WO2011145016A1 - Autofocus imaging - Google Patents
Autofocus imaging Download PDFInfo
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- WO2011145016A1 WO2011145016A1 PCT/IB2011/051963 IB2011051963W WO2011145016A1 WO 2011145016 A1 WO2011145016 A1 WO 2011145016A1 IB 2011051963 W IB2011051963 W IB 2011051963W WO 2011145016 A1 WO2011145016 A1 WO 2011145016A1
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- WIPO (PCT)
- Prior art keywords
- image sensor
- autofocus
- primary image
- pixels
- imaging system
- Prior art date
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- 238000003384 imaging method Methods 0.000 title claims abstract description 45
- 238000005070 sampling Methods 0.000 claims abstract description 24
- 230000003287 optical effect Effects 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims description 18
- 238000005286 illumination Methods 0.000 claims description 10
- 230000010354 integration Effects 0.000 claims description 5
- 238000001429 visible spectrum Methods 0.000 claims description 4
- 238000004590 computer program Methods 0.000 claims description 2
- 230000007170 pathology Effects 0.000 abstract description 5
- 230000006870 function Effects 0.000 description 7
- 230000005855 radiation Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000012120 mounting media Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
- G02B21/241—Devices for focusing
- G02B21/245—Devices for focusing using auxiliary sources, detectors
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- 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
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N23/00—Cameras or camera modules comprising electronic image sensors; Control thereof
- H04N23/45—Cameras or camera modules comprising electronic image sensors; Control thereof for generating image signals from two or more image sensors being of different type or operating in different modes, e.g. with a CMOS sensor for moving images in combination with a charge-coupled device [CCD] for still images
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N23/00—Cameras or camera modules comprising electronic image sensors; Control thereof
- H04N23/60—Control of cameras or camera modules
- H04N23/67—Focus control based on electronic image sensor signals
- H04N23/673—Focus control based on electronic image sensor signals based on contrast or high frequency components of image signals, e.g. hill climbing method
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N3/00—Scanning details of television systems; Combination thereof with generation of supply voltages
- H04N3/10—Scanning details of television systems; Combination thereof with generation of supply voltages by means not exclusively optical-mechanical
- H04N3/14—Scanning details of television systems; Combination thereof with generation of supply voltages by means not exclusively optical-mechanical by means of electrically scanned solid-state devices
- H04N3/15—Scanning details of television systems; Combination thereof with generation of supply voltages by means not exclusively optical-mechanical by means of electrically scanned solid-state devices for picture signal generation
- H04N3/1581—Scanning details of television systems; Combination thereof with generation of supply voltages by means not exclusively optical-mechanical by means of electrically scanned solid-state devices for picture signal generation using linear image-sensor
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N25/00—Circuitry of solid-state image sensors [SSIS]; Control thereof
- H04N25/70—SSIS architectures; Circuits associated therewith
- H04N25/701—Line sensors
Definitions
- the present invention relates to the field of digital pathology, notably.
- the present invention relates to an autofocus imaging system for a microscope, a microscope comprising an autofocus imaging system, a method for autofocus imaging of a microscope, a computer-readable medium and a program element.
- Line sensors may be used for scanning a whole tissue slide. These slide scanners may perform a continuous mechanical scanning, thereby reducing stitching problems and allowing for the use of so-called time delay integration (TDI) line sensors in order to accommodate for low brightness of the illumination.
- TDI time delay integration
- focusing focus maps may be used. Before the actual scanning the optimum focus position is determined at a number of positions on the slide. This results in a "focus map". This procedure may be necessary because the axial position of the tissue layer may vary with several micrometers across the slide, as may be seen in Fig. 1. The variation of the tissue layer may thus be more than the focal depth of the microscope objective. During scanning the focus position of the objection is set on a trajectory that interpolates between the measured optimum focus settings on the selected measurement locations. This procedure may be both prone to errors and be also time-consuming, thereby limiting the throughput of the system.
- WO 2005/010495 A2 describes a system and a method for generating digital images of a microscope slide, the microscope comprising a main camera and a focus camera which is tilted with respect to the optical axis.
- the performance of the autofocus function may be insufficient. It may be desirable to have an autofocus imaging system with improved performance.
- an autofocus imaging system for a microscope which comprises a primary image sensor and an autofocus image sensor.
- the primary image sensor is adapted for acquiring primary image data of an object of interest, such as a tissue slide.
- the autofocus image sensor is adapted for acquiring autofocus image data of an oblique section of the object of interest.
- the primary image sensor is further adapted for sampling a first number of pixels per distance in object space and the autofocus image sensor is further adapted for sampling a second number of pixels per distance in object space, wherein the second number is between one quarter and three quarters of the first number.
- the autofocus image sensor samples a smaller number of pixels per distance in object space than the primary image sensor.
- the computational load and also the sampling time may be reduced.
- the quality of the autofocus sensor signal may be optimized.
- the second number is half of the first number.
- the autofocus image sensor samples half the numbers of pixels per distance in object space in the primary image sensor.
- the primary image sensor assembly may comprise one line sensor or may comprise more than one line sensor, for example three or even more line sensors.
- Each line sensor may detect a different wavelength or wavelength range. For example, one line sensor may detect green light, a second red light and a third line sensor may detect blue light (only).
- the autofocus image sensor is tilted with respect to an optical axis of radiation from the object of interest towards the autofocus image sensor, e.g. tilted with respect to an optical axis of the primary image sensor.
- the position of the tissue layer on the sensor is a measure for the amount of de focus.
- the autofocus image sensor is adapted for acquiring the autofocus image data at a light frequency outside the frequency of the visible spectrum.
- the autofocus imaging system is adapted for dark field illumination of the autofocus image sensor.
- the object of interest may be illuminated with a beam comprising a set of directions of propagation, such that the angle of these directions of propagation is larger than the angle sub-tended by the detection aperture of the autofocus imaging sensor.
- a beam comprising a set of directions of propagation, such that the angle of these directions of propagation is larger than the angle sub-tended by the detection aperture of the autofocus imaging sensor.
- light reflected from various surfaces air, cover slip, cover slip- tissue layer, tissue layer-slide, slide-air
- all low object spatial frequencies may be blocked and only signal emanating from the tissue (which has sufficiently high spatial frequencies) may be detected at the autofocus image sensor. This may improve the robustness and accuracy that the axial position of the tissue layer can be measured.
- a microscope comprising an above and below described imaging system is provided.
- the microscope is adapted as a slide scanner for digital pathology.
- a method for autofocus imaging of a microscope in which primary image data of an object of interest is acquired by a primary image sensor, autofocus image data of an oblique section of the object of interest is acquired by an autofocus image sensor, a first number of pixels per distance in object space are sampled, the first number of pixels being pixels of the primary image sensor, and a second number of pixels per distance in object space is sampled, the second number of pixels being pixels of the autofocus image sensor.
- the second number is between one quarter and three quarters of the first number.
- a computer-readable medium in which a computer program for autofocus imaging of a microscope is stored which, when executed by a processor of a microscope, causes the processor to carry out the above and/or below described method steps.
- a program element for autofocus imaging of a microscope which, when being executed by a processor of a microscope, causes the processor to carry out the above and/or below described method steps.
- a computer-readable medium may be a floppy disk, a hard disk, a CD, a DVD, an USB (Universal Serial Bus) storage device, a RAM (Random Access Memory), a ROM (Read Only Memory) and an EPROM (Erasable Programmable Read Only Memory).
- a computer-readable medium may also be a data communication network, for example the Internet, which allows downloading a program code.
- the autofocus imaging sensor which may be a two-dimensional sensor, samples a smaller number of pixels per distance in object space and the primary sensor, which may be a line sensor or which may comprise more than one line sensors.
- the auto focus sensor samples half the number of pixels than the primary sensor.
- Fig. 1 shows a cross-section of a tissue slide assembly.
- Fig. 2 shows a tilted autofocus image sensor.
- Fig. 3 shows the effect of defocus on MTF.
- Fig. 4 shows the ratio of defocus MTF to zero defocus MTF.
- Fig. 5 shows a microscope with an autofocus imaging system according to an exemplary embodiment of the invention.
- Fig. 6 shows a microscope with an autofocus imaging system according to another exemplary embodiment of the invention.
- Fig. 7 shows a microscope with an autofocus imaging system according to another exemplary embodiment of the invention.
- Fig. 8 shows a microscope system according to an exemplary embodiment of the invention.
- Fig. 9 shows a flow-chart of a method according to an exemplary embodiment of the invention.
- the character prime (') associated to a symbol will mean that the image space is considered (e.g. sensor reference) while a symbol without prime character will mean that the object space is considered (typically the sample reference).
- a rotation in image space and, as will be described more specifically, a rotation of the physical sensor, will be indicated.
- an angle Beta ( ⁇ without prima) will indicate a rotation in object space, and as will be described more specifically a rotation of an oblique cross section of the sample that is imaged by the autofocus sensor.
- Fig. 1 shows a schematic cross-section of a tissue slide assembly, comprising a microscope slide 1 , having a typical thickness of 1 mm, a cover slip 2, with a typical thickness of 0,17 mm, a mounting medium 3 for fixing and sealing off a tissue layer 4.
- the tissue layer is typically around 5 ⁇ thick
- the mounting layer includes the tissue layer and is typically 10-15 ⁇ thick.
- the mounting medium may be applied to the slide with tissue layer in liquid form before a cover slip is attached to the slide, subsequently the mounting liquid solidifies, thus mechanically fixing the tissue layer and sealing it off from the outside environment in order to provide stability against deterioration.
- the axial position of the tissue layer may vary within several ⁇ across the slide.
- the focus may have to be adjusted continuously, since the axial position of the tissue layer varies.
- An alternative for the use of the "focus map"-method is the use of a continuous autofocus system, i.e. an additional system that continuously measures the optimum focus position and adapts the axial position of the objective lens during the actual scan for acquiring the digital image.
- the autofocus system may be based on optimizing the contrast in the obtained image.
- a variety of matrix may be used for contrast optimization.
- the sine of the focus error (above or below focus) can not be determined in this manner, i.e. the focus error signal is not polar. This may be disadvantageous for a continuous autofocus system that needs permanent updates on the optimum focus setting.
- the autofocus system may use the line reflected at a reference surface at or near the object plane, such as in optical disks.
- a drawback of this method when applied to tissue slides may be that the relevant interface (between microscope slide and tissue layer and between tissue layer and cover slip) may have a low reflectance and that the reflection signal is distorted by scattering arising from the nearby tissue layer, thus comprising robustness.
- This autofocus image sensor makes an image of an oblique section of the object, as depicted in Fig. 2. This section may cut through the tissue layer at some point depending on the axial position of the tissue layer or relative to the focal plane of the objective lens. In this way the position of the tissue layer on the sensor is a measure for the amount of defocus.
- the reader may refer to the European patent application N°09306350.
- the tilted autofocus image sensor makes an image of an oblique cross-section 5 of the tissue slide assembly. The tilt is in the scanning direction 6.
- the sensor has N x pixels and samples the object in the scan direction with ⁇ per pixel and in the axial direction with ⁇ per pixel.
- the autofocus imaging system operates using wavelengths outside the visible spectrum so as not to spoil the white light imaging of the tissue layer.
- the autofocus system operates using wavelengths on the infrared side of the visible spectrum, because ultraviolet radiation may damage the tissue and may require more complicated and/or expensive optical components than infrared radiation.
- the additional autofocus image may be provided by using a so-called dark field illumination.
- the sample is illuminated with a beam comprising a set of directions of propagation, as already described above.
- TDI-line sensor primary image sensor
- Such a TDI-line sensor records each object pixel L times, where the number of stages L can be typically up to 128. This has the effect that the total integration time, and hence signal level, increases by a factor L compared to a conventional single line sensor. This is used to increase the scanning speed of the system.
- a reasonable starting point in the design of such a system may entail having a resolution R af of the autofocus sensor approximately equal to the resolution Ri m of the (TDI- based) image sensor in order to be able to test the same level of sharpness in the image.
- the novel insight of the inventors is that this implies a problem with signal level on the autofocus image sensor as will be apparent from the following considerations. Taking a linear scan speed v the line rate of the image sensor is:
- i m is the image sensor (quantum) efficiency and T
- the signal level at the autofocus sensor is much smaller than the signal level at the image sensor.
- the autofocus sensor signal will be relatively noisy, which compromises the accuracy of the focus error signal.
- MTF Modulation Transfer Function
- Fig. 3 shows the MTF for the nominal in- focus situation 301 and for a case with defocus 302.
- the x-axis 303 depicts the normalized spatial frequency and the y-axis 304 depicts the MTF values.
- Fig. 4 shows the ratio 401 of the two MTF-functions 301, 302.
- the y-axis 403 depicts the MTF ratio; a minimum 402 can be observed the x value equal to 1.
- Both MTF- functions show a cut-off at 2 ⁇ / ⁇ (this is the so-called 'diffraction limit'), which is the ultimate resolution limit for a conventional microscope.
- the ratio of the two MTF-functions shows a dip for the middle spatial frequencies. From this analysis we may conclude that:
- the autofocus image sensor sampling (pixels/m in object space) is selected 3/4 to 1/4 or, e.g., at least a factor two smaller than the image sensor sampling. This gives a good compromise between defocus sensitivity and autofocus to image signal ratio.
- the beam splitter fraction ⁇ is adapted such that the autofocus sensor signal is sufficiently high compared to the image sensor signal.
- the parameter settings are such that the TDI-based line sensor has sufficient redundancy to maintain a sufficiently high image sensor signal, i.e. T
- the depth range Az tot of the autofocus system must be sufficiently large for realistic settings of other parameters.
- the autofocus image sensor has N x pixels in the scan direction, with pixel size b.
- the sensor is tilted over an angle ⁇ ' so that the lateral and axial sampling is given by:
- the lateral and axial sampling at the object (the tissue slide) is given where M is the magnification and n the refractive index of the object.
- the axial sampling at the object now follows as:
- Table 1 shows an example of parameter settings according to the invention.
- the autofocus resolution is 2x0.9 ⁇
- the image resolution is preferably about 2x0.25 ⁇ (taking a 20x/NA0.75 microscope objective).
- Fig. 5 shows part of a microscope and in particular the imaging branch of the light path.
- An embodiment for epi-mode dark field illumination is shown in Fig. 6.
- a colour splitter 22 splits off the white light which is imaged by a tube lens 23 onto the image sensor arrangement, which may comprise a first, a second and a third primary image sensor 24, 32, 33, which may be adapted in the form of line sensors, 24 for generating the digital tissue image.
- the infrared light is imaged by a second tube lens 25 onto the auto focus image sensor 26, which is tilted with respect to the optical axis 31 of radiation from the object of interest towards the auto focus image sensor 26.
- tilted with respect to the optical axis of the primary image sensor means that the radiation from the object of interest which impinges on the auto focus image sensor does not impinge on the autofocus image sensor perpendicularly.
- the radiation which travels from the object of interest towards the primary image sensor may impinge perpendicularly on the primary image sensor, although this is not required already described herein above.
- Rays scattered by the tissue can pass through the aperture 21 and are imaged onto the autofocus image sensor 26.
- Fig. 6 shows an optical layout for epi-mode dark field illumination of a microscope with an autofocus imaging system 500 having a laser diode 14 , the illumination being integrated with the imaging branch.
- Two crossed gratings 15 are arranged after the laser diode 14 for generating diffraction orders, for example a 0th diffraction order SO, a + l st order S'+i and a -1 st order S'_i .
- a field stop 16 is arranged close to the gratings 15 for limiting the width of the dark field illumination beams, and a collimator lens 17 the collimates the light from the laser diode 14.
- a polarizing beam splitter 28 is provided to split the beam after it has passed the collimator lens 17. Furthermore, the microscope comprises quarter- wave plate 29. Both elements 28 and 29 take care of directing the beam originating from the laser towards the objective lens and erecting the scattered light originating from the tissue towards the autofocus image sensor.
- Fig. 7 shows an optical layout for multi-spot illumination of a microscope 500, which illumination is integrated with the imaging branch.
- the lens 17 collimates the beam which is incident on a spot generator for generating an array of spots 30. By tilting the whole assembly, the spot array can be tilted so that the resulting incident spot array and the slide is tilted as well.
- the spot generator 29 generates an array of low-NA beams, which can pass the beam splitter 27 without introducing significant aberrations.
- an array of spots is used to illuminate the oblique section 5 that is imaged by the autofocus image sensor.
- the spots that are focused on the tissue may experience time-dependent scattering as the absorption and refractive index of the region into which the spot is focused changes with scanning.
- the axial position of a tissue layer may be located. Namely, close to focus the high resolution information is visible, away from focus this is blurred.
- the signal variations on a comparatively small time scale may be maximum when the tissue layer coincides with the focal plane.
- Fig. 8 shows a microscope system 802 comprising a microscope with an autofocus imaging system 500 connected to a processor or processing unit 800 which is connected to a user interface 801, such as a computer.
- Fig. 9 shows a flow-chart of a method according to an exemplary embodiment.
- primary and secondary i.e. autofocus image data of an object of interest are acquired by a primary image sensor and an autofocus image sensor, respectively.
- the pixels of the primary image sensor are sampled.
- step 903 (which can be before, after or at the same time as step 902) a certain number of pixels per distance in object space of the autofocus image sensor is sampled. This number is smaller than the sampled number of pixels of the primary image sensor.
- the focus of the microscope is adjusted based on the sampling.
- the focus of the primary image sensor may be adjusted automatically.
- the principles of the present invention may be advantageously applied to a sensor which the applicant of the present invention has already proposed under European patent application N°09306350, and which is hereby incorporated by reference.
- the primary image sensor and the autofocus image sensor may share a same sensing area.
- the primary image sensor and the autofocus image sensor may together form a unique sensor with a sensing area (typically formed of pixels) that is both used for autofocus and for image acquisition.
- the larger autofocus pixels may be either actual physical pixels located next to or intermixed in the array or arrays of primary image pixels, or the autofocus pixels may be virtual pixels obtained by combining two or more of the primary image pixels into a larger virtual autofocus pixel. Such a combination may be done on the sensor itself, or in a separate processing unit.
- the described autofocus system finds application in digital pathology and other fields of rapid micro scanning.
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Abstract
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Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR112012029152A BR112012029152A8 (en) | 2010-05-18 | 2011-05-04 | AUTOFOCUS IMAGE FORMATION SYSTEM FOR A MICROSCOPE, METHOD FOR AUTOFOCUS IMAGE FORMATION OF A MICROSCOPE, COMPUTER READABLE MEDIA AND PROGRAM ELEMENT FOR AUTOFOCUS IMAGE FORMATION OF A MICROSCOPE |
EP11723679A EP2572226A1 (en) | 2010-05-18 | 2011-05-04 | Autofocus imaging |
CN201180024191.XA CN102893198B (en) | 2010-05-18 | 2011-05-04 | Automatic focus imaging system, formation method and microscope |
JP2013510699A JP5809248B2 (en) | 2010-05-18 | 2011-05-04 | Autofocus imaging |
US13/697,681 US10061108B2 (en) | 2010-05-18 | 2011-05-04 | Autofocus imaging for a microscope |
US15/922,003 US10371929B2 (en) | 2010-05-18 | 2018-03-15 | Autofocus imaging |
US15/922,988 US10365468B2 (en) | 2010-05-18 | 2018-03-16 | Autofocus imaging |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP10305520.8 | 2010-05-18 | ||
EP10305520 | 2010-05-18 | ||
EP10305559.6 | 2010-05-27 | ||
EP10305559A EP2390706A1 (en) | 2010-05-27 | 2010-05-27 | Autofocus imaging. |
Related Child Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/697,681 A-371-Of-International US10061108B2 (en) | 2010-05-18 | 2011-05-04 | Autofocus imaging for a microscope |
US15/922,003 Division US10371929B2 (en) | 2010-05-18 | 2018-03-15 | Autofocus imaging |
US15/922,988 Division US10365468B2 (en) | 2010-05-18 | 2018-03-16 | Autofocus imaging |
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WO2011145016A1 true WO2011145016A1 (en) | 2011-11-24 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/IB2011/051963 WO2011145016A1 (en) | 2010-05-18 | 2011-05-04 | Autofocus imaging |
Country Status (6)
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US (3) | US10061108B2 (en) |
EP (1) | EP2572226A1 (en) |
JP (1) | JP5809248B2 (en) |
CN (1) | CN102893198B (en) |
BR (1) | BR112012029152A8 (en) |
WO (1) | WO2011145016A1 (en) |
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US10330910B2 (en) | 2013-04-26 | 2019-06-25 | Hamamatsu Photonics K.K. | Image acquisition device and method and system for acquiring focusing information for specimen |
US10348954B2 (en) | 2013-04-26 | 2019-07-09 | Hamamatsu Photonics K.K. | Image acquisition device and method and system for creating focus map for specimen |
US10634894B2 (en) | 2015-09-24 | 2020-04-28 | Leica Biosystems Imaging, Inc. | Real-time focusing in line scan imaging |
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CN103592754B (en) * | 2013-11-07 | 2016-03-09 | 麦克奥迪实业集团有限公司 | A kind of digital slices real time scan automatic focus tracking |
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Also Published As
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BR112012029152A2 (en) | 2017-12-12 |
US20180203220A1 (en) | 2018-07-19 |
CN102893198B (en) | 2015-11-25 |
BR112012029152A8 (en) | 2018-02-06 |
JP2013531270A (en) | 2013-08-01 |
US20130057674A1 (en) | 2013-03-07 |
CN102893198A (en) | 2013-01-23 |
EP2572226A1 (en) | 2013-03-27 |
US20180210182A1 (en) | 2018-07-26 |
US10371929B2 (en) | 2019-08-06 |
JP5809248B2 (en) | 2015-11-10 |
US10061108B2 (en) | 2018-08-28 |
US10365468B2 (en) | 2019-07-30 |
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