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
• • 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
  • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
• • 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
• • 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
  • G02B21/0048—Scanning details, e.g. scanning stages scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
• • 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
  • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence

Definitions

• the present invention relates to a scanning optical microscope and a use of a scanning optical microscope for three-dimensional scanning of a sample to be examined according to the preamble of claim 1 and according to claim 20.
• optical scanning microscopes which in particular as confocal microscopes can be formed, therefore, can be characterized by a like confocal structure of lenses and pinholes, ie, that the pinhole and the imaging lens are arranged such that they have mutually conjugate foci.
• the sample itself is located in a focal point downstream of the imaging objective, which is preferably scanned there in all three spatial directions. Consequently, such a confocal designed microscope allows spot illumination and detection of the sample.
• the sample can be imaged in several spatial dimensions. Consequently, with conventional confocal microscopes, a spatial resolution of at best approximately 200 nm laterally and, for example, 500 nm longitudinally can be achieved.
• the confocal microscopes known from the general state of the art enable the non-destructive three-dimensional recordings of structures of a sample to be examined, whereby this sample is scanned pointwise, for example by means of a laser beam, in order, for example, to excite fluorescence in the sample. This fluorescence is detected for each point of the sample, so that in particular the time resolution of a confocal microscope over the recording time per Biid Vietnamese is coupled to the number of pixels.
• the optical scanning microscope according to the invention is a confocal microscope or a confocal microscope.
• confocal microscopes are known to a great extent as well, which could realize, for example, an increased time resolution, however, they also have considerable disadvantages, as described below:
• RSOM Nipkow disc
• the incident laser beam decomposes into many parallel beams, which scan the sample along circular segments by the rotation of the disk.
• the resulting confocal image is then written directly onto a CCD chip (charge coupled device chip - charge coupled device chip).
• the laser beam can also be split by means of an array of microlenses, whereby at least a little more than 100 images per second can be achieved.
• such constructed accesses or systems can very easily become interchangeable tions, so that fluorescent light from the focus of a beam can enter into an adjacent detection path, whereby the resolution is significantly limited, especially in a scanning of strongly fluorescent specimens.
• resonant mirrors or acousto-optic deflectors are used in the corresponding commercially available confocal microscopes, the confocal information being written on a point detector. Consequently, the temporal resolution is already limited by the speed of the point detector, so that the fastest confocal microscopes of this type allow only about 50 two-dimensional images with a maximum of about 1000x1000 pixels per second. In an attempt to enable a higher time resolution, the detector would have to be operated near saturation, which in turn has negative effects on the image quality, so that highest speeds are associated with only a low image resolution.
• a scanning optical microscope for scanning a sample to be examined is claimed in preferably up to three spatial directions, with at least
• a beam divider separating a read beam path and a detection beam path
• an image scanning unit arranged upstream in the reading beam path of the sample and at least one imaging objective for scanning the sample by the reading radiation
• a detection scan unit arranged downstream of the beam splitter in the detection beam path, with detection optics in order to focus the detection radiation onto a detector.
• the optical scanning microscope according to the invention in particular in the embodiment of a confocal microscope, has a detection beam path which is optically conjugated in the detection beam path and to a focus of the imaging objective.
• Aperture on which, in particular in the case of a reflection or single-photon fluorescence measurement advantageously increases the resolution.
• the light source has the possibility to change the wavelength of the reading radiation or the reading beam substantially quickly.
• the light source emits a reading radiation substantially coming from a point, which can for example also be referred to as punctiform reading radiation, it being also possible for the light source to emit parallel light beams.
• a further arranged in the reading beam path of the light source arranged aperture namely the reading beam aperture plate for generating a substantially punctiform reading radiation.
• the read radiation applied by the light source to the read beam aperture is directed only through a primarily circular aperture or throughbore of the read beam aperture to produce a defined shaped read radiation.
• the opening of the reading beam aperture has a different shape, such as a slit-like shape, an oval shape, an angular shape, etc.
• the optical scanning microscope according to the invention has a beam expander arranged downstream of the read beam aperture in order to expand the punctiform and / or parallel read radiation.
• the beam splitter is also designed to be wavelength-selective, in particular when a different wavelength than for the read beam or the read radiation or the exciter radiation is selected for the detection or the detection radiation.
• the image scanning unit is preferably suitable for moving a focus of the reading beam or reading radiation through the sample in at least one and preferably in two and particularly preferably in three spatial directions.
• the first three-dimensional scanning unit or the three-dimensional scanning unit A scanning unit, a first deflection unit, namely the two-dimensional imaging deflection unit whose axes are operated at different frequencies in order to scan a plane spanned by a fast and a central axis, has a downstream optics, namely the imaging optics, and / or a second deflection unit, namely the second deflection unit. mensional image deflection unit which realizes a slow axis oriented perpendicular to the plane.
• the imaging optics downstream of the two-dimensional image deflection unit which is preferably a component of the two-dimensional image scanning unit, is preferably based on the geometry of the entire structure of the image scanning unit, the beam properties of the reading and the detection beam and the reading and detection radiation, the properties of the image deflection unit (s ) and / or the properties of the imaging lens.
• the imaging optics arranged downstream of the two-dimensional image deflection unit are designed such that the sample is scanned in a plane perpendicular to the steel direction (xy plane), whereby this plane can also be displaced in the beam direction (z direction) by the one-dimensional image deflection unit.
• the second scanning unit or the detection scanning unit preferably has a third deflection unit, namely the detection deflection unit and preferably one of these detection deflection units arranged downstream in the detection beam path, namely the detection optics, for the detection radiation to a detector arranged downstream of the detection scan unit in the detection beam path focus on.
• the one- or two-dimensional detector is preferably located in the focal plane of the detection optics and preferably consists of several pixels along each usable spatial direction (preferably approximately 500 ⁇ 500, better 1000 ⁇ 1000 pixels).
• the detection deflection unit and the detector have a comparable number of dimensions. That is, for example, a one-dimensional detection deflection unit with a one-dimensional detector (line detector), but preferably a two-dimensional detection deflection unit with a two-dimensional detector (area detector) can be used.
• the detection optical unit downstream of the detection deflection unit or its configuration is advantageously substantially unaffected, but this is preferably adapted, for example, to the geometry of the structure, the beam properties of the detection beam and / or the properties of the detection deflection unit.
• this detection optics is designed in such a way that the detection beam is focused in a punctiform manner on the detector, whereby it is primarily possible by means of the detection deflection unit to snap this focal point over the detector.
• the focus of the detection optics is always in the plane of the detector. It is advantageous if the diameter of the focal point of the detection radiation on the detector is comparable to the size of a pixel of the detector.
• the raster movement of the focal point in the sample advantageously results in that the axes of the two-dimensional image deflection unit are preferably operated at different frequencies.
• this line movement is supplemented by a further axis, which moves the focus to an axis oriented perpendicularly to the fast axis and thus from individual scanned lines to a scanned plane (FIG.
• this second axis is referred to as the "middle" axis. That is, it is preferable to scan, by means of the two-dimensional image deflection unit, a plane which is preferably spanned by the fast axis and the central axis in the xy direction.
• the detection scanning unit if it is designed at least two-dimensionally, preferably also has a fast and a slow axis.
• the detector preferably has the ability to convert the incident photons into an electrically measurable signal, to integrate this signal per pixel, and to Save readout process. Consequently, this information is primarily passed on to a control electronics or control device in a read-out process for all pixels.
• Examples of such detectors include CCD sensors (Charge Coupled Device Sensors) or CMOS sensors (Complimentary Metal Oxide Semiconductor Sensor).
• the objective downstream of the detection deflection unit or the detection optics is an F-theta optic, which is distinguished, in particular, by the detection beam focus in the entire angular range (the scan field) accessible to the detection scan unit in a plane and preferably in the plane of the Detector is held so that this detection optics, for example, also called plan field optics.
• both the image scanning unit and the detection scanning unit move the associated focus in a plane.
• telecentric F-theta lenses can also be used in the optical scanning microscope according to the invention, by means of which the laser beam focus is not kept in the entire scan field in one plane, but furthermore the laser beam is kept perpendicular to the scan field.
• a high time resolution and in particular also a high spatial resolution is made possible if, for example, the light to be detected, that is, e.g. the fluorescence radiation emitted by the sample or the radiation reflected by the sample, which is also referred to as detection radiation, by means of a detection scanning unit arranged at a distance from the image scanning unit and in particular a detection deflection unit and optimized detection optics, such as the above-mentioned F-theta Optics focused on a line or area detector or applied.
• the light to be detected that is, e.g. the fluorescence radiation emitted by the sample or the radiation reflected by the sample, which is also referred to as detection radiation
• a detection scanning unit arranged at a distance from the image scanning unit and in particular a detection deflection unit and optimized detection optics, such as the above-mentioned F-theta Optics focused on a line or area detector or applied.
• imaging scan unit and detection scan unit advantageously has the effect that each pixel of the line or area detector corresponds to a position in the sample plane. That is, as soon as a two-dimensional image of the sample has consequently been rastered onto the area detector, this stored information is read out as an entire two-dimensional image from a corresponding arithmetic unit.
• the image of the sample is scanned or written line by line on the line detector and read out after each line. This advantageously divides the clock rate for reading the data by the number of pixels on the line or area detector.
• the line detector or the area detector itself need not necessarily be based on a CCD or CMOS technology, but rather that any one- or two-dimensional matrix of point detectors with sufficient sensitivity and spatial and temporal resolution can be used.
• the line detector or the area detector are designed in two parts, it is possible to avoid dead times, which can arise due to the readout of the line detector or the area detector and can unfavorably lead to a reduction or reduction of the frame rate.
• the line detector or the area detector advantageously has at least two (partial) areas which can be read out separately, so that, for example, the time duration required to describe the first area of the detector can be used, by the second one already described area of the detector. If the first area of the detector is described and the second area of the detector is read out, the second area of the detector is in turn written on and substantially the same time a readout of the first area of the detector takes place.
• the beam splitter is preferably realized as a wavelength-selective dichroic beam splitter or as a dichroic mirror.
• a detection beam path of the detection tion scanning unit arranged upstream blocking filter, which filters out the wavelength of the reading radiation or excitation radiation, arranged in the optical scanning microscope according to the invention.
• the imaging optics associated with the imaging scanning unit preferably comprise at least one telecentric lens or a telecentric lens, which is characterized in that the entrance and exit pupil lies at infinity.
• the imaging scanning unit is advantageously arranged downstream of at least one imaging objective in the read beam path.
• the focal plane is known to be changed in such a way that, for example, the imaging objective or the sample is moved or moved along the beam direction, so that at a high frame rate, mechanical disturbances of the sample caused by vibrations along the beam direction occur can.
• the change of the z-component of the image scanning unit can be realized, for example, by means of a zoom lens, which is preferably arranged between the telecentric optical system and the imaging lens and the imaging objective, in order to guide a convergent or divergent reading beam into the imaging objective to shift a longitudinal position (z-position) of a focal plane of the sample to be examined.
• the zoom lens therefore preferably replaces the one-dimensional image deflection unit and the associated optics or realizes the one-dimensional image deflection unit and the associated optics. It is conceivable that the lenses of this zoom lens are mounted, for example, on automatic, such as piezomotor operated positioning units.
• the lenses of the zoom lens can be displaced, for example, by means of movable elements moved by the driving of a piezoelectric motor.
• an electric lens which is formed with or without an associated correction optics, is located between the imaging optics and the imaging optics.
• tion lens arranged to guide a convergent or divergent reading beam in the imaging lens to move a longitudinal position (z position) of a focal plane of the sample to be examined, so that advantageously a movement of the last optical unit before the sample, such as Imaging lenses can be avoided.
• the electrical lens preferably replaces the one-dimensional imaging deflection unit and the associated optics or realizes the one-dimensional imaging deflection unit and the associated optics.
• the light source is a laser, for example, a coherent illumination.
• the light source is, for example, an LED (light emitting diode), a mercury vapor lamp, a xenon lamp, an arc lamp for incoherent illumination or another light beam emitting element.
• light in the sense of the optical scanning microscope described here is not necessarily limited to the visible part of the electromagnetic spectrum, but it is also possible to use another, invisible part of the electromagnetic spectrum as exciter or reading radiation In such a case, for example, it is possible to deviate different optical components, such as lenses, that is, in a corresponding embodiment, the entire scanning optical microscope instead of lenses, for example, curved mirrors, such as For example, concave mirror, or Fresnel zone plates.
• the optical scanning microscope has a first lens arranged in the detection beam path between the beam splitter and the detection beam aperture in order to enable confocality of the detection beam aperture to a focal plane of the specimen.
• the optical scanning microscope preferably has a second detection beam path in the detection beam path between the detection beam aperture and an element which For example, a blocking filter or a wavelength-dispersive deflection can be arranged lens, which primarily parallelizes the detection radiation, for forwarding the detection radiation in the detection scan on.
• a blocking filter or a wavelength-dispersive deflection can be arranged lens, which primarily parallelizes the detection radiation, for forwarding the detection radiation in the detection scan on.
• the detection scanning unit or the detector are preferably coupled to the image scanning unit in such a way that the movement (s) of the detection radiation or the detection beam along the axis or the axes, ie the fast and / or slow axis of the detection scanning unit, at least in frequency and phase are substantially rigidly coupled to the corresponding movement of the reading radiation or of the reading beam along the axes, ie the fast and / or middle and / or slow axis of the image scanning unit.
• the raster motion of the reading beam through the sample causes an axis to oscillate at the highest frequency. This fast axis is always advantageous or one of the coupled in frequency and phase axis or axes.
• the coupling of the movement of the one or more axes of the image scanning unit and the detection scanning unit in frequency and phase includes, in particular, that the frequencies in both scanning units may be substantially different, but advantageously at least one substantially fixed frequency. and phase relationship between one or more axes of the image scanning unit and the detection scanning unit. It is preferably conceivable to select the frequency ratio in particular of the corresponding axes as an integer, so that it is possible, for example, to convert two line movements of the image scanning unit to one line movement of the detection scanning unit.
• the detection scan unit which preferably applies the image to a non-point-shaped detector and in particular a line or area detector, and whose axis or axes are to move substantially synchronously with the image scanning unit
• the fast axis of the detection scan unit or of the line or area detector is synchronized with the fast axis of the imaging scan unit
• Such a synchronization takes place in particular in the case when a one-dimensional array or line detector is used as the detector and also the imaging and / or or the detection scan unit is formed one-dimensionally.
• the fast axis of the detection scan unit or of the area detector is synchronized with the fast axis of the image scan unit
• the slow axis of the detection scan unit or the area detector is synchronized with the slow axis or even the center axis of the image scan unit
• the fast axis of the image scan unit is synchronized with the fast axis of the detection scan unit, and the center axis of the image scan unit is synchronized with the slow axis of the detection scan unit.
• the fast axis of both scan units can write an axis perpendicular to the read beam to an axis of the detector, while the second axis of the detection scan unit can be primarily perpendicular to or parallel to the optical axis Axis can be assigned in the sample.
• the image scanning unit and / or the detection scanning unit each have a deflection unit for adjusting and adjusting the beam in order to select the radiation emerging from the imaging scanning unit and / or the detection scanning unit in a defined and essentially free angle, at least within a defined range to divert the radiation entering the scanning unit.
• the angle range of the respective deflection unit, such as the imaging deflection unit and / or the Detektionsabliegatti, emerging beam can be centered around a defined and substantially preferred pre-definable direction of arrival around or even rotated against this.
• the deflection unit of the light beam should preferably not depend on the wavelength of the light, since otherwise a detection radiation having a different wavelength than the reading beam, such as in the case of fluorescent light, is deflected at a different angle than the reading radiation itself, so for example excitation light. Such a difference in the deflection of reading beam and detection beam would lead to a reduction of the resolution, which in turn has a negative effect on the use of the corresponding scanning unit.
• the image scanning unit and / or the detection scanning unit which are based, for example, on the diffraction of the light beam, have an acousto-optic or electro-optical deflector.
• the image scanning unit and / or the detection scanning unit preferably has a low mass inertia, thereby advantageously allowing a high line rate or frame rate by movements that can be executed substantially quickly.
• the imaging scanning unit and / or the detection scanning unit preferably have a resonant mirror with the highest possible resonance frequency, so that in particular the use of acoustic-optical deflectors (AOD) could be dispensed with.
• AOD acoustic-optical deflectors
• the deflection of the imaging scan unit along the slow or central axis is preferably replaceable by a dynamic wavelength variation of the excitation radiation or the read radiation.
• a polychromatic light source and a wavelength-dispersive element arranged downstream of the light source in the reading beam and arranged upstream of the beam splitter are advantageously required.
• Such a dispersive element can be implemented, for example, as an acousto-optic deflector or a rotating grating
• a detector is connected upstream of the detection scanning unit in the detection beam path arranged wavelength-dispersive deflection used to spectrally split the detection radiation, so that advantageously thereby a dynamic variation of the detected radiation can take place.
• the slow axis of the detection scan unit is not primarily used for imaging a spatial dimension, but preferably for imaging different wavelengths on the detector or the detector unit.
• the slow axis of the detection scan unit is preferably substantially rigidly connected and synchronized in frequency and phase with the introduced dispersive element.
• this wavelength-dispersive deflection element replaces the blocking filter which is otherwise preferably arranged, for example, for the detection of fluorescence or fluorescence radiation.
• a corresponding embodiment of the optical scanning microscope according to the invention is particularly advantageous if, for example, the detection radiation does not consist solely of radiation reflected in the sample.
• the use of a scanning optical microscope according to the invention, as described above, claimed for three-dimensional scanning of a sample to be examined, the use of such a scanning optical microscope is not limited to the microscopy itself, but in all areas of image production or representation application Can be found.
• the resolution and the size of the field of view is determined by the imaging lens and the corresponding associated optics and thus essentially depend on the imaging properties of these elements or devices, wherein substantially all those optics can be used, the rear focal plane lie at infinity , Further advantages, objects and characteristics of the present invention will be explained with reference to the following description of the appended drawing, in which an example of a construction of an embodiment of the optical scanning microscope according to the invention is shown. 1 shows a schematic representation of the beam path of the reading radiation and the
• FIG. 1 shows a schematic representation of the beam path of the reading radiation 2 (solid line) and the detection radiation 3 (dashed line) through individual units 5 to 21 of an embodiment of the optical scanning microscope 1 according to the invention.
• the first apertured diaphragm, namely the read beam aperture 6 is preferably arranged in the read beam path 2 and preferably between the light source 4 and a beam expander 7, while the second apertured diaphragm namely, the detection radiation aperture 16 is preferably arranged in the detection beam path 3 between a first lens 15 and a second lens 17.
• the reading radiation 2 generated by the light source 4 is thrown onto the reading beam aperture 6, so that only a single through a defined hole or through a defined shaped opening (not shown here) of the reading beam aperture 6 urgent reading beam 2 on a beam widening device. 7 can be projected.
• a wavelength-dispersive element 5 is shown, which is arranged in the reading beam 2 between the light source 4 and the reading beam aperture 6 and, as already indicated above, for example, an acousto-optic deflector or a rotating grating or a prism can be.
• This wavelength-dispersive element 5 advantageously serves to replace the deflection of the imaging scanning unit along the slow or central axis, preferably by a dynamic wavelength variation of the excitation radiation or the read radiation.
• the image quality is advantageously not due to interactions between individual read or pickup beams 2 or individual read or pickup radiation 2 negatively impaired, the image quality known from commercially available confocal microscopes is obtained at least by the use of the optical scanning microscope according to the invention with only a read or excitation beam 2 or a reading or Anregerstrahlung 2 and advantageously even significantly improved.
• the reading radiation is focused into the sample to be examined or refocused onto the sample to be examined, and the detection radiation reflected or fluorescently emitted by the sample from this focus is imaged by the same objective onto a second pinhole, from there on the detection radiation a radiation detector (for example, a photomultiplier or an avalanche photodiode) passes.
• a radiation detector for example, a photomultiplier or an avalanche photodiode
• the reading radiation 2 which is widened by the beam widening device 7 or beam widening device 7 is also fed to an imaging scanning unit 9, which i.a. a first two-dimensional image deflection unit 10 and an imaging optics 11 which can be assigned to this first two-dimensional imaging deflection unit 10, which can be, for example, a telecentric optical system, and a scanning of the sample area 14 lying in the sample plane or focal plane performs.
• An imaging objective 13 is additionally arranged between the imaging scanning unit 9 and the sample 14 or the sample planes.
• the reading radiation 2 entering the image scanning unit 9 is scanned not only in a certain angle range over the first two-dimensional image deflection unit 10 but also on average - for example at an angle of approximately 90 ° - deflected out the two-dimensional image deflection unit 10 emerges.
• a one-dimensional imaging scan unit 12 In order to enable a shifting of the scanned focal plane along the beam direction, it is possible to supplement the two-dimensional imaging deflection unit 10 and the associated imaging optics 11 by a one-dimensional imaging scan unit 12, wherein the one-dimensional imaging scan unit 12 primarily serves the focal plane along the beam direction (z direction) move.
• the radiation or detection radiation 3 reflected or fluorescently emitted by the sample 14 located in the sample plane is then guided again by the imaging scanning unit 9 in the direction of the beam splitter 8, which can also be in the form of a dichroic mirror, which already transmits the radiation transmitted by the beam expander 7 Read radiation 2 has forwarded to the image scanning unit 9.
• the reading and detection radiation separating beam splitter 8 is distinguished by a defined and substantially determinable ratio of transmitted and reflected radiation.
• the proportion of the transmitted radiation is preferably smaller than the proportion of the detection radiation reflected to the detection scanning unit
• the beam splitter 8 is preferably designed as a polarization-independent element.
• the beam portion 8 is preferably realized by a dichroic mirror.
• Dichroic mirrors are known, in particular, for the fact that these mirrors, for example made of specially vaporized glass, selectively reflect certain wavelengths and transmit others.
• the excitation radiation 2 here excitation radiation, since this is intended to stimulate individual molecules of the sample to be examined to fluoresce
• the detection radiation 3 is preferably completely reflected.
• the reading beam or exciter beam can also be reflected thereon and the detection beam transmitted, in which case the components of the light generation and the detection must be correspondingly positioned.
• the detection radiation 3 coming from the beam splitter 8 or dichroic mirror 8 is irradiated onto a detection beam aperture 16 via a first lens 15, which preferably concentrates the incoming detection radiation 3 as far as possible, with only the radiation passing through the hole or the recess of the detection beams.
• Aperture 16 urgent detection radiation 3 is transmitted to a further, second lens 17 which on the one hand parallelises or collimates the transmitted detection radiation such that as little scattered radiation as possible, and on the other hand (if fluorescence is to be detected) this parallelized or collimated Detection radiation 3 to a barrier filter 18 passes.
• the blocking filter 8 which is used primarily in optical fluorescence scanning microscopes, preferably serves to filter out or absorb the remaining excitation radiation 2 or the resulting and unwanted scattered radiation, which would be substantially detrimental to image formation of the sample, as this, for example, the Image quality would be increasingly degraded.
• the barrier filter 18 is primarily not used.
• the detection filter 2 transmitted by the blocking filter 18 to a detection scan unit 19, which is thus arranged in the detection beam path 3 of the detection beam aperture 16 and preferably in frequency and phase for all axes, ie
• the two-dimensional detection scan unit 19 for the fast axis and the slow axis it is substantially rigidly coupled to the respective axis of the imaging scan unit 9.
• Such a coupling of the different axes of the two scanning units 9 and 19 with each other for example, by the use of non-resonant systems feasible, so that, for example, acousto-optical deflectors (AODs) or electro-optical deflectors (EODs), which have a correspondingly required dynamics and Precession, but at the same time have higher losses are preferably used for the fast axis, while, for example, a galvanic mirror for the slow or middle axis is used. Due to the wavelength dependence of the deflection angle of acousto-optical deflectors or electro-optical deflectors already mentioned above, it is also advantageous to use mirrors for the fast axes. It is conceivable that these mirrors with an associated control electronics according to the requirements of the optical scanning microscope according to the invention in frequency and phase substantially rigidly coupled to each other.
• the detection scanning unit 19 has, in particular, a detection deflection unit 20 for which the above-described couplings or at least one of the above-described coupling exists in frequency and phase with the corresponding axes or with the respective corresponding axis of the imaging deflection unit a detection optics 21.
• the use of the detection scan unit 19, which has, for example, an F-theta optical system, makes it possible to scan a one- or two-dimensional image of the sample to be examined according to the focal plane pixel by pixel on a detector 22 and in particular on a line or area detector 22.
• optical elements such as adjustable mirrors (not shown here) are arranged at different areas of the scanning microscope construction.
• the optical elements as a lens through which the light is transmitted and thereby bundled or expanded by refraction effects, or as a mirror which, for example, in a non-planar design, such as.
• the reflected beam can also be bundled or widened, or embodied as another beam-shaping element, such as, for example, Fresnel zone plates.

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• 2012
  • 2012-02-20 DE DE201210101344 patent/DE102012101344A1/de not_active Withdrawn
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