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
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
  • G01N21/6458—Fluorescence microscopy
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/06—Means for illuminating specimens
  • G02B21/08—Condensers
  • G02B21/10—Condensers affording dark-field illumination
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
  • G02B21/365—Control or image processing arrangements for digital or video microscopes
  • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
  • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems

Definitions

• SPIM technology Selective Plane Illumination Microscopy
• SPIM allows the acquisition of 3D objects in the form of optical sections as a widefield technique, the advantages being the speed, the low bleaching of the sample and an extended penetration depth.
• fluorophores in the sample are excited with laser light in the form of a light sheet for this purpose. The light sheet can be scanned through the sample.
• the SPIM method is described with a periodically structured light sheet.
• the periodically structured light sheet fluorescence excitation takes place at the high intensity locations.
• the structuring is used to suppress scattered light from out-of-focus planes and to increase the resolution through structured illumination (see below in the text, Heintzmann et al.).
• WO2006 / 127692 describes the so-called PALM process (photoactivated light microscopy). The method is based on the photoactivation of individual molecules separated according to the extents of the detection PSF and their highly accurate localization by fluorescence detection.
• the PALM method as described in WO2006 / 127692 uses essentially the following primary steps to produce a microscopic image with an increased optical resolution compared to the standard microscope: 1.) Photoactivation of Single Molecules: Activation alters the fluorescence properties of the molecules Switching on / off, changing the emission spectrum, etc.), the activation being such that the distance between activated molecules is greater than or equal to the optical resolution of the standard microscope (given by Abbe's resolution limit). 2.) excitation of the activated molecules and localization of the molecules with a spatially resolving detector. 3.) deactivating the activated molecules.
• the activation preferably takes place in wide-field illumination and distributed statistically. By choosing the activation energy, it is attempted to achieve as few / no molecules (1) as possible with overlapping diffraction disks (2) on the camera (see Fig. 1a). However, overlapping diffraction disks are accepted and can not be evaluated ((3) in Fig. 1 b). This results in regions in which the distance between the activated molecules is larger or much larger than the diffraction disc on the camera (4). Due to the statistical activation of the molecules, about 10,000 individual images must be evaluated for the generation of a high-resolution image to determine the positions of the molecules. As a result, large amounts of data must be processed and the measurement slowed down (about 1 min per high-resolution image). The billing of the single images to a high-resolution image requires about 4 hours.
• Focusing plane is in WO2006 / 127692 the use of a
• Another method used to avoid the autofluorescence problem is to combine the PALM method with the TIRF technology, where the
• Waves is kept very small. With TIRF, however, no 3D imaging is feasible.
• PALM initially only offers one due to the spatially resolved detection
• the axial resolution is determined primarily by the extent of the detection PSF used. This is another
• Heintzmann et al. (R. Heintzmann, TM Jovin and C. Cremer, "Saturated patterned excitation microscopy - a concept for optical resolution improvement", JOSA A 19, 1599-1609 (2002).) Suggest as a further concept for increasing resolution a nonlinear process in the form of direct saturation of a fluorescence transition.
• the increased resolution is based on a periodically grid-like patterned illumination of the sample, whereby a transfer of high spatial frequencies of the object takes place in the range of the optical transfer function of the microscope. The transfer can be followed indirectly by theoretical post-processing of the data.
• the object of the invention is to avoid the disadvantages of the method described above.
• the invention describes methods and arrangements for realizing a PALM microscope with optimized photoactivation to realize a higher frame rate. Compared to PALM, high resolution 3D imaging without nonlinear photoactivation is achieved. A PALM-TI RF combination to reduce the extra-focal autofluorescence is not required.
• the advantageous use of the MultiView method multiple illumination angles on the sample) for obtaining an increased penetration depth and an isotropic optical triggering in x, y, z can take place.
• Fig. 2 An activation according to Fig. 2 occurs as many molecules as possible without “gaps" ((4) in Fig. 1b) and without the diffraction disks of the molecules being superimposed on the camera ((3) in Fig. 1b.) If the diffraction disks are set of the individual molecules to each other so preferably results in a dense sphere packing. This achieves an increase in the speed of the PALM process and a reduction in the number of individual images.
• Fig. 3 shows schematically from the side (direction of incidence equal viewing direction) a planar light sheet LB, also called a lens, which is generated for example with a cylindrical lens (02 - ZL) and passes through the sample (P).
• a planar light sheet LB also called a lens
• the sample passes through the sample (P).
• an objective O of a microscope for example a wide-field microscope with a CCD camera, a laser scanning microscope or a microscope with structured illumination.
• the light sheet LB irradiated laterally into the sample in the form of the SPIM light sheet, which lies substantially exactly in the focal plane (F) of the objective O, is photoactivated in FIG. 3 perpendicular to the optical axis of fluorescence excitation and detection.
• the fluorescence excitation can take place here only within the focal plane (F).
• the fluorescence excitation and the fluorescence detection takes place according to WO2006 / 127692 via the microscope objective O.
• a localized excitation in the z-direction takes place and samples can be examined three-dimensionally without nonlinear photoactivation with the PALM method.
• the width of the light beam for photoactivation i. its extension in the z direction is adapted to be advantageously less than or equal to the axial extent of the PSF given by the numerical aperture of the objective O.
• fluorescence molecules are activated and bleached outside the focal plane.
• fluorescence can only come from this plane defined by the activation beam.
• this arrangement is inherently 3D-resolving.
• Detection is carried out by conventional means, such as classical wide-field microscopy, confocal microscopy or structured illumination (ZEISS APOTOM).
• the excitation beam is able to excite autofluorescence over the entire sample space. This can be prevented in Fig. 3 in addition, the light beam for excitation of the fluorescence (after photoactivation) with a light sheet (LB) perpendicular to the detection via 02 - ZL is irradiated. This ensures that no out-of-focus autofluorescence signals are generated that interfere with imaging.
• the fluorescent light can be detected particularly efficiently separated from the excitation light, since no spectral separation by means of a dichroic beam splitter is required. For some switchable dyes, such as DENTRA, photoactivation and fluorescence excitation occur at the same wavelength. This can be realized particularly easily hereby.
• Non-focal autofluorescence which is generated by the activation beam, can be separated spectrally, but in particular also over time (the fluorescence excitation occurs after activation) from the actually interesting fluorescence signal.
• the recording of the high-resolution image is carried out for the arrangements according to the invention as described in WO2006 / 127692 with the abovementioned steps 1-4.
• the activatable fluorescent dyes used are preferably fluorescent proteins known from the prior art, such as PA-GFP or DRONPA.
• the photoactivation takes place here with 405 nm, the fluorescence excitation at 488 nm and the detection in the range above 490 nm.
• reversibly switchable synthetic dyes such as Alexa / Cyan constructs can be used.
• Fig. 4a a further arrangement according to the invention with a lighting means of two light sheets LB 1 and LB 2 is shown schematically.
• the interferometric superimposition of light sheets from several directions results in an interference pattern along the marked x direction in the focal plane.
• the light sheets may in turn contain laser light for photoactivation and / or fluorescence excitation.
• the superimposition forms a so-called standing wave field (SW), which is shown schematically in FIG. 4b as a stripe pattern. If, for example, two sheets of light are used for the photoactivation, it can be achieved that the distance of the fluorescence emitters to be activated is greater than or equal to the width of the PSF of the detection (O in FIG. 4a).
• interference causes stripe-shaped activation located in x and z.
• a dot pattern PM 1 is produced, as shown in FIG. 5, ie an activation localized in x, z and y_, so that the distance (1) the activated fluorescence emitter is greater than or equal to the width of the PSF of detection (O in Fig. 4a).
• the fluorescence excitation can preferably also take place via one or more light sheets, which in turn avoids extra-focal autofluorescence.
• the fluorescence excitation from the direction O in FIG. 4 a can also be structured in one or more directions and, for example, also have a hole pattern (structuring in x and y). In this way it can be ensured that the distance of the fluorescence emitters is greater than or equal to the width of the PSF of the detection.
• Such structuring can be carried out, for example, with a lattice structure (DE 10257237A1) or with a multispot excitation (DE102006017841).
• the photoactivation can take place via the objective O. and the fluorescence excitation can be realized with a plurality of light disk beams which form an interference pattern of the type described above.
• Molecules that have been activated with overlapping diffraction disks are stimulated to different degrees. In this way, gaps in the camera image are avoided. Autofluorescence generated by the activation beam can be separated temporally and / or spectrally. After recording a layer, you must deactivate it for the 3D image over the sample area.
• the structured activation can also be realized by a specific imaging (eg of a grating - as described above) or by a scanning mechanism.
• the light beam can, for example, scan the image field and its intensity during movement is changed by, for example, a fast AOTF so that an activation pattern, for example, corresponding to FIG. 5, arises in the focal plane.
• molecules are also activated outside the focal plane.
• the intensity of the activation beam is ideally chosen so that on average only one molecule per activation spot (equivalent to approx. PSF size) is excited. This reduces the likelihood that 2 molecules with overlapping diffraction slices will be activated simultaneously. In the course of image acquisition, the activation pattern must of course be phase-shifted, so that all molecules are activated evenly. Autofluorescence generated by the activation beam can be separated temporally and / or spectrally. The activation intensities are usually so small that autofluorescence should not play a role.
• the photoactivation and fluorescence excitation beam can be exchanged here. This has the advantage that no photoactivation occurs outside the focal plane. For this, activation can be unstructured across the light sheet and the excitation matched to the PSF structured across the lens be performed. Molecules that have been activated with overlapping diffraction disks are stimulated to different degrees. In this way, gaps in the camera image can be avoided. However, there is the problem of extra-focal autofluorescence.
• a problem with all variants described is the axial resolution, which is generally determined by the width of the light sheet used in the SPIM method. Since the NA used to generate it is usually much smaller than the NA of the detection objective, the problem immediately arises of a highly elongated system PSF (lateral expansion determined by the resolution of the PALM method (nanometer range), axial expansion This is disadvantageous in the case of 3D imaging With the aid of the multiview technique known from the prior art (recording of stacks from different angles), this problem can be circumvented and an effective, largely homogeneous spatial resolution corresponding to the lateral PALM resolution are generated.
• the photoactivated molecules in the edge regions of the light sheet used for activation are deactivated again by a further structured light sheet in the sense of a nonlinear interaction in order to achieve a higher z resolution.
• a light sheet may also be used as the deactivation beam, but is structured such that it has a zero point in the focal plane over the area of the image area to be observed, as shown in FIG.
• the pupil intensity distribution (I) of the light sheet beam corresponds here to a line which has been previously generated by a suitable optical system (eg Powell lens).
• a phase plate (II) is introduced, which has over half of the line a region (III), which generates a pi-phase jump.
• the light sheet (V) extended in the xy plane is generated by a suitable optical system (IV).
• a zero-point plane (VI) results parallel to the focal plane of the detection optics (VII), in which no molecules are excited.
• Figure 7 shows a general optical embodiment for using the described advantageous methods and applications with photoactivation / deactivation over (x-direction structured) light sheet and CCD wide-field detection.
• the sample is marked, for example, with Dronpa and can be turned on (activated) above 405 nm and excited at 488 nm or switched off again.
• the lasers (1) are designed for 405 nm (photoactivation) and 488 nm (fluorescence excitation and photodeactivation) and are combined via a beam union (2) and dichroic mirrors.
• photoactivation and fluorescence excitation it is also possible to use one and the same wavelength as explained above using the example of DENTRA.
• An AOTF (3) is used for wavelength selection and fast switching / attenuation of the laser wavelengths.
• PBS pole splitter
• the light is transmitted via cylinder optics (7) to produce a light sheet and imaging optics (8) via beam paths (10) or (11) for photoactivation (or else deactivation or excitation) via the single-mode fibers (6) to the sample (12). irradiated.
• the optical path lengths are adjusted accordingly.
• a displacement of the standing wave field in the focal plane to illuminate the sample in the intensity minima is done by adjusting the relative phase between the two sheets, for example with a phase modulator (PH).
• the light generated in the sample (12) is detected via a detection objective (13) (microscope objective) in a detection beam path (14) via a tube lens (15) and emission filter (16) by means of a CCD camera. Dashed in the detection beam path is an optional (swivelable) color splitter (18) for reflection of a laser (22) or a wide-field light source (23). shown if the fluorescence excitation by the detection lens.
• a laser or the wide-field light source (23) can also be used for activation, wherein instead of a wavelength of 488 nm, a wavelength of 405 nm is to be provided when DRONPA is used as the dye.
• a scanner unit (21) is provided for the laser beam path (22), which allows a punctiform scanning of the image field. Between the scanner unit (21) and the color divider (18), an imaging optical system (19) adapted for this purpose is provided.
• phase plates (9) can be introduced into the illumination beam paths.

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• 2008
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