EP1709473A1 - Systeme microscope et procede de correction de la tache des systemes optiques du systeme microscope - Google Patents

Systeme microscope et procede de correction de la tache des systemes optiques du systeme microscope

Info

Publication number
EP1709473A1
EP1709473A1 EP04804992A EP04804992A EP1709473A1 EP 1709473 A1 EP1709473 A1 EP 1709473A1 EP 04804992 A EP04804992 A EP 04804992A EP 04804992 A EP04804992 A EP 04804992A EP 1709473 A1 EP1709473 A1 EP 1709473A1
Authority
EP
European Patent Office
Prior art keywords
wavelength
microscope system
light beam
brightness distribution
pixel
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04804992A
Other languages
German (de)
English (en)
Inventor
Frank Olschewski
Jochen Nickel
William Hay
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leica Microsystems CMS GmbH
Original Assignee
Leica Microsystems CMS GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leica Microsystems CMS GmbH filed Critical Leica Microsystems CMS GmbH
Publication of EP1709473A1 publication Critical patent/EP1709473A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the invention relates to a microscope system.
  • the invention relates to a microscope system with at least one optics present in the microscope system that defines an illumination field, at least one light source that emits an illuminating light beam that illuminates a sample through the optics, at least one detector that pixel-by-pixel a detection light beam originating from the sample detects an electronic circuit downstream of the detector with a memory unit in which a wavelength-dependent brightness distribution of the illumination field of the optics present in the microscope system is stored.
  • the invention relates to a method for shading correction of at least one optical system present in the microscope system, which defines an illumination field, at least one light source which emits an illuminating light beam which illuminates a sample through the optical system and comprises at least one detector.
  • the U.S. Patent 6,355,919 discloses a method for calibrating a scanning microscope.
  • the scanning microscope can be calibrated as often as required.
  • the means for calibration are arranged in one plane of an intermediate image and can be scanned by the scanning light beam.
  • the means for calibration are arranged outside the current image field and are designed as reference structures. However, this does not compensate for the curvature of the field of view.
  • the invention has for its object to provide a microscope system with which imaging and illumination can be achieved, which compensates for a correction of the darkening of the edge caused by the field curvature.
  • the object is achieved by a microscope system which comprises the features of claim 1.
  • the invention has for its object to provide a method with which shading effects of the optics of a microscope system can be eliminated.
  • the objective problem is solved by a method which has the features of patent claim 17.
  • the microscope system is provided with at least one optical system that defines an illumination field and that is present in the microscope system.
  • at least light source is provided in the microscope system, which emits an illuminating light beam that illuminates a sample through the optics.
  • at least one detector is implemented which detects a detection light beam emanating from the sample pixel by pixel.
  • An electronic circuit downstream of the detector is used to process the image data recorded by the detector.
  • a wavelength-dependent brightness distribution of the illumination field of the optics present in the microscope system is stored in a storage unit.
  • a controllable element is provided in the illuminating light beam, which controls the intensity of the illuminating light beam pixel by pixel as a function of the stored, wavelength-dependent brightness distribution, so that the illuminating field is homogeneously illuminated.
  • the electronic circuit calculates the stored, wavelength-dependent brightness distribution pixel by pixel in such a way that a homogeneously illuminated image field is created.
  • the controllable element in the illuminating light beam is an LCD matrix, the individual pixels of which are controlled in accordance with the stored, wavelength-dependent brightness distribution.
  • the detector is a CCD chip.
  • a scanning device is in the
  • Illumination light beam of the microscope system which guides the illumination light beam pixel by pixel over or through the sample.
  • the controllable element in the illuminating light beam is an acousto-optical one Element that can be controlled depending on the wavelength-dependent brightness distribution stored in the memory unit such that the illumination field composed of the individual pixels has a homogeneous brightness distribution.
  • the acousto-optical element is an AOTF, or an AOBS, or an AOM.
  • the microscope system can be used with different light sources, e.g. a laser, a multi-line laser or a laser which emits a continuous wavelength spectrum.
  • different light sources e.g. a laser, a multi-line laser or a laser which emits a continuous wavelength spectrum.
  • the detector of the microscope system is at least one light-sensitive element which serially records the pixels of the illumination field on the sample.
  • the electronic circuit assembles the individual pixels into an image field that can be offset against the corresponding wavelength-dependent brightness distribution.
  • the method for shading correction is characterized by the following steps: • depositing the wavelength-dependent brightness distribution in a RAM table • pixel-by-pixel control of an element with a wavelength-dependent brightness distribution of the illumination field of the optics, such that the illumination field is homogeneously illuminated; • pixel-by-pixel recording of the detection light beam emanating from the sample; and • Computing the image field recorded with the optics with the wavelength-dependent brightness distribution of the illumination field of the optics.
  • Figure 1 is a schematic representation of a microscope with a controllable element in the illuminating light beam.
  • 2 shows a schematic representation of a scanning microscope with a controllable element in the illuminating light beam;
  • FIG. 3 shows a schematic illustration of a further embodiment of the scanning microscope with a control circuit for changing the intensity of the illuminating light beam emanating from the light source;
  • 4a shows a schematic representation of the brightness distribution for an image field
  • 4a shows a schematic representation of the brightness distribution for an illumination field
  • 5 shows a schematic illustration of an area sensor for recording the detection light beam emanating from the sample
  • Fig. 6 is a schematic representation of the controllable element, in which the individual pixels are driven according to the brightness distribution for a wavelength.
  • the embodiment of the microscope system shown here comprises an incident light microscope. It goes without saying that the microscope system 1 likewise comprises a transmitted light microscope or a switchable configuration from both types of illumination. In the schematic representation of the microscope, only those components are shown that are essential for the description. All other elements or components, such as tripod, nosepiece, tube eyepiece, camera, camera tube, etc., are well known to a person skilled in the art and therefore do not need to be expressly mentioned here.
  • the microscope comprises at least one light source 3 that emits an illuminating light beam 5, which is shown in FIG. 1 as a solid line.
  • a steel deflecting means 7 is provided which directs the illuminating light beam 5 onto an optic 9 in the working position.
  • the optics 9 are arranged over a sample 10, which in turn is located on a slide 11 carrier. It is well known to a person skilled in the art that the slide 11 can be positioned on a movable table. For the sake of simplicity, the table is not shown here. In the embodiment described here, it is an incident light microscope, so that the light emanating from the sample 10 is imaged by the optics 9 onto at least one detector 20. The detector 20 and the optics 9 are arranged in the detection light beam 12. The detection light beam 12 is shown as a dashed line. If you ignore all aberrations, such as astigmatism, longitudinal color errors, transverse color errors, etc., in an optic, a so-called lens system, only one curved surface is always imaged on another curved surface.
  • FIG. 4a An image field 40 is shown in FIG. 4a.
  • each image field 40 becomes ever darker, ie the intensity of the recorded image field 40 or also the intensity of the illumination field decreases towards the outside from the center 41. This drop in intensity from the center 41 towards the outside is also dependent on the wavelength.
  • the image field 40 is, for example, rectangular and the intensity decreases toward first and second long sides 42a and 42b or towards first and second short sides 43a and 43b.
  • a controllable element 13 is provided in the illuminating light beam 5 and in the detection light beam 12.
  • the controllable element 13 is connected to an electronic circuit 14, which is provided with a storage unit 15.
  • a wavelength-dependent brightness distribution, such as, for example, in FIG. 4 a), of the illumination field of the optics 9 present in the microscope system is stored in the memory unit 15.
  • the electronic circuit 14 can also be connected to the optics 9 or the revolver so that the electronic circuit 14 constantly receives information via the optics 9 currently located in the illumination or detection light beam. It is clear to every specialist that different optics show different shading.
  • the electronic circuit 14 serves to control the intensity of the illuminating light beam 4 pixel by pixel as a function of the stored, wavelength-dependent brightness distribution.
  • the controllable element 13 is controlled with the stored, wavelength-dependent brightness distribution in such a way that the illumination field 46 (see FIG.
  • the detection light beam 12 emanating from the sample 9 is offset against the wavelength-dependent brightness distribution stored in the storage unit 15 in such a way that a homogeneously illuminated image field 40 is produced.
  • the detection light beam 12 emanating from the sample is detected pixel by pixel by the detector 20, which is a CCD chip. This data is supplied to the electronic circuit 14, which then uses the wavelength to calculate the stored brightness distribution against the measured brightness distribution.
  • the illuminating light beam 5 coming from at least one light source 3 is directed onto a controllable element 13.
  • the illuminating light beam 5 passes from the controllable element 13 to a scanning device 16.
  • the scanning device 16 comprises a gimbal-mounted scanning mirror 18, which guides the illuminating light beam 3 through a scanning optics 19 and an optics 9 over or through a sample 10.
  • the illuminating light beam 5 is guided over the sample surface.
  • biological samples 10 (specimens) or transparent samples 10 the illuminating light beam 5 can also be guided through the sample 15.
  • non-luminous preparations may be prepared with a suitable dye (not shown, since the state of the art is established).
  • the dyes present in the sample 10 are excited by the illuminating light beam 5 and emit light in their own characteristic area of the spectrum. This light emanating from the sample defines a detection light beam 12.
  • This passes through the optics 9, the scanning optics 19 and via the scanning device 16 to the controllable element 13, passes through it uninfluenced and reaches, for example via a detection pinhole 21, at least one detector 20 which acts as a photomultiplier is executed.
  • detection components such as diodes, diode arrays, photomultiplier arrays, CCD chips or CMOS image sensors, can also be used.
  • the detection light beam 12 originating or defined by the sample 10 is shown in FIG. 2 as a dashed line in FIG. 1. Electrical detection signals proportional to the power of the light emanating from the sample 9 are generated in the detector 20. Since, as already mentioned above, not only light of one wavelength is emitted by the sample 9, it makes sense to insert a selection means for the spectrum emanating from the sample 9 in front of the at least one detector 20.
  • the selection means is an SP module 23.
  • the SP module 23 is designed such that it can record a complete lambda scan, that is to say that all the wavelengths emitted by the light source 3 can be recorded. Likewise, several wavelengths emanating from the sample 9 can be spatially separated and possibly also recorded in parallel in time.
  • the data generated by the detector 20 are forwarded to the electronic circuit 14.
  • At least one peripheral device 27 is assigned to the electronic circuit 14.
  • the peripheral device 27 can be, for example, a display on which the user receives information on setting the scanning microscope or the current setup and can also take the image data in graphic form.
  • the memory unit 15 is connected to the electronic circuit 14, in which, as already described, the wavelength-dependent brightness distribution, as for example in FIG. 3 b, of the illumination field 46 of the optics 9 present in the microscope system 1 is stored.
  • the detection light beam 12 is spatially spectrally split with a prism 31.
  • Another possibility of spectral splitting is the use of a reflection, or Transmission grating.
  • the spectrally split light fan 32 is focused with the focusing optics 33 and then strikes a mirror diaphragm arrangement 34, 35.
  • the mirror diaphragm arrangement 34, 35, the means for spectral, spatial splitting, the focusing optics 33 and the detectors 36 and 37 are combined as an SP module 23 (or mutiband detector).
  • the scanning device 16 guides the illuminating light beam 5 pixel by pixel over or through the sample 9.
  • the controllable element 13 in the illuminating light beam 5 is an acousto-optical element which, depending on the wavelength-dependent brightness distribution stored in the storage unit 15, can be controlled in such a way that it consists of the individual pixels composite lighting field 46 has a homogeneous brightness distribution.
  • the acousto-optical element 13 is an AOTF, or an AOBS, or an AOM.
  • the light source 3 consists of at least one laser that generates the illuminating light beam 5.
  • the at least one laser can be a multi-line laser. It is also conceivable that the laser emits a continuous wavelength spectrum, so that the sample 9 is either illuminated with a continuous wavelength spectrum or the user selects any wavelengths from the continuous wavelength spectrum for illuminating the sample 9.
  • FIG. 3 shows a schematic illustration of a further embodiment of the scanning microscope 100 with a control circuit 60 for changing the intensity of the illuminating light beam 5 emanating from the light source 3.
  • the same elements as already described in the description of FIG. 2 are identified by the same reference numerals ,
  • the light source 3, which is designed as a multi-line laser, is provided with the control circuit 60, so that the intensity of the illuminating light emanating from the laser is controlled as a function of the stored, wavelength-dependent brightness distribution.
  • the illumination field 46 (see FIG. 4b) is then homogeneously illuminated and shows no decrease in intensity towards the long sides 42a, 42b and / or the short sides 43a, 43b.
  • the dyes present in the sample 10 are excited by the illuminating light beam 5 and emit light in their own characteristic area of the spectrum. This light emanating from the sample defines a detection light beam 12. This passes through the optics 9, the scanning optics 19 and via the scanning device 16 to a wavelength-selective element 63, passes through this uninfluenced and reaches via at least one detection pinhole 21 at least one detector 20, which is designed as a photomultiplier. Other versions of the detector have already been mentioned in the description of FIG. 2.
  • FIG. 5 shows a schematic illustration of the detector 20, which in this embodiment is designed as a surface sensor 44 for recording the detection light beam 12 emanating from the sample 10.
  • Area sensor 44 comprises several pixels 45 ⁇ , 45 1
  • the light emanating from the sample 10 is transmitted from the optics 9 to the surface sensor 44.
  • the individual pixels 45 ⁇ , ⁇ , 45 ⁇ 2 , 45 ⁇ , 3 , ..., 45 n ⁇ m- ⁇ , 45 n , m register the intensity, this being superimposed on the shading effect of the optics 9.
  • the wavelength-dependent brightness distribution is stored in the memory unit 15 and the electronic circuit 14 calculates the data recorded with the detector 20 against the wavelength-dependent brightness distribution, so that the recorded image field 40 is illuminated homogeneously at the wavelength ⁇ ⁇ .
  • FIG. 6 shows a schematic representation of the controllable element 13, in which the individual pixels 50 ⁇ , ⁇ ; 50 ⁇ ,; 50 ⁇ , 3 ; ...; 50 n , m - ⁇ ; 50 ⁇ , m are controlled according to the brightness distribution for one wavelength.
  • 50 n , m assigned gray values represent the shading effect caused by the optics 9. This is the negative representation for the control of the individual pixels 50 ⁇ ; 50 ⁇ , 2 ; 50 1
  • the pixels 50 ⁇ , ⁇ ; 50 ⁇ , 2 ; 50 1 3 ; ...; 50 n, m - ⁇ ; 50 n , m are inversely controlled depending on the gray value, ie the darker the pixels 50 ⁇ , ⁇ ; 50 ⁇ , 2 ; 50 1 ⁇ 3 ; ...; 50 n , m - ⁇ ; 50 n , m , the lower the shadowing caused by the activation.
  • the actuation of the controllable element 13 is such that the illumination field 46 on the sample 10 is homogeneously illuminated. If a laser steel illuminates the sample 10 in a meandering manner, so that the sample illuminates 10, ie the illumination field one after the other, is covered with a large number of pixels.
  • a scanning device 16 guides the illuminating light beam 5 pixel by pixel over or through the sample 10 in order to obtain a homogeneously illuminated illuminating field 46, the controllable element 13 in the illuminating light beam 5 is an acousto-optical element 13.
  • the acousto-optical element 13 also becomes inversely controlled. This means that the greater the gray value due to the shading, the higher the intensity of the laser beam when it reaches this position. This takes place as a function of the wavelength-dependent brightness distribution stored in the memory unit 15 such that the illumination field 46 composed of the individual pixels has a homogeneous brightness distribution. As already mentioned above, the shading effect occurs.
  • the shading effect can be described with f (x, y, ⁇ ).
  • X is the x position and y is the y position of the individual pixel in the lighting field.
  • y is the y position of the individual pixel in the lighting field. It is particularly advantageous if the wavelength-dependent brightness distribution is represented as a model.
  • the wavelength-dependent brightness distribution or the attenuation can be represented as functional for each wavelength. Doing so
  • G (x, y) F ⁇ l (x-x0, y - y0) which increases the lighting characteristics where the optics cause attenuation, the shift x0, y0 stands for the quality of the adjustment. If, for example, the laser does not run centrally on the sample 10 in the illumination field 46, this misalignment can also be used when determining the wavelength-dependent brightness distribution are taken into account. The system then applies the appropriate correction to illuminate sample 10.

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  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Physics & Mathematics (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Studio Devices (AREA)

Abstract

L'invention concerne un système microscope (1) comprenant un système optique (9) qui définit un champ d'éclairage. Un rayon lumineux de détection (12), émanant de l'échantillon (10), est détecté pixel par pixel. Un circuit électronique (14), implanté en aval du détecteur (20), comprend une unité mémoire (15) dans laquelle est enregistrée une répartition de luminance, fonction de la longueur d'onde, d'un champ d'éclairage (46) du système optique (9) présent dans le système microscope (1). Un élément (13) contrôlable, placé dans le rayon lumineux d'éclairage (5), commande l'intensité du rayon lumineux d'éclairage (5) pixel par pixel en fonction de la répartition de luminance fonction de la longueur d'onde, de telle manière que le champ d'éclairage (46) soit éclairé de manière uniforme et que le circuit (14) calcule pixel par pixel la répartition de luminance fonction de la longueur d'onde et enregistrée de manière à obtenir un champ image (40) éclairé de manière uniforme.
EP04804992A 2004-01-28 2004-12-22 Systeme microscope et procede de correction de la tache des systemes optiques du systeme microscope Withdrawn EP1709473A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102004004115.6A DE102004004115B4 (de) 2004-01-28 2004-01-28 Mikroskopsystem und Verfahren zur Shading-Korrektur der im Mikroskopsystem vorhandenen Optiken
PCT/EP2004/053659 WO2005073776A1 (fr) 2004-01-28 2004-12-22 Systeme microscope et procede de correction de la tache des systemes optiques du systeme microscope

Publications (1)

Publication Number Publication Date
EP1709473A1 true EP1709473A1 (fr) 2006-10-11

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP04804992A Withdrawn EP1709473A1 (fr) 2004-01-28 2004-12-22 Systeme microscope et procede de correction de la tache des systemes optiques du systeme microscope

Country Status (5)

Country Link
US (1) US20070076232A1 (fr)
EP (1) EP1709473A1 (fr)
JP (1) JP2007519962A (fr)
DE (1) DE102004004115B4 (fr)
WO (1) WO2005073776A1 (fr)

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Publication number Priority date Publication date Assignee Title
EP2387959B1 (fr) 2008-04-21 2013-11-13 Covidien LP Dispositifs emboliques à balle tressée et systèmes de mise en place
WO2019070226A1 (fr) 2017-10-02 2019-04-11 Nanotronics Imaging, Inc. Appareil et procédé pour réduire le vignettage dans une imagerie microscopique
CN112714887B (zh) 2018-09-28 2024-02-23 仪景通株式会社 显微镜系统、投影单元以及图像投影方法
JP7150868B2 (ja) 2018-09-28 2022-10-11 株式会社エビデント 顕微鏡システム、投影ユニット、及び、画像投影方法
EP3988987A4 (fr) * 2018-09-28 2023-09-20 Evident Corporation Système de microscope, unité de projection et procédé de projection d'image
DE102020120114A1 (de) 2020-07-30 2022-02-03 Abberior Instruments Gmbh Detektionseinrichtung für ein Laserscanning-Mikroskop

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JPH0685022B2 (ja) * 1984-07-05 1994-10-26 オリンパス光学工業株式会社 内視鏡用照明光学系
US5749830A (en) * 1993-12-03 1998-05-12 Olympus Optical Co., Ltd. Fluorescent endoscope apparatus
JPH07155291A (ja) * 1993-12-03 1995-06-20 Olympus Optical Co Ltd 蛍光観察装置
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JP4512278B2 (ja) * 2000-02-04 2010-07-28 オリンパス株式会社 顕微鏡システム
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Also Published As

Publication number Publication date
DE102004004115B4 (de) 2016-08-18
US20070076232A1 (en) 2007-04-05
JP2007519962A (ja) 2007-07-19
DE102004004115A1 (de) 2005-08-11
WO2005073776A1 (fr) 2005-08-11

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