WO2015044035A1 - Microscope optique confocal et procédé d'analyse d'un échantillon au moyen d'un microscope optique confocal - Google Patents

Microscope optique confocal et procédé d'analyse d'un échantillon au moyen d'un microscope optique confocal Download PDF

Info

Publication number
WO2015044035A1
WO2015044035A1 PCT/EP2014/069953 EP2014069953W WO2015044035A1 WO 2015044035 A1 WO2015044035 A1 WO 2015044035A1 EP 2014069953 W EP2014069953 W EP 2014069953W WO 2015044035 A1 WO2015044035 A1 WO 2015044035A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
light
light source
different
detector device
Prior art date
Application number
PCT/EP2014/069953
Other languages
German (de)
English (en)
Other versions
WO2015044035A8 (fr
Inventor
Peter Westphal
Alexander Gaiduk
Helmut Lippert
Original Assignee
Carl Zeiss Microscopy 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 Carl Zeiss Microscopy Gmbh filed Critical Carl Zeiss Microscopy Gmbh
Publication of WO2015044035A1 publication Critical patent/WO2015044035A1/fr
Publication of WO2015044035A8 publication Critical patent/WO2015044035A8/fr

Links

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
    • G02B21/004—Scanning details, e.g. scanning stages fixed arrays, e.g. switchable aperture 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
    • G02B21/006—Optical details of the image generation focusing arrangements; selection of the plane to be imaged
    • 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
    • G02B21/008—Details of detection or image processing, including general computer control
    • 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
    • G02B21/0064—Optical details of the image generation multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling
    • 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
    • G02B21/0068—Optical details of the image generation arrangements using polarisation

Definitions

  • the present invention relates in a first aspect to a light microscope according to the preamble of claim 1.
  • the invention relates to a method for examining a sample with a light microscope according to the preamble of claim 12.
  • a generic light microscope includes light source means for emitting illumination light toward a sample, detector means for recording sample images of the sample, and optical imaging means for focusing illumination light as a spot pattern onto a height plane on the sample and for directing sample light from the sample to the detector means.
  • the light source device and the optical imaging means are configured to illuminate a plurality of spaced-apart lateral regions of the sample during a camera integration time of the detector device with the light spot pattern.
  • illumination light is emitted in the direction of a sample with a light source device, that sample images of the sample are recorded with a detector device and that illuminating light is focused as a light spot pattern onto a height plane on the sample with optical imaging means and sample light is passed from the sample to the detector device.
  • the light source device and the optical imaging means are designed to illuminate a plurality of spaced-apart lateral regions of the sample simultaneously or successively during a camera integration time of the detector device with the light spot pattern. Autofocusing is carried out in particular with such light microscopes and methods. In this case, a sample table on which the sample is held, adjusted in a height direction.
  • DE 197 13 362 A1 describes a confocal scanning microscope with a confocal measurement of the dimensions.
  • This problem can be alleviated by illuminating a plurality of spaced-apart lateral regions of the sample with one light spot each. As a result, it is probable that at least some of the illuminated lateral regions reflect a quantity of sample light which neither saturates nor is too low for the detector device.
  • the light source device, the optical imaging means and the detector device are designed to enable images of at least two sample images in which the light spot patterns are focused on different height planes and / or in which different height planes are imaged onto the detector device wherein the light source means, the optical imaging means and the detector means are spatially equally spaced for each of the images of the different sample images.
  • the same lateral areas of the sample are illuminated for the recordings of the at least two sample images.
  • electronic evaluation means are provided and designed to calculate height information of the sample with the aid of the at least two sample images.
  • the light source device, the optical imaging means and the detector device are arranged spatially the same during recording of at least two sample images, the light spot patterns being focused on different height planes in the at least two sample images and / or Different height levels are imaged on the detector device, and wherein for the at least two sample images each same lateral regions of the sample are illuminated. Furthermore, height information of the sample is calculated with the aid of the at least two sample images.
  • a basic idea of the invention can be seen in that a spatially identical arrangement is used for recording the at least two sample images. It is therefore carried out between the images of these images no scanning movement in the height direction.
  • Such a scanning movement is carried out in conventional light microscopes, in particular by moving a sample stage relative to the beam path or by changing an illumination or detection plane relative to the sample.
  • a speed advantage is achieved.
  • improved precision is possible since inaccuracies of mechanical movements are eliminated and vibrations of optical components have less effect.
  • a spatially fixed arrangement can be used. In this case, no movement of the light source device, the optical imaging means or the detector device takes place during or between the recordings of the two sample images.
  • a lateral area should be understood to mean a sample area with an extension transverse, in particular vertical, to a height direction.
  • the height direction may correspond to the optical axis from one objective to the next.
  • the lateral areas illuminated by the light spot pattern are preferably separated from one another, that is, there are non-illuminated sample areas between them.
  • the light spot pattern causes the sample to have a spatially inhomogeneous light intensity distribution is irradiated.
  • a grid or stripe image can be generated as a light spot pattern.
  • a light spot pattern consists of a plurality of mutually separate light spots, which are each round, for example.
  • the lateral region which is illuminated by a light spot during the recording of the first sample image illuminates with the lateral region which illuminates from a light spot during the recording of the second sample image will, at least overlapped.
  • the centers of these two lateral regions are identical.
  • the extent of the two lateral areas may be different. At a common center, therefore, one lateral area is completely contained in the other.
  • the two light spots for the two images of the two sample images can be focused in different height levels.
  • the light spots generated on the sample surface, that is, the illuminated lateral areas are of different sizes, but have a common center.
  • the invention is particularly suitable for the investigation of samples with a height profile, ie a non-uniform height.
  • structures within a particular liquid sample can also be investigated. In both cases, the exact heights of regions of interest of the sample are unknown.
  • One goal is the determination of these heights, ie either the surface profile or a determination of the course and position of sample parts, for example cell structures of biological samples. More generally, therefore, the profile of sample parts within a sample can also be understood by a height profile, and the sample surface can be understood as the surface of a sample part.
  • the light spots are focused on at least one height level on the sample.
  • a height level refers to a plane which is transverse, in particular perpendicular, to an optical axis from an objective to the sample. Since the position, geometry or structure of the sample are initially unknown, such a plane may be either spaced from the sample, that is, between the objective and the sample, the plane may be within the sample, or the plane may intersect, ie partially within, the sample surface and partially outside the sample.
  • the focusing of the light spots on a height plane indicates that the height plane is an optically conjugate plane to a plane in which the light spot pattern is generated. This may be the light source itself or, for example, a pinhole disk, which is irradiated by the illumination light.
  • the size of an illuminated lateral region depends on the height of the elevation plane at which the light spot is focused relative to that lateral region. Depending on the surface profile, differently sized spots of light on the sample surface are therefore produced with one height level.
  • sample light is received by the illuminated lateral areas.
  • a relatively small illuminated lateral area a relatively small area with high sample light intensity is illuminated on the detector device.
  • a larger illuminated lateral area leads to a comparatively large area with lower sample light intensity being illuminated on the detector device.
  • the light spot patterns are focused on different height levels for the two images of the sample images.
  • the detector elements which record the two sample images can be located in the same image plane for the recording of the two images. Whether a lateral area illuminated for the second sample image is larger or smaller than the corresponding lateral area illuminated for the first sample image depends on whether the height of this lateral area is above or below the first height plane on which the light spot pattern focuses on the first sample image becomes. Therefore, from a comparison of the measurement information, the same Lateral area are obtained in both sample images, it can be determined whether the height of this lateral area is above or below the first height level.
  • an identical light spot pattern is generated on the sample surface for the acquisition of both sample images.
  • different altitude levels are mapped to the detector device.
  • the detector device accordingly receives at least two sample images in which a detector plane in which the detector elements of the detector device are located is optically conjugate to a first height level and a second height level different therefrom.
  • the height level to which the light spot pattern is focused may be identical to the first or second height level or to both.
  • the image planes are different with respect to the detector plane of the detection direction and are measured separately, with which the two sample images are recorded.
  • the height level is thus imaged differently sharply on the detector plane.
  • it can then be determined from a comparison of the measurement information obtained at the same lateral area in both sample images, whether the height of this lateral area is above or below the first height level.
  • height information can be obtained precisely by the invention, without mechanical movements would be required.
  • the sample light to be detected may be illumination light reflected by the sample, in particular reflected or scattered illumination light.
  • the sample light can also be luminescent light, ie fluorescent or phosphorescent light, which is produced by excitation by means of the illumination light.
  • the light source device used can in principle be designed as desired. It may comprise one or more light source units, for example lasers or LEDs.
  • a plurality of light sources may be arranged next to each other in a corresponding pattern.
  • a light source may illuminate a mask that generates the light spot pattern.
  • the mask can be formed for example by pinhole or mirror.
  • an electronically adjustable micromirror array (DMD, English: Digital Mirror Device) can be used.
  • Other electronically controllable light modulators can be used which, for example, can be based on switchable liquid crystal regions, such as an LCoS (English: Liquid Crystal on Silicon).
  • a grid can also be used. This can be adjustable transversely and / or in the propagation direction of the light. This increases the number of different lateral areas that can be illuminated.
  • the height level can be changed, in which the light spot pattern is sharply imaged.
  • the grating may have a periodic structure with one or two lattice constants.
  • the grid may also have an irregular structure, whereby light spots of different dimensions are generated simultaneously.
  • the light source device or the optical imaging means can also have a scanner, by means of which an illumination light beam illuminates different lateral areas one after the other and thus generates the light spot pattern.
  • the light spots of a light spot pattern have different sizes and / or shapes.
  • at least one lateral area of the sample is illuminated with a light intensity which is suitable for this sample area, in order to avoid the detector device
  • a light intensity or an image sharpness are preferably determined in the at least two sample images for each of the lateral regions.
  • the light intensities or image sharpnesses which were determined in the at least two sample images at the same lateral area then become the
  • Quantitative statements are possible with the help of pre-stored reference data. These may have been determined in a previous reference measurement on an object with a known height profile. By comparing the image sharpening, the reference data can be used to make a statement as to how far the height of the examined lateral area is from the two height levels.
  • the light intensity for the different lateral areas can also be evaluated.
  • the light source device for focusing the light spot pattern on different height levels comprises a first and at least one second light source unit.
  • the first light source unit is arranged in a plane that is optically conjugate to a first height level on the sample
  • the second light source unit is arranged in a plane that is optically conjugate to a second height level on the sample.
  • a spatially rigid arrangement can be used to generate light spot patterns in the different elevation planes.
  • the at least two light source units can be switched on one after the other.
  • one and the same detector region of the detector device that is, for example, the same camera sensor elements, can be used to receive the at least two sample images.
  • the at least two sample images can also be recorded simultaneously.
  • sample light is split into at least two spatially different detection beam paths.
  • the detector device in each case comprises a detector area for each of the different detection beam paths.
  • the at least two sample images can be recorded simultaneously. As a result, a speed gain can be achieved.
  • the beam splitting means may in principle be of any type and comprise, for example, a semitransparent mirror.
  • the beam splitting means are formed by a light diffractive element, wherein the two detection beam paths are generated by different diffraction orders of the light diffractive element.
  • the illumination light emitted by the at least two light source units may differ in wavelength.
  • a dichroic element may be present which reflects or transmits light as a function of wavelength. Losses of light are thereby reduced compared to merging the illumination light of the light source units through a semitransparent mirror.
  • polarization means may be present, by means of which the illumination light emitted by the at least two light source units is differently polarized.
  • a polarization beam splitter may be present, which reflects or transmits incident illumination light as a function of its polarization.
  • the light source device may also be configured to illuminate light from at least send out two different spectral ranges.
  • the optical imaging means in this case have a longitudinal chromatic aberration by which illumination light is wavelength-dependent focused at different height levels on the sample.
  • the light source device can have different light source units.
  • a single spectrally broadband light source unit can also be provided.
  • two sample images can also be taken, in which a light spot pattern is focused on one and the same height level, but different height levels are imaged on the detector device.
  • beam splitting means can be present between the sample and the detector device, with which sample light is split up to at least two spatially different detection beam paths.
  • the detector device in each case has a detector area for each of the different detection beam paths. Different altitude levels are mapped to the different detector areas.
  • an image of the illuminated height plane is generated in each case one image plane, wherein the image planes of the different detection beam paths are generated in different planes relative to the respective detector area.
  • the two detector areas can be formed, for example, by different cameras. However, it is particularly preferred that the different detector areas are different sections of a common camera, that is, a common camera chip.
  • the beam splitting means with which sample light is directed to at least two spatially different detection beam paths, preferably comprises a dichroic element which reflects or transmits light in a wavelength-dependent manner. As a result, light losses when dividing the sample light onto the different detection beam paths are low, which results in improved measurement accuracy.
  • the beam splitting means with which sample light is directed to at least two spatially different detection beam paths preferably comprises a polarization beam splitter which reflects incident sample light depending on its polarization direction or transmitted.
  • a three-dimensional image of the sample can be obtained with the obtained height information.
  • a height-scanning movement can also be carried out, for example a height adjustment of a sample table.
  • at least two sample images are taken again by the method described. This allows height information to be collected over a larger altitude range.
  • the main camera may be different from the detector device used for the autofocus method.
  • a scanning device may be present. This can be used to shift a focus of illumination light in its propagation direction or to shift an imaging plane from the sample light. be guided.
  • the electronic evaluation means are designed to control the scanning device in the autofocus method so that the displacement takes place in accordance with the determined adjustment direction to achieve a sharp image. This means that the sample is moved in the adjustment direction or the focus of the illumination light is shifted in the opposite direction to the adjustment direction.
  • a dynamic range of the detector device is preferably larger than a dynamic range of the main camera.
  • the dynamic range indicates the respective range of measurable light intensities without saturation occurring.
  • a particularly large dynamic range is especially important for the autofocus method. This is done before the actual sample measurement with the main camera, so that sample properties are still largely unknown. Therefore, a predetermined light intensity of the light source device is used, which may be relatively high or low for the still unknown sample. As a result, the light intensity received by the detector means may also have very low or high values. Therefore, a higher dynamic range is advantageous for the detector device used for the autofocus method.
  • the detector device preferably has at least one HDR camera (HDR: High Dynamic Range).
  • HDR High Dynamic Range
  • This can take, for example, several raw images with exposure times of different lengths in succession and charge them to a sample image.
  • the HDR camera can also be designed to record the raw images simultaneously with different camera sensors or different camera pixels of the same camera sensor. In this case, the exposure times may in turn be different for the raw images.
  • the sensitivities of the camera sensors may differ.
  • an HDR camera can also be formed with a camera chip whose photosensitivity is not linear but, for example, logarithmic.
  • a light microscope can also be understood as any optically operating topology measuring device.
  • observation means such as a camera or an eyepiece
  • optical imaging means such as a lens
  • Fig. 1 shows a first embodiment of a light microscope according to the invention, in which two light source units are arranged in different planes.
  • FIG. 2 shows a second exemplary embodiment of a light microscope according to the invention, in which two different height levels are imaged side by side on a detector device.
  • 3 shows a third exemplary embodiment of a light microscope according to the invention, in which two different height levels are imaged onto two detector units.
  • FIG. 4 shows a fourth exemplary embodiment of a light microscope according to the invention, in which two light source units of different wavelengths are arranged in different planes.
  • FIG. 5 shows a fifth exemplary embodiment of a light microscope according to the invention, in which two light source units of different wavelengths are arranged in different planes and in which two different height planes are imaged side by side on a detector device.
  • FIG. 6 shows a sixth exemplary embodiment of a light microscope according to the invention, in which two light source units are arranged in different planes.
  • Fig. 7 shows a seventh embodiment of a light microscope according to the invention, in which two light source units of different wavelengths are arranged in different planes and in which two different height levels be imaged side by side on a detector device.
  • FIG. 8 shows an eighth exemplary embodiment of a light microscope according to the invention, in which two light source units focus illuminating light of different wavelengths on different height levels and in which two different height levels are imaged side by side on a detector device.
  • FIG. 9 shows a ninth exemplary embodiment of a light microscope according to the invention, in which a light spot pattern is generated by a scanning illumination light beam and in which two different height planes are imaged side by side on a detector device.
  • FIG. 10 shows a tenth exemplary embodiment of a light microscope according to the invention, in which a light spot pattern is generated by a scanning illumination light beam and in which two different height levels are imaged onto two detector units.
  • FIG. 1 shows a first exemplary embodiment of a light microscope 100 according to the invention.
  • This first comprises observation means 28, which may comprise an eyepiece and / or a main camera, a lens 22 and further imaging means 27. With these, a focal plane 24 is imaged onto the observation means 28. in the
  • a sample 26 can be arranged on, for example, a sample table or other sample holding means.
  • a basic object of the invention is to obtain height information about the sample 26. In contrast to conventional measuring methods, no scanning movement in the height direction is required for this purpose.
  • altitude direction the direction of the sample 26 to the lens 22 are understood, that is, the direction of the optical axis.
  • the light source device 10 for recording altitude information, a light source device 10 and a detector device 30 are used.
  • the light source device 10 in this embodiment includes two light source units 1 1 and 12. Each of them emits illuminating light, which is passed to the sample 26 as a light spot pattern.
  • the light source units 1 1 and 12 are arranged in different optical planes, which are not optically conjugate to each other.
  • the light source unit 1 1 is located behind a confocal plane 14 and the light source unit 12 in front of the confocal plane 14, which is a plane conjugate to the focal plane 24.
  • the light spot patterns of the two light source units 1 1 and 12 are imaged or focused on different planes on the sample.
  • the illumination light of the light source units 1 1 and 12 is guided via beam combination means 15 onto a common beam path.
  • beam combination means 15 For this purpose, for example, serve a partially transparent mirror.
  • the illumination light is further guided to a coupling element 21, which is arranged in the beam path from the sample 26 to the main camera 28.
  • the coupling element 21 may for example be a mirror, which is either partially transparent or covers only a portion of a lighting light cross section.
  • the Einkop- ele- element 21 may be adjustable to sample light either in the direction of
  • Main camera 28 or in the direction of the detector device 30 to guide.
  • the detector device 30 comprises a single camera 31 on which an image of a sample plane is generated by imaging means 36.
  • This sample plane can be the focal plane 24, with which the imaged sample plane differs from the two altitude planes, which are illuminated in a focused manner by the light source units 11 and 12.
  • the two light source units 1 1 and 12 may each successively generate a light spot pattern on the sample 26.
  • This allows the camera 31 to successively record two sample images, with no mechanical movements required.
  • no height scan is carried out by adjusting the light source device 10, the detector device 30 or optical imaging means 22, 36 between them and the sample 26 for receiving these two sample images.
  • the light source device 10, the optical imaging means 22, 36 and the detector device 30 are arranged spatially the same for each of the images of the different sample images.
  • the sample 26 may have an unknown height profile. This may be either the surface of the sample 26, the surface of sample parts or the arrangement of certain sample parts.
  • incident illumination light sample light is emitted.
  • the sample light may be surface reflected, scattered or diffracted illumination light.
  • the sample light may also be luminescent light emitted from illuminated sample parts.
  • a sharp image of a light spot pattern is generated in different height levels. Accordingly, in the respective height plane, light spots of the light spot pattern have minimum cross-sectional dimensions, while as the distance from the respective height plane increases, the cross-sectional dimensions become larger.
  • the cross-sectional dimensions of the light spots generated on the sample profile therefore depend on how the sample profile extends to a height plane.
  • two light spot patterns are generated on the sample profile that differ in the cross-sectional dimensions of their light spots.
  • the different cross-sectional dimensions of the light spots can be detected.
  • the sample light intensity emanating from a sample point can be determined in the two recorded sample images.
  • the light spots of different light spot patterns illuminate the same sample areas. Different light spot patterns are thus not laterally offset from one another. Rather, equal lateral areas of the sample are illuminated with both light spot patterns. That is, the lateral area of the sample illuminated by any light spot of a light spot pattern is at least overlapping with the lateral area illuminated by a light spot of the other light spot pattern. Preferably, these lateral areas are concentric.
  • the light source device 10 here comprises only one light source unit 11.
  • the same light spot pattern of the light source unit 11 is imaged in the same height level on the sample. The lighting is therefore identical for both sample images.
  • the light source unit is located in the confocal plane 14, with which the light spot pattern is sharply imaged in the focal plane 14.
  • this embodiment differs from the previous one additionally in the detection beam path.
  • beam splitting means 33 may be a partially transmissive mirror, which preferably reflects and transmits sample light in equal proportions.
  • sample light is forwarded to the detector device 30 on two different beam paths.
  • the detector device 30 comprises exactly one camera 31.
  • the two beam paths lead sample light to different detector areas 41, 42 of this camera 31, that is, to different camera elements of the same camera 31.
  • the focal plane 24 is imaged via the two beam paths into two different image planes 34, 35 which are offset relative to each other in the propagation direction of the sample light.
  • these two image planes 34, 35 are different in the propagation direction of the sample light with respect to a detection plane in which the camera 31 with its two detector regions 41, 42 is arranged.
  • different imaging means 36, 37 or the same imaging means can be arranged at different locations in the two beam paths.
  • optical paths of different lengths can also be used by the beam-splitting means 33 to the detector regions 41, 42.
  • the detection plane of the detector area 41 coincides with the image plane 34. With this detector region 41, therefore, a sample image is recorded which is a sharp image of the focal plane 14. However, because the image plane 35 is different from the detection plane of the detector region 42, the sample image recorded with the detector region 42 is a blurred image of the focal plane. In other words, the detection plane of the detector area 41 is optically conjugate to the focal plane 14, while the detection plane of the detector area 42 is optically conjugate to another altitude plane. Thus, different height levels are imaged onto the detector device 30 in the two sample images. In the two sample images, the image of a lateral area illuminated by a light spot differs depending on the height of this lateral area relative to the focal plane 14. Thus, as described with reference to FIG. 1, a comparison can be made by comparing the sharpness or light intensity in the two sample images. whether and how far the illuminated lateral areas each lie above or below the focal plane 14.
  • the light source device 30 generates a light spot pattern sharply in the focal plane 14.
  • the light spot pattern can also be generated sharply in any height level different from the focal plane 14.
  • the detection plane of the detector region 41 does not have to be optically conjugate to the focal plane 14. Rather, it is sufficient that the two detection levels of the two detector areas 41 and 42 are conjugate to two different height levels.
  • the light spot pattern is preferably sharply imaged, wherein the light spot pattern can, however, in principle also be sharply imaged into another elevation plane.
  • Sample light is again divided by beam splitting means 33 into two different beam paths, which lead to two detector areas 41 and 42.
  • the two detector regions 41 and 42 are also arranged differently with respect to an image plane 34.
  • the two detector regions 41 and 42 are not formed by the same camera but by two different cameras 31 and 32.
  • the embodiment of Fig. 2 offers the advantage that only a single camera is required, whereby lower costs and a smaller space requirement are possible.
  • the embodiment of FIG. 3 offers the advantage that two smaller camera chips suffice instead of one large camera chip.
  • the optical axes of the illumination light are perpendicular to the respective camera surface, whereby not distorted images are generated on the camera surfaces.
  • the embodiment of the invention shown in FIG. 4 corresponds largely to the embodiment of FIG. 1.
  • Two light source units 1 1 and 12 are arranged in different planes, one of which is located in front of and behind a confocal plane 14. While in the embodiment of FIG. 1 the light source units 1 1 and 12 emit illumination light of the same spectral range, in the embodiment of FIG. 4 the two light source units 1 1 and 12 emit illumination light of different spectral ranges.
  • the light source unit 11 may comprise an array of LEDs or lasers emitting 800nm light while the light source unit 12 comprises an array of LEDs or lasers emitting 850nm light.
  • the arrangements of the LEDs or lasers is the same for both light source units 1 1 and 12 and corresponds to the light spot pattern to be generated.
  • a dichroic optical element is used as the beam combining means 15. This has a cut-off wavelength between transmission and reflection, which lies between the two spectral ranges of the light emitted by the light source units 1 1 and 12 illumination light. As a result, light losses are less than, for example, a semipermeable mirror as beam combining means. Due to the different spectral ranges of the illumination light of the two light source units 11 and 12, sample light with two different spectral ranges is also emitted. While in FIG. 4 the detection beam path is similar to that of FIG. 1, in the embodiment of FIG. 5 the different spectral ranges of the sample light are used. Thus, the illumination beam path of the embodiment of Fig.
  • the sample light with the light source units 1 1, 12 is similar to the structure of Fig. 4.
  • the sample light with beam splitting means 33 which comprise a dichroic element, divided into two beam paths and guided to different detector areas.
  • the detector areas may be different areas of the same camera 31 (as shown), or may be formed by different cameras (not shown). These designs allow measurements to be made with high light intensity and low light losses, increasing measurement accuracy.
  • the two sample images can be recorded simultaneously.
  • two light source units 1 1 and 12 are arranged in different planes here.
  • a polarization beam splitter 15 is used here. This reflects or transmits incident light depending on its polarization.
  • a polarizer 16, 17 is located between the polarization beam splitter 15 and the two light source units 1 1 and 12.
  • the illumination light of the two light source units 1 1 and 12 is polarized differently by the polarizers 16, 17, so that the light from one of the light source units at the polarization beam splitter 1 1 and 12 largely completely transmitted and the light from the other of the two light source units 1 1 and 12 is largely completely reflected.
  • a combination of the two beam paths is achieved with low light losses.
  • polarized illumination described with reference to FIG. 6 is used.
  • a polarization beam splitter is used in the detection beam path as beam splitting means 33. This transmits or reflects incident sample light depending on its polarization. This structure is suitable if the sample light emitted thereon is also polarized by a polarization of illumination light. This is the case, for example, when illumination light is reflected on the sample surface as sample light. Depending on its polarization, the sample light is passed through the polarization beam splitter 33 onto two different beam paths and guided to different detector areas of the detector device 30. In this embodiment, both sample images can be recorded simultaneously with only slight light losses.
  • FIG 8 shows a further embodiment in which two light source units 1 1 and 12 are used, which are arranged in geometrically identical planes 14.
  • the illumination light emitted by the two light sources 1 1 and 12 separates in wavelength and is brought via dichroic beam combination means to a common beam path to the sample 26.
  • the optical imaging means 22, 40 are preferably achromatic. That is, the refractive power and thus imaging are identical for at least two wavelengths. For other wavelengths, differences in the refractive power of the optical imaging means 22, 40 are only small.
  • chromatic optical imaging means 22, 40 are used, preferably a chromatic objective 22.
  • This has a longitudinal chromatic aberration, by means of which illumination light is focused on different height levels as a function of wavelength.
  • the light spot patterns of the two light source units 1 1 and 12 are imaged on different height planes 24 and 25, although both light source units 1 1 and 12 are arranged in the same confocal plane 14.
  • the detection of the sample light can take place as shown and with reference to FIG. 1 or to FIG. 5.
  • the beam splitting means 33 the sample light is guided as a function of the wavelength to different beam paths (not shown).
  • sample light due to the light spot pattern imaged in the elevation plane 24 may be distinguished from the sample light due to the spot pattern imaged in the elevation plane 25.
  • an image is respectively generated in two image planes 34 and 35.
  • These two image planes 34 and 35 are different from a detection plane, which is formed in Fig. 8 by a single camera. As a result, different height levels can be examined, as described for FIG. 2.
  • FIG. 1 Another embodiment of the invention is shown in FIG.
  • the detection of sample light is carried out as described for Fig. 2.
  • the illumination is effected with a light source device 10, which generates a light spot pattern with the aid of lateral scanning means 48, 49.
  • a single illumination light beam of the light source device 10 thus successively generates the light spots of a light spot pattern.
  • the lateral scanning means 48, 49 thus carry out a scan in the lateral direction, that is to say perpendicular to the height direction, and not in the vertical direction. The scan takes place during the recording of the sample images, which are recorded simultaneously in the example shown.
  • the imaging means 40 are designed as adjustable focusing means. These can be used to change to which height level a light spot pattern is focused. An adjustment of the focusing means 40 is performed after two sample images have been taken and evaluated. The direction of the adjustment may be selected so that the height level in which a light spot pattern is focused is brought closer to the height of one or more of the lateral areas of the sample.
  • the design of the imaging means 40 as adjustable focusing means can be supplemented in each of the other embodiments.
  • FIG. This is scanned laterally with an illumination light beam, as described for Fig. 9.
  • sample light is split into two beam paths and guided to different cameras 31, 32, as described for FIG. 3.
  • two sample images are generated and evaluated. This should generally be understood as at least two sample images.
  • the two light source units and the division of the sample light into two beam paths to two detector areas or two cameras are also to be understood as "at least two.”
  • the invention includes two sample images in which different altitude information is contained
  • the light microscope according to the invention is spatially identical for the recording of both sample images.
  • no height scan is required for the recording of these images, which is associated with a time and a measurement accuracy advantage.

Landscapes

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

Abstract

L'invention concerne un microscope optique comprenant une source lumineuse qui émet une lumière d'éclairage en direction d'un échantillon, un dispositif de détection qui enregistre des images de l'échantillon et un moyen de reproduction optique qui concentre la lumière d'éclairage sous la forme d'un motif de taches lumineuses sur un niveau de hauteur de l'échantillon et qui dirige la lumière provenant de l'échantillon vers le dispositif de détection. La source lumineuse et le moyen de reproduction optique sont conçus pour, pendant un temps d'intégration de la caméra du dispositif de détection, éclairer avec le motif de taches lumineuses plusieurs zones latérales de l'échantillon espacées les unes des autres. Le microscope optique selon l'invention est caractérisé en ce que la source lumineuse, le moyen de reproduction optique et le dispositif de détection sont conçus pour permettre la prise de vue d'au moins deux images de l'échantillon pour lesquelles le motif de taches lumineuses est concentré sur différents niveaux de hauteur et/ou pour lesquelles différents niveaux de hauteur sont reproduits sur le dispositif de détection, la source lumineuse, le moyen de reproduction optique et le dispositif de détection étant agencés dans l'espace de manière identique pour chaque prise de vue des différentes images de l'échantillon. Des zones latérales respectivement identiques de l'échantillon sont alors éclairées dans les deux ou plus de deux images de l'échantillon. En outre, des moyens d'évaluation électroniques sont conçus pour calculer des informations de hauteur de l'échantillon à l'aide des deux ou plus de deux images de l'échantillon. L'invention concerne par ailleurs un procédé associé permettant d'analyser un échantillon au moyen d'un microscope optique.
PCT/EP2014/069953 2013-09-30 2014-09-19 Microscope optique confocal et procédé d'analyse d'un échantillon au moyen d'un microscope optique confocal WO2015044035A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102013016367.6 2013-09-30
DE201310016367 DE102013016367A1 (de) 2013-09-30 2013-09-30 Lichtmikroskop und Verfahren zum Untersuchen einer Probe mit einem Lichtmikroskop

Publications (2)

Publication Number Publication Date
WO2015044035A1 true WO2015044035A1 (fr) 2015-04-02
WO2015044035A8 WO2015044035A8 (fr) 2015-04-30

Family

ID=51846612

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2014/069953 WO2015044035A1 (fr) 2013-09-30 2014-09-19 Microscope optique confocal et procédé d'analyse d'un échantillon au moyen d'un microscope optique confocal

Country Status (2)

Country Link
DE (1) DE102013016367A1 (fr)
WO (1) WO2015044035A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106443995A (zh) * 2016-11-25 2017-02-22 中国科学院上海技术物理研究所 用于空间生物样品荧光观察的光纤共聚焦显微成像仪器
CN108885336A (zh) * 2016-04-08 2018-11-23 徕卡显微系统复合显微镜有限公司 用于研究样品的方法和显微镜
CN111638596A (zh) * 2020-07-10 2020-09-08 宁波蔡康光电科技有限责任公司 一种共聚焦显微镜
CN114460020A (zh) * 2022-01-30 2022-05-10 清华大学深圳国际研究生院 一种基于数字微反射镜的高光谱扫描系统及方法
CN117232792A (zh) * 2023-11-14 2023-12-15 南京木木西里科技有限公司 基于图像信息的显微镜缺陷检测系统

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106970047A (zh) * 2017-05-12 2017-07-21 中国工程物理研究院激光聚变研究中心 反射率分布测量系统及方法
LU101084B1 (de) * 2018-12-21 2020-06-22 Abberior Instruments Gmbh Verfahren und Vorrichtung zum punktförmigen Beleuchten einer Probe in einem Mikroskiop
DE102023101782B3 (de) 2023-01-25 2024-06-13 Leica Microsystems Cms Gmbh Vorrichtung und Verfahren zum Erzeugen eines zusammengesetzten Bildes einer Probe

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19713362A1 (de) 1997-03-29 1998-10-01 Zeiss Carl Jena Gmbh Konfokale mikroskopische Anordnung
US6399942B1 (en) * 1999-03-23 2002-06-04 Takaoka Electric Mfg. Co., Ltd. Active confocal image acquisition apparatus and method of three-dimensional measurement using same
US20020167723A1 (en) * 2000-09-11 2002-11-14 Olympus Optical Co., Ltd. Confocal microscope and height measurement method using the same
US6657216B1 (en) * 2002-06-17 2003-12-02 Nanometrics Incorporated Dual spot confocal displacement sensor
DE10242373A1 (de) 2002-09-12 2004-04-15 Siemens Ag Konfokaler Abstandssensor
DE102004014048A1 (de) 2004-03-19 2005-10-20 Sirona Dental Systems Gmbh Vermessungseinrichtung und Verfahren nach dem Grundprinzip der konfokalen Mikroskopie
DE102005052743A1 (de) * 2005-11-04 2007-05-10 Precitec Optronik Gmbh Messsystem zur Vermessung von Grenz- oder Oberflächen von Werkstücken
JP2008268387A (ja) * 2007-04-18 2008-11-06 Nidec Tosok Corp 共焦点顕微鏡
US7570795B2 (en) 2006-07-18 2009-08-04 Mitutoyo Corporation Multi-region autofocus tool and mode
US20100033811A1 (en) 2006-06-16 2010-02-11 Carl Zeiss Microimaging Gmbh Autofocus device for microscopy
US7858911B2 (en) 1999-03-23 2010-12-28 Kla-Tencor Corporation Confocal wafer inspection system and method
US20110017902A1 (en) 2009-07-27 2011-01-27 Sensovation Ag Method and apparatus for autofocus

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19713362A1 (de) 1997-03-29 1998-10-01 Zeiss Carl Jena Gmbh Konfokale mikroskopische Anordnung
US6399942B1 (en) * 1999-03-23 2002-06-04 Takaoka Electric Mfg. Co., Ltd. Active confocal image acquisition apparatus and method of three-dimensional measurement using same
US7858911B2 (en) 1999-03-23 2010-12-28 Kla-Tencor Corporation Confocal wafer inspection system and method
US20020167723A1 (en) * 2000-09-11 2002-11-14 Olympus Optical Co., Ltd. Confocal microscope and height measurement method using the same
US6657216B1 (en) * 2002-06-17 2003-12-02 Nanometrics Incorporated Dual spot confocal displacement sensor
DE10242373A1 (de) 2002-09-12 2004-04-15 Siemens Ag Konfokaler Abstandssensor
DE102004014048A1 (de) 2004-03-19 2005-10-20 Sirona Dental Systems Gmbh Vermessungseinrichtung und Verfahren nach dem Grundprinzip der konfokalen Mikroskopie
DE102005052743A1 (de) * 2005-11-04 2007-05-10 Precitec Optronik Gmbh Messsystem zur Vermessung von Grenz- oder Oberflächen von Werkstücken
US20100033811A1 (en) 2006-06-16 2010-02-11 Carl Zeiss Microimaging Gmbh Autofocus device for microscopy
US7570795B2 (en) 2006-07-18 2009-08-04 Mitutoyo Corporation Multi-region autofocus tool and mode
JP2008268387A (ja) * 2007-04-18 2008-11-06 Nidec Tosok Corp 共焦点顕微鏡
US20110017902A1 (en) 2009-07-27 2011-01-27 Sensovation Ag Method and apparatus for autofocus

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108885336A (zh) * 2016-04-08 2018-11-23 徕卡显微系统复合显微镜有限公司 用于研究样品的方法和显微镜
CN108885336B (zh) * 2016-04-08 2021-12-14 徕卡显微系统复合显微镜有限公司 用于研究样品的方法和显微镜
CN106443995A (zh) * 2016-11-25 2017-02-22 中国科学院上海技术物理研究所 用于空间生物样品荧光观察的光纤共聚焦显微成像仪器
CN106443995B (zh) * 2016-11-25 2019-01-01 中国科学院上海技术物理研究所 用于空间生物样品荧光观察的光纤共聚焦显微成像仪器
CN111638596A (zh) * 2020-07-10 2020-09-08 宁波蔡康光电科技有限责任公司 一种共聚焦显微镜
CN114460020A (zh) * 2022-01-30 2022-05-10 清华大学深圳国际研究生院 一种基于数字微反射镜的高光谱扫描系统及方法
CN114460020B (zh) * 2022-01-30 2023-11-17 清华大学深圳国际研究生院 一种基于数字微反射镜的高光谱扫描系统及方法
CN117232792A (zh) * 2023-11-14 2023-12-15 南京木木西里科技有限公司 基于图像信息的显微镜缺陷检测系统
CN117232792B (zh) * 2023-11-14 2024-01-30 南京木木西里科技有限公司 基于图像信息的显微镜缺陷检测系统

Also Published As

Publication number Publication date
WO2015044035A8 (fr) 2015-04-30
DE102013016367A1 (de) 2015-04-02

Similar Documents

Publication Publication Date Title
DE102013016368B4 (de) Lichtmikroskop und Mikroskopieverfahren zum Untersuchen einer mikroskopischen Probe
WO2015044035A1 (fr) Microscope optique confocal et procédé d'analyse d'un échantillon au moyen d'un microscope optique confocal
EP3108281B1 (fr) Procédé et système de microscopie à feuille de lumière
DE102015001421B4 (de) Vorrichtung und Verfahren zur Strahldiagnose an Laserbearbeitungs-Optiken (PRl-2015-001)
EP2137488B1 (fr) Procédé et dispositif de reproduction optique à discrimination en profondeur
DE102013001238B4 (de) Lichtmikroskop und Mikroskopieverfahren
WO2013171309A1 (fr) Microscope optique et procédé de prise de vues au moyen d'un microscope optique
EP3058414B1 (fr) Microscope à balayage et procédé de détermination de la fonction d'étalement ponctuel d'un microscope à balayage
DE102007019267A1 (de) Messanordnung sowie Verfahren zum dreidimensionalen Messen eines Objekts
WO2016193037A1 (fr) Procédé pour la détermination d'une information de hauteur à résolution spatiale d'un échantillon au moyen d'un microscope à champ large et microscope à champ large
EP3948392A1 (fr) Procédé et dispositif de détection de déplacements d'un échantillon par rapport à un objectif
DE112015002582T5 (de) Einstellsystem für ein Phasenmodulationselement und Einstellverfahren für ein Phasenmodulationselement
EP3101385B1 (fr) Dispositif et procede de detection de topographies de surface
DE10118463A1 (de) Verfahren und Anordnung zur tiefenaufgelösten optischen Erfassung einer Probe
DE102008001473B3 (de) Optische Anordnung zur Beleuchtung eines Messobjektes, interferometrische Anordnung zur Vermessung von Flächen eines Messobjektes
DE102008005356B4 (de) Autofokusvorrichtung und Autofokussierverfahren für eine Abbildungsvorrichtung
DE102019102330C5 (de) Optisches System für ein Mikroskop, Mikroskop mit einem optischen System und Verfahren zur Abbildung eines Objekts unter Verwendung eines Mikroskops
WO2020207795A1 (fr) Microscope à feuille de lumière et procédé de détermination de l'indice de réfraction d'objets dans la chambre à échantillons
EP2767797B1 (fr) Interféromètre à faible cohérence et procédé de mesure optique à résolution spatiale du profil de surface d'un objet
DE102006023887B3 (de) Anordnung und Verfahren zur konfokalen Durchlicht-Mikroskopie, insbesondere auch zur Vermessung von bewegten Phasenobjekten
DE10138656B4 (de) Oberflächenprofilmesseinrichtung
WO2004055570A2 (fr) Microscope coherent
DE102009021096A1 (de) Verfahren und Vorrichtung zum Bestimmen des Polarisationszustandes elektromagnetischer Strahlung
AT500501B1 (de) Vorrichtung zur messung von teilstrecken am auge mittels fourier-domain kurzkohärenz-interferometrie
DD279962B5 (de) Konfokales Laserrastermikroskop

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14793023

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14793023

Country of ref document: EP

Kind code of ref document: A1