WO2007019895A1 - Microscope optique a balayage equipe d'un mecanisme de mise au point automatique - Google Patents

Microscope optique a balayage equipe d'un mecanisme de mise au point automatique Download PDF

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Publication number
WO2007019895A1
WO2007019895A1 PCT/EP2006/004433 EP2006004433W WO2007019895A1 WO 2007019895 A1 WO2007019895 A1 WO 2007019895A1 EP 2006004433 W EP2006004433 W EP 2006004433W WO 2007019895 A1 WO2007019895 A1 WO 2007019895A1
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Prior art keywords
line
detector
scanning microscope
plane
spot
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PCT/EP2006/004433
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German (de)
English (en)
Inventor
Peter Westphal
Daniel Bublitz
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Carl Zeiss Microimaging Gmbh
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Publication of WO2007019895A1 publication Critical patent/WO2007019895A1/fr

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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
    • 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

Definitions

  • the invention relates to a laser scanning microscope with an autofocus device, in particular a light scanning microscope with an excitation and a detection beam path, means for scanning scanning of an object by moving an imaged spot or multispot area over the object and a spot or multispot area imaging lens, wherein a verstellmechanismus is provided for the lens.
  • Light-scanning microscopes are known in the prior art, for this purpose reference is made, for example, to DE 197 02 753 A1 or DE 102 57 237 A1, which describes a light scanning microscope designed as a laser scanning microscope.
  • the term "light” here means the entire region of the radiation which obeys the laws of optics.
  • Scanning microscopes typically reach an object image by imaging the aforementioned spot or multispot area with a detection that does not resolve a structure of the spot or the multispots (e.g., confocal detection). Moving the spot or multispot area over the object provides the picture. At the detector there is always only radiation information for the respective spot or multi-spot area, and an electronic combination of this image information taking into account the displacement of the spot or multi-spot area leads to the desired image.
  • Confocal detection is a way to achieve a very high depth resolution. The signal evaluation is then restricted substantially to the focal plane, since areas lying outside the focal plane do not provide any significant signal information in the case of confocal detection; they are displayed in front of or behind the confocal aperture.
  • Adjusting the focal plane position is thus extremely important for a scanning microscope, especially when working with confocal detection. This is especially true if you have a Sample is used, which is thicker than the depth of field of the lens. It is then necessary to approach the plane which is to be measured before the measurement in the sample.
  • the focus adjustment mechanisms are highly precise in conventional scanning microscopes, which would make it possible first to approach a known reference surface and then to set the focal plane to the desired distance from the reference surface, the distance between the current focal plane and the reference surface can be due to thermal effects, vibrations or temporally changed by other disturbances. One would then intermittently repeatedly check the distance to the reference surface, which would be very expensive.
  • Triangulations In order to obtain precise images of a sample or a sample section by means of imaging optics in laser scanning microscopy, it is necessary to place the sample exactly in the focal position of the imaging position of the optics and secondly the position of the focal plane in the sample to know.
  • triangulations imaging methods with contrast evaluation and position determination by means of obliquely arranged convocal slit are known.
  • Triangulationsvon a collimated laser beam is reflected in the pupil plane of an objective and closed from the course of this laser beam relative to the imaging beam path to the z position of the laser light reflected from the sample.
  • image errors occur so that the autofocus quality varies greatly over a given depth of field.
  • fluctuations are to be determined as to whether the measurement result from the center or at the edge of the sample or the detector used is determined.
  • a triangulation method is therefore performed iteratively, which is relatively time consuming.
  • the sample is illuminated with a specific intensity distribution, usually by placing a grid in a field stop plane of an illumination beam path.
  • the disadvantage of this is that for recording the image series different z-positions must be approached with high accuracy, which in turn is time consuming.
  • a slit diaphragm When determining the position by means of an obliquely placed concave slit diaphragm, a slit diaphragm is placed in a field stop plane of the illumination beam path and imaged onto the sample. The light reflected from the specimen is directed to a CCD line inclined relative to the slit and the position on the CCD line where the reflected light has a maximum is determined.
  • This procedure is very fast, though Problems with impurities on the sample or sample surface that can cause intensity fluctuations. Also, a very large Justieraufwand in the image of the gap on the CCD line to apply, because the gap must be very narrow in order to achieve high accuracy can be.
  • An improvement of the position determination by means of obliquely placed convocal slit aperture is described in DE 10319182A1.
  • the invention is therefore based on the object of specifying a laser scanning microscope, with which not only the position of the measurement plane can be determined exactly, but at the same time the distance to a reference plane can be detected.
  • a repeated adjustment of the sample in the z-direction should be avoided for the determination of the reference plane.
  • an accurate determination of the position of the focal plane should be possible with little effort.
  • This object is achieved by a light scanning microscope of the type mentioned above, which has an autofocus device for detecting a position of the focal plane of the objective, which images different depth ranges on the imaged spot or multispot area to different locations of a spatially resolving detector.
  • the light scanning microscope according to the invention thus forms a depth cutout on the spot-resolving detector at each spot, so that the position not only of the focal plane but also of a reference plane, for example a transitional glass / sample material can be determined.
  • the light scanning microscope thus determines the position of the focal plane relative to the reference plane.
  • the verstellmechanismus is then controlled so that the distance of the focal plane, which represents the measurement plane, is kept constant by the reference plane to a certain extent, or changed according to application-specific specifications.
  • the invention thus for the first time uses a depth-resolved image of the spot or multispot area for autofocusing.
  • a one-dimensional detector element suffices.
  • the coordinate of the detector element scales the depth information.
  • the intensity of the imaged radiation from the spot or multi-spot region depends on the one spatial coordinate of the detector element, which in turn is assigned taking into account the imaging conditions of the depth coordinate.
  • a detector line is sufficient, to which the depth-dependent spatially resolved imaging takes place such that different depth ranges are brought to different areas of the detector row for imaging.
  • the center of the detector line is in a plane conjugate to the focal plane rather than the otherwise confocal aperture.
  • the deep-resolution image can be realized by means of an anamorphic optical system arranged in front of the detector line, which focuses a line focus on the detector line, which lies in one plane with the detector line and intersects the detector line.
  • a tilting of the anamorphic optics or the detector line realizes this geometric arrangement particularly simple.
  • the anamorphic optics can be realized for example as a cylindrical lens, as a toric lens or as a combination of a one-dimensional holographic diffuser with a spherical lens.
  • the excitation or the detection beam path can be used in the light-scanning microscope in order to decouple the radiation for the autofocus device.
  • the integration takes place in the illumination beam path.
  • illumination light reflected back from the focal plane of the object or from the reference plane is used in the autofocus device.
  • An increase in radiant power, with the Lighting light is directed to the object is not necessary because the autofocus device has no effect on the radiation intensity in the detection beam path.
  • the decoupling of the radiation for the autofocus device in the beam path between the detector module or excitation module and the scanner, ie when the beam is at rest, advantageously causes the depth information to be averaged over the entire imaged area of the object. Individual object areas in the focal plane, which do not scatter radiation, then do not interfere.
  • the autofocus device now allows the light scanning microscope to control the focus adjustment mechanism as desired. Therefore, a development is preferred in which a control unit is provided which reads out the signals of the autofocus device and actuates the focus adjustment mechanism.
  • control unit will use the signals to determine the position of the focal plane with respect to a reference plane. It is therefore preferred that the control unit from the signals determines a measure of a distance between the focal plane of the lens and a reference plane on the object and optionally taken into account in the control of the verstellmechanismus.
  • the signals of the autofocus device can be the signal of the detector line in the mentioned embodiments.
  • the signal will usually have at least two intensity maxima: the first maximum corresponds to the position of the current measuring position, ie the position of the focal plane, the second maximum is the position of the reference plane, for example, to assign a glass / sample material interface.
  • the distance between the two maxima provides the distance between focal plane and reference plane, wherein the function with which the depth resolution is transmitted to the spatial resolution of the detector is taken into account.
  • the angle between the line focus and the longitudinal axis of the detector line, the depth of field of the objective and the image scale can be used to convert the distance between the maxima into the distance between the focal plane and the reference plane.
  • the width of the maxima is usually determined by the depth of field of the lens.
  • the anamorphic optic produces the mentioned line focus.
  • the intensity distribution along the line focus is rarely constant, or only when considerable effort is driven. It is simpler to consider the intensity distribution along the line focus when determining the maxima.
  • the intensity distribution along the line thereby corresponds to the intensity distribution of the spot illumination with excitation light.
  • a laser scanning microscope which directs illumination radiation through an illumination beam path onto a sample and detects radiation emitted on the sample in a detection beam path
  • the microscope has a beam splitter which can be used in the illumination or detection beam path the beam splitter in the inserted state at the sample reflected illumination radiation from the illumination or detection beam path along an optical axis of the autofocus device decouples, a the beam splitter with respect to the decoupled illumination radiation downstream, anamorphic optical element which focuses the decoupled illumination radiation in a line focus and a the Optical element subordinate line or area detector which has an area sensitive to illumination radiation, which lies in a plane which au of the line focus and the optical axis is biased, wherein the optical element and the detector are arranged to each other and tilted against each other so that the line focus with the sensitive area forms an angle greater than 0 ° and less than 90 °.
  • the autofocus device of the microscope which can be provided either as a standalone module or permanently installed in the laser scanning microscope, uses reflected or backscattered illumination radiation for the focusing on the sample.
  • this illumination radiation is excitation radiation, which is why these terms are also used interchangeably below.
  • the use of the illumination radiation has the advantage that, in the case of a radiation-sensitive sample, there is no additional exposure to radiation of radiation which is only required for autofocus. If you wanted in the prior art this To avoid stress, illumination radiation for the autofocus device would have to be selected spectrally so that it does not damage the specimen. However, this would mean that the chromatic demands on the microscope rise significantly. Both problems avoids the device according to the invention or the microscope according to the invention by the use of the already irradiated for microscopy illumination radiation.
  • the autofocus device couples illuminating radiation returning from the specimen either in the illumination beam path or in the detection beam path and directs it onto a detector line.
  • An anamorphic optical element is used, which bundles the decoupled radiation into a focal line.
  • a detector detects the radiation, wherein the anamorphic element and the detector are aligned in a certain manner to each other such that a radiation-sensitive region of the detector is oblique to the longitudinal extent of the focal line.
  • the detector has a sensitive area that is line or line shaped. This can be realized by using a line detector. Alternatively, of course, an area detector comes into question, which is read in the corresponding line or line-shaped section. Furthermore, the detector allows a spatial resolution along the line-shaped, sensitive area.
  • the anamorphic element and the beam splitter are adjusted with respect to the arrangement in the illumination or detection beam path so that different levels in the
  • Focal lines which are assigned to spaced planes in the sample, at different longitudinal position of the sensitive detector area and an evaluation of the detector signals provides a direct statement about the distance of the planes in or on the sample, taking into account the geometry and imaging conditions used.
  • the detector will be aligned so that the sensitive area lies in the plane defined by the longitudinal direction of the focal line and the optical axis of the autofocus beam path and at an angle to the optical axis.
  • Detector with its line-shaped sensitive area is inclined to the optical axis, is particularly easy to implement.
  • the focal line extend obliquely to the optical axis and the sensitive area is, for example, perpendicular to the optical axis.
  • the focal line is the sensitive detector area and both lie substantially in the same plane as the optical axis.
  • the point of intersection between the focal line and the sensitive detector area depends, with regard to its position along the sensitive detector area, on the position of the focal line on the optical axis and thus on the position of the associated plane in the sample.
  • Essential for the anamorphic optical element is that it generates the aforementioned focal line, that is, has a line focus.
  • this can be achieved by using a cylindrical lens, a toric lens or a one-dimensional, holographic diffuser.
  • a control device which reads these signals of the detector and determines the layers of intensity maxima along the sensitive area. Intensity maxima are associated with intersections of focal lines, so that taking into account the angle to which the line focus and sensitive range are tilted against each other, the distance of the focal lines along the optical axis of the autofocus device can be determined very simply. Taking into account the reproduction scale of the microscope, the control device thus calculates the distance of the focal plane from a reference plane.
  • the anamorphic optical element usually generates the focal line with a certain intensity distribution along the line.
  • this intensity distribution will cause the intensity maxima to vary, depending on which portion of the focal line intersects the sensitive area of the detector.
  • the inhomogeneity of the focal line is therefore expediently taken into account by the control device in determining the positions of the intensity maxima, for example in which a correction function is used.
  • the autofocus device can be used as a module in the illumination or detection beam path of a laser scanning microscope or can already be provided there in the microscope.
  • no blocking of illumination radiation must occur between the sample and the beam splitter, i. It must be provided a color neutral beam splitter and any block filter must be arranged downstream of the beam splitter relative to the sample.
  • the autofocus device evaluates averaged radiation over an image when the detector integrates over a period of time that is longer than the scanning of at least one section of the sample takes.
  • the autofocus device or the microscope according to the invention converts a plane lying in the sample into a focal line, with spaced planes in the sample corresponding to spaced focal lines, which in turn are spaced according to the magnification on the optical axis of the autofocus devices.
  • a particularly large measuring range is obtained by adjusting the beam splitter and the anamorphic optical element so that the focal plane of the objective is conjugate to a focal line which intersects the optical axis in the sensitive region of the detector.
  • the sensitive area of the detector, the optical axis and the focus plane of the objective associated focal line thus ideally intersect as possible in one point.
  • the autofocus device or the microscope according to the invention is particularly suitable, as mentioned, to determine the distance between the current focal plane and a reference plane.
  • a reference plane for example, a glass / sample transition is possible, which is present in conventional samples either at the bottom of the sample (i.e., at the slide) or at the top of the sample (on the coverslip).
  • the control device is expediently assigned the positions of the intensity maxima of such interfaces in or on the sample.
  • Fig. 1 is a schematic representation of a light scanning microscope with a
  • FIG. 3 is a simplified sectional view of an object detected by the light scanning microscope of FIG. 1,
  • Fig. 4 is a simplified representation of a signal waveform, as with the
  • FIG. 5 is a schematic diagram of the objective together with the autofocus device of FIG. 1; and
  • FIG. 6 is a schematic diagram for explaining geometrical relationship within FIG.
  • FIG. 1 schematically shows a light scanning microscope designed as a laser scanning microscope (LSM) 1.
  • LSM laser scanning microscope
  • the laser scanning microscope 1 is essentially subdivided into an illumination or excitation module 3, a detection module 4 and a microscope module 5.
  • the excitation module 3 provides excitation radiation and feeds it into the microscope module 5, so that it is directed to the object 2 as spot-shaped illumination.
  • the spot-shaped illumination is guided by the microscope module 5 in a scanning manner over the object 2.
  • the spot area illuminated at the object 2 with excitation radiation from the excitation module 3 is confocally detected by the detection module 4 via the microscope module 5, e.g. in the form of a fluorescence analysis.
  • the microscope module 5 has an objective 6, which can be changed by means of a drive A in a focus adjustment FV with respect to the position of the focal plane in the object 2. This focus adjustment is explained in more detail for example in DE 197 02 753 A1.
  • the objective 6 is preceded by a tube lens 7.
  • the radiation coming from the excitation module 3 is guided via the objective 2 by means of a scanning optics 8 and a scanner 9 through the tube lens 7 and the objective 6 as a scanning spot.
  • the scanner 9 causes a so-called de-scanning in the reverse direction of the beam towards the detection module 4, so that after the scanner 9 in the detection module 4 there is again a stationary beam.
  • the excitation beam path of the excitation module 3 and the detection beam path of the detection module 4 are combined via a main color splitter HFT.
  • a color-neutral divider can also be used, as described, for example, in DE 102 57 237 A1.
  • the radiation is divided by further, unspecified color divider into individual detection channels, which are each constructed of a photomultiplier 14 with upstream Pinhole 15 and Pinholeoptik 16.
  • the pinhole 15 narrows the detection area almost completely to the theoretical focal plane; Radiation generated outside this focal plane can not pass through the pinhole 15.
  • the main beam splitter HFT thus transmits the detection radiation, for example due to suitable spectral filter properties or by suitable geometric formation in the form of partial mirroring, as is known in the prior art.
  • the radiation is conducted via secondary divider and the Pinholeoptiken 16 and the Pinholeblenden 15 to the PMT detectors.
  • the LSM 1 is a laser scanning microscope of known design.
  • the excitation module 3 causes an illumination of the spot with radiation of different wavelengths.
  • different illumination channels are provided, which are each constructed in the embodiment of a laser terminal 10 and 12 for coupling the radiation of a laser and coupling optics 11 and 13 and which are combined via an unspecified mirror staircase.
  • a telescope 17 is provided, which ensures the coupling according to conditioned radiation at the main color divider HFT.
  • the LSM 1 additionally has an autofocus device 22, which is installed in the illustrated embodiment in the illumination beam path.
  • a beam splitter sits as an output coupler 18 in the illumination beam path 2, which decouples radiation backscattered from the object 2 into the excitation beam path of the excitation module 3 at the excitation module 3 and bundles optics 19 formed here as anamorphic optics into a line focus LF.
  • the optic 19 is an anamorphic photon.
  • a spherical lens 19 can be used, since this then already provides a line focus.
  • the line focus LF is directed to a detector line 20, which forms an angle ⁇ with the optical axis OA and is cut by the line focus LF.
  • the line focus LF and the detector line 20 are thus in one plane.
  • the anamorphic 19 and the detector line 20 form an autofocus device 22, whose operation will be explained with reference to Figures 3 and 4.
  • the present geometry is described below with reference to Figures 5 and 6 in more detail. It is essential here that the anamorphic photodiode 28 produces a linear focus.
  • FIGS. 2a-c A possible embodiment for the optics 19 are shown in FIGS. 2a-c.
  • the line focus LF or, in FIG. 2c, the longitudinal axis L of the detector line 20 is shown by way of example in FIG. 2b.
  • the optics 19 can be a combination of one-dimensional holographic diffuser 26 with upstream spherical optics 25 (FIG. 2a), toric lens 24 (FIG. 2b) or cylindrical lens 23 (FIG. 2c), if the laser scanning microscope uses a spot spot for scanning.
  • FIGS. 1 and 2 show an oblique position of the detector line 20 in order to ensure that the line focus LF lies obliquely to the longitudinal axis L of the detector line 20.
  • this mutual skew can also be achieved without tilting the detector line 20 with respect to the optical axis OA, for example by means of a suitable holographic element or an obliquely arranged cylinder optic. It is therefore essential, as will be explained below, a tilting of the longitudinal direction of the focal line to the longitudinal direction of the detector line 20. This could also be achieved, for example, by the Anamorphot 19 is tilted.
  • a decoupling on the detection module 4 can take place when the main color splitter HFT allows backscattered excitation radiation to pass through.
  • a possible attachment point for the autofocus device 22 is indicated in Figure 1 at 30 and indicated by a dashed line.
  • Figure 3 shows a schematic sectional view through the object 2, which is detected by the laser scanning microscope 1 of Figure 1.
  • the object 2 has a slide 27, on which, covered by a cover glass 28, a cell layer 29 to be microscoped is arranged. Further, by way of example, the position of the focal plane F corresponding to the plane indicated by the confocal condition of the detection module 4, i. is determined by the selective action of Pinholes 15. Above the cell layer 29 there is a transition to the cover glass 28. Such a glass / cell layer transition has a refractive index jump. As is known, radiation is fundamentally reflected at a refractive index jump. The refractive index jump of the transition between cover glass 28 and cell layer 29 can thus be used as a reference plane R.
  • the imaging of the line focus LF in the autofocus device 22 onto the detector array 20 causes radiation incident on the detector array 20 to be fanned out of different regions along the optical axis OA on which the spot is illuminated.
  • the result of this fanning in the signal of the detector line 20 is shown in FIG. 4.
  • the reflection at the refractive index jump of the reference plane R leads to an increased radiation intensity at a certain point of the detector line 20.
  • a corresponding peak which in FIG. 4 is a reference plane peak PR is designated.
  • the structure of the cell layer measured in the measurement plane, ie the focal plane likewise leads to a reflection which occurs elsewhere on the detector line 20, ie at another x-coordinate in FIG. 4 and likewise leads to an increase in the intensity I (in FIG referred to as focal plane peak PF).
  • the detector line 20 whose corresponding x-coordinate is usually given by the pixel number, there are thus two intensity maxima as peaks, namely focal plane peak PF and reference plane peak PR, which are spaced apart by one pixel spacing d.
  • the pixel spacing d can be converted into the distance D between the focal plane F and the reference plane R in a simple manner.
  • the magnification of the optical image has to be considered.
  • each peak is determined by the depth of field of the lens 6. It can enter into the determination of the center of gravity of the focal plane peak PF and the reference plane peak PR. Furthermore, in the case of the determination of the peak or center of gravity, a basic course of the signal S resulting from the intensity distribution which is fundamentally given in the line focus LF can be taken into account. For example, with a Gaussian illuminated spot, e.g. find this Gaussian distribution also in the line focus LF. The same applies of course to other intensity distributions in the illuminated spot.
  • the determination of the distance D is made in the embodiment of Figure 1 by a control unit 21, which reads both the signal of the detector row 20, as well as the drive A for focus adjustment of the lens 6 controls accordingly.
  • the controller further determines the peak centroids, the peak distance d and controls the setting of the distance D to a certain extent.
  • FIG. 5 schematically shows the mode of operation of the autofocus device 22.
  • the optical axis OA and the elements 2, 6, anamorphot 19 and detector line 20 lying thereon are shown in FIG. 5.
  • Folds of the optical axis OA, as they occur in particular at the beam splitter 18 in Fig. 1, are not shown in order to keep the figure clear.
  • the objective 6, together with the anamorphic 19, images planes spaced apart in the sample 2 into spaced focal lines.
  • the object focal plane 37 is imaged, for example, in a focal line 38, and a plane 36 located farther in the object 2, ie further away from the objective 6, is imaged into a focal line 39, which is seen along the optical axis OA in the imaging direction in front of the focal line 38, ie closer to Anamorphic 19 is located. Due to the inclination of the Detector line 20 with respect to the optical axis OA cut the focal lines 38 and 39, the sensitive portion of the detector line 20 at various intersections 40, 41, which are spaced along the detector line. Thus, at the points of intersection 40, 41 associated points of the longitudinally of the sensitive area spatially resolving detector line 29 form the already mentioned intensity maxima. This is illustrated in FIG. 6.
  • FIG. 6 shows in its upper half the detector row 20 and the optical axis OA lying obliquely at an angle ⁇ and the focal lines 38, 39 intersecting the detector row 20. Due to the different points of intersection, signal I of detector line 20 as a function of pixel number n of the detector line results in the already mentioned intensity curve S which has peaks as maxima 43 and 44 which correspond to intersection points 40 and 41 of focal line 38 and 39, respectively, with the detector line 29 are assigned. By the corresponding coordinates 45 and 46 of the maxima, as described with reference to FIG.
  • the distance of the focal lines 39 and 38 along the optical axis OA can be easily calculated by the distance d between the coordinates 45 and 46 is multiplied by the cosine of the angle ⁇ . Together with the imaging ratio, which is achieved in the microscope module and in particular taking into account the objective 6, one thus obtains the distance D between the planes 37 and 38 in the sample 17.
  • the two intensity maxima occurring on the detector line 29 are assigned to such excellent planes in the sample 17.
  • the maximum 43 corresponds to the focal plane of the lens 16, d. H. the current measuring position or plane, from which the confocal image in the sample 17 takes place.
  • the second maximum 44 can be assigned as the reference plane of an interface in the sample between the sample material and the substrate (for ease of illustration, the sample 17 in Figures 1, 4 and 5 is not shown structured, so that the interface is not shown, for example, exactly the dashed line 36 would be).
  • control unit After calculating the distance between the planes, the control unit can set or keep constant the distance of the measurement plane from the interface which serves as the reference plane. Also, simply calibrate the sample stage adjustment mechanism if it exists.
  • the focal lines 38 and 39 are usually not homogeneous along their longitudinal direction in terms of intensity, since a conventional anamorphic photopotential focus focuses the light on the focal line.
  • the resulting zero curve 47 is shown in Fig. 6 for the intensity.
  • this zero curve 47 is considered, for example, subtracted from the signal of the intensity curve S.
  • the illustrated construction shows a laser scanning microscope 1 with punctiform screening.
  • the autofocus method can also be used with linear screening, in which case the line shape of the radiation without anamorphic imaging already exists.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
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Abstract

L'invention concerne un microscope optique à balayage présentant un trajet optique d'excitation et un trajet optique de détection, des moyens (9), destinés à balayer un objet (2) en déplaçant une zone spot, linéaire ou multispot reproduite au-dessus de cet objet (2), et un objectif (6) reproduisant la zone spot, linéaire ou multispot, un mécanisme de réglage de mise au point (A) étant prévu pour cet objectif (6). Selon la présente invention, un dispositif de mise au point automatique (22) est prévu pour enregistrer une position d'un plan de mise au point (F) de l'objectif (6), lequel dispositif reproduit différentes zones de profondeur sur la zone spot, linéaire ou multispot reproduite en différents endroits d'un détecteur à résolution locale (20).
PCT/EP2006/004433 2005-05-12 2006-05-11 Microscope optique a balayage equipe d'un mecanisme de mise au point automatique WO2007019895A1 (fr)

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DE102005022125.4 2005-05-12
DE200510022125 DE102005022125A1 (de) 2005-05-12 2005-05-12 Lichtrastermikroskop mit Autofokusmechanismus

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DE102007055530A1 (de) * 2007-11-21 2009-05-28 Carl Zeiss Ag Laserstrahlbearbeitung
CN116754565A (zh) * 2023-08-04 2023-09-15 哈尔滨工业大学 一种光学元件全口径表面微缺陷光致荧光检测用自动对焦检测方法

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DE102007017598A1 (de) * 2007-04-13 2008-10-16 Carl Zeiss Microimaging Gmbh Verfahren und Anordnung zum Positionieren eines Lichtblattes in der Fokusebene einer Detektionsoptik
DE102010000550A1 (de) 2010-02-25 2011-08-25 KLA-Tencor MIE GmbH, 35781 Verfahren zum Fokussieren einer Objektebene und optische Anordnung
DE102011083354B3 (de) 2011-09-23 2013-03-28 Carl Zeiss Ag Anzeigevorrichtung und Anzeigeverfahren
DE102011083353A1 (de) 2011-09-23 2013-03-28 Carl Zeiss Ag Abbildungsvorrichtung und Abbildungsverfahren
DE102012012281A1 (de) 2012-06-21 2013-12-24 Carl Zeiss Meditec Ag Augenchirurgie-mikroskop mit einrichtung zur ametropie-messung
DE102013021974B3 (de) 2013-12-20 2015-03-19 Carl Zeiss Meditec Ag Vorrichtung zur Bestimmung einer Ametropie eines Auges
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