advantageous. If the operator drives the viewing window 44, for example, continuously or multiply in one direction, a meander-shaped scanning route 50 is selected instead of the spiral scan route 50, the propagation direction of which - analogous to the screening route 54 - is selected in the direction of displacement of the viewing section 44. In this way, a direction of propagation of the scan route 50, which may confuse the operator and which precludes the direction of displacement of the viewing section 44, is avoided.
• tissue recognition is not performed on the basis of an overview image, it is possible to run a random rapid scan over the sample field 48, in which individual images, ie images 52 spaced apart from one another, are produced. These island-like images 52 are now being examined for tissue 56. If tissue 56 has been found in a figure 52, additional images can now be appended to this "find" image 52 in order to image the tissue area, either immediately or only after the completed quick scan.
• a type of image such as a type of tissue
• the viewing cutout 44 not only lies in a tissue region 56 but also covers a special type of tissue, which is indicated in FIG. 6 by lines lying inside one another.
• the control unit 28 can select similar image categories or tissue categories from, for example, the overview image or another earlier image. Such areas now receive an even higher range category.
• the scan route 50 now passes hierarchically through the area categories. First, areas with the highest category are scanned. Then areas with the next lower area category are scanned and so on.
• the tissue region 56 covers the already scanned highest categorized region and only at the end the tissue-free region, in the event that the visual excision also partially covers it.
• the control unit controls the scan route 50 so that a field having at least the size and shape of the current view section 44 is scanned around the most categorized area, for example, 1.5 times the area size, as shown by a dotted line in FIG is represented by a possibly future viewing section 44. If the operator moves the viewing section 44 to the next highly categorized area, he can immediately display the entire field of view 44 without delay. Another future parameter is that earlier movements of the field of view 44 by an operator are taken into account. Previous movements may be directional or sectoral movements. Directional movements are, for example, screening directions. Area related moves are movements from one area to the next. For example, in earlier examinations of other samples, if the operator has preferred certain areas found by the control unit 28 in the current sample 6, those areas may be categorized higher than other areas so that the area categories can be used as future parameters.
• the scanning route 50 has been further scanned outside the visible cutout 44, ie a plurality of images 52 have been recorded outside the visible cutout 44. Instead of leaving the operator unaware of where these mappings 52 lie within the sample field 48, this may be indicated to the operator, as shown in FIG.
• FIG. 7 shows the view of the viewing section 44 on the screen 36.
• the sample field 48 is displayed in the form of a rectangle, in which the viewing section 44 is also reduced in size and displayed in its position within the sample field 48.
• the already recorded images 52 are displayed in their position within the sample field 48, so that the operator can recognize which areas of the sample field 48 have already been scanned. He can now preferably place the viewing section 44 over such areas so that it can be displayed completely or partially from already recorded pictures 52. With automatic recognition of prioritized areas, such as tissue areas 56, the images 52 will be laid over such areas, so that the operator additionally gets an indication of where the prioritized areas are located. As a result, he can shift the viewing section 44 in a more targeted manner and examine the sample 6 efficiently.
• the operator is thus the location of the recorded images 52 of the scan route 50 displayed, which are outside the current view section 40.
• Each recorded image 52 is displayed as a surface corresponding to its position on the sample 6 or the sample field 48. It is likewise shown in FIG. 7 that the respectively last recorded image 52 is displayed, for example next to the display field of the field of view 44.
• Each image 52 is covered by the following image 52 so that the images 52 recorded outside the field of view 44 are only briefly shown become. However, this is sufficient to give the operator the opportunity to know interesting images 52 as such.
• he has the option of stopping the sequence of illustrations in Figure 52 in order to be able to view the current Figure 52 for a longer time. However, the scanning continues unchanged, except that the recorded images 52 are no longer displayed until the operator cancels the display stop again.
• an autofocus method that may be performed provides an autofocus plane, it may be that the autofocus plane is not optimal with respect to the examination to be performed by the operator or the operator manually moves the focus position out of the autofocus plane for other reasons.
• Images 52 from the old focal plane then become in-picture mappings 52. They are expediently identified as such, for example colored, and the scan route 50 is newly defined in the current focal plane.
• the current focal plane expediently runs parallel to the autofocus plane.
• the scan route 50 is guided vertically by the control unit 28.
• a stack of overlapping images 52 is recorded, which are thus superimposed in the x and y directions and are spaced from one another by a predetermined distance in the z direction only.
• the height of the stack is determined by the control unit 28 and is for example a fixed number of images 52 on both sides of the autofocus plane.
• Other parameters, such as the position of the cover glass 10 or of a sample carrier 8 can also be taken into account so that the focus positions remain within the material to be analyzed. This process is shown by way of example in FIG.
• FIG. 8 shows a stack of images 52 superimposed in the z-direction, of which the bold-framed image 52 lies in the autofocus plane.
• the control unit 28 controls the recording of the images 52 above and below the focal plane, for example, three planes above the focal plane and four planes below the focal plane, as shown in FIG. All images 52 are conveniently located centrally in the current field of view 44. If the operator attempts to manually adjust the focus, he can focus through the previously recorded image stack and select the optimal focus plane without the central image 52 moving as the focus is moved must be recreated. This leads to a very pleasant adjustment of the focus. Only when the image stack has been completely recorded, a focus plane is searched out and the scanning route 50 in this focal plane running horizontally through.
• the scan route 50 extends spirally outward around the central image 52 as described for FIG. Illustrations 52 of the inactive focal plane can be shown here, but are expediently marked as inactive, as described in the example of FIG. 4.
• the scan is completed in the visible section 44 in the selected focal plane, for example the dashed plane of FIG. 8, it can be continued in an adjacent focal plane so that the scan route 50 thus jumps into an adjacent focal plane.
• the adjacent focal planes are indicated in dotted lines in FIG. If the focus is readjusted by the operator, it is possible to resort directly to already taken pictures and the viewing section 44 can be displayed immediately.
• the scan route 50 leaves the current image plane, for example to record one or more images 52 in an adjacent focal plane, the scan route in the sense of the invention leads out of the viewing section 44 since the current view section 44 is a two-dimensional view section 44 in the current image plane.
• Each image plane change of the scan route 50 ie focal plane change,
• the completion of the scan in a focal plane need not relate to the complete sample field 48. It is sufficient if the viewing section 44 is completely scanned so that it then jumps into the adjacent focal plane and the viewing section 44 is further scanned in this plane. Instead of the field of view 44, highly categorized areas may also be scanned first, jumping to a different focal plane when all the most categorized areas have been scanned. In the event that the operator has chosen a high magnification and consequently the image stack, as exemplified in FIG. 8, was created, but the operator has not actually moved the focal plane out of the autofocus plane within a predetermined time, then a scanning process or the Scan route 50 in a plane outside the focal plane can be aborted, and the scanning can be continued in the autofocus plane.
• the autofocus plane is to be preferred over other planes in the selection of the scan route 50.
• the following parameters can be taken into account or changed, wherein - if necessary, except for the changes caused by the fluorescence analysis - can be fully recourse to the above method steps from the bright field method.
• the digital microscope 2 contains one or more spectral filters 58, which can be introduced into the beam path 60 and limit the radiation to the desired spectrum.
• the beam path of the illumination or excitation expediently takes place at least between microscope objective 18 and sample 6 in imaging beam path 60.
• a beam path which is coupled into the imaging beam path via a semitransparent mirror is expedient. possibly also in front of the microscope objective.
• the spectral filter or filters 58 are advantageously introduced into the illumination beam path 60 in front of the sample 6, so that the sample 6 is illuminated as gently as possible.
• Each spectral channel can be seen as an image plane, analogous to the focal planes, so that the number of possible image planes results from a multiplication of the number of spectral channels by the number of focal planes.
• an overview image in the transmitted light and / or bright field is expediently created in order to give the operator a first overview of the sample 6.
• an overview image can be produced in the fluorescence spectrum, wherein the scanning process is expediently initially limited to one spectral channel, in particular the channel of the most stable staining, e.g. the most stable DAPI staining.
• the scan route 50 can be selected both horizontally and vertically analogous to the bright field method.
• the selection of the visible section 44 can then take place, as described for the bright field method, by the operator, wherein the magnification can be preset, for example to 10x.
• the fluorescence analysis then presumably leads to a manual focus adjustment by the operator, so that initially the imaging stack is created in the z-direction, preferably in several fluorescence channels, in particular in all fluorescence channels.
• the operator can set an exposure time, in particular for all fluorescence channels, wherein the exposure time can be displayed in the field of view 44 for easier orientation, for example under the central image 52 in the field of view 44.
• the anticipatory scan can take place, for example as described above, wherein also several fluorescence channels the range categories can be used.
• the fluorescence channels can be categorized.
• all fluorescence channels can be scanned for range category by area category, ie the scan route 50 only changes the range or range category when the range has been traversed in all fluorescence channels.
• scan route 50 first pivots in the vertical z-direction and picks up the image stacks in all fluorescence channels before resuming the horizontal scan at a desired focal plane. Since area categorization may be more difficult in fluorescence mode than in bright field mode, it may be useful to perform the leading scan using a scan speed acceleration parameter such as shorter exposure time with or without pixel binning and / or skipping mappings along the scan route 50.
• the anticipatory scan can take place in all fluorescence channels, in each case using at least one acceleration parameter.
• the operator can look over the sample 6 relatively quickly and find the range of interest for him, for example an area with a particularly good coloration.
• the use of an acceleration parameter should, however, only take place at a magnification below a limit magnification, for example only up to 10x. If the operator adjusts the magnification, eg 20x, the continuous scan without acceleration parameter will be executed to provide the best image quality.
• parameter changes may occur that make a change to the scan route 50 meaningful. For example, if we shake the digital microscope, there is a clear risk that the images taken during the shake will be "blurred” and out of focus.
• the shock is registered by the control unit 28, which is connected to an acceleration sensor 62 For example, if a threshold is detected, the period of that high acceleration is detected, and all the shots taken during that period are repeated, so the scan route 50 is interrupted and resumed at an earlier route point.
• temperature fluctuations in the area around the microscope objective 18 are detected by the control unit 20 by means of a temperature sensor. If a temperature fluctuation rises above a predetermined threshold value, this triggers a check of the autofocus.
• the threshold value can refer to the temperature at which the autofocus was last performed. For example, in the winter we open a window in a laboratory where the digital microscope 2 is located and it quickly cools down, or sunlight suddenly falls directly onto the digital microscope 2 as a result of the solar movement, so that it suddenly heats up significantly, a critical temperature change can occur easily occur.
• the autofocus process may result in a new autofocus plane being selected, which will alter the depth of focus in the specimen. Again, this is a change in a shot parameter and results in an image plane change of the shots, so the scan route 50 is recalculated, aborted, and resumed.

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• 2016
  • 2016-06-15 DE DE102016110988.6A patent/DE102016110988A1/de not_active Withdrawn
• 2017
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  • 2017-06-12 JP JP2018566418A patent/JP2019527375A/ja active Pending

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