Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/32—Micromanipulators structurally combined with 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

Definitions

• the object of the invention is therefore to develop an arrangement with which the manipulation in restricted sample areas can be carried out in a simple manner substantially in the focal plane of the microscope.
• confocal sample manipulation should also be possible without the use of multiphoton effects.
• the arrangement should also be able to enable a spatial, isotropic imaging of the sample before, after or during the manipulation.
• This object is achieved in an optical arrangement of the type described above in that the means for photomanipulation comprise a first manipulation optics, is coupled by means of the light of a first manipulation light source in the illumination beam path for forming a substantially planar manipulation light sheet.
• the manipulation light source can be a laser that emits light of a different wavelength or a different wavelength range than the actual illumination light source.
• the illumination light source itself can also be used as a manipulation light source, in which case the manipulation optics and the coupling-in elements can be dispensed with. It is also conceivable to combine several lasers in one light source module, whereby, depending on the selected method, illumination or Pulation, one or more of the light sources are selected and coupled into the illumination beam path.
• the first manipulation optics may also comprise means for structuring the light sheet.
• the light sheet itself has a substantially perpendicular to the optical axis of the microscope objective in the coordinates X and Y, adapted to the sample field to be examined matched length and width and extending in the direction of the optical axis of the imaging lens thickness, which is in the range of a few micrometers .
• This light sheet can be spatially structured, for example, by means of a slit, wherein the slit comprises one or more slots for spatial structuring.
• the structuring of the light sheets by means of grids, screens or the like makes it possible, for example, to trigger photobleaching processes limited to the bright areas and then to observe FRAP processes since no bleaching processes were triggered in the dark areas.
• a significant advantage of the SPIM technology is the ability to generate spatial images of the sample, and in the present arrangement, therefore, the sample and / or the sample holder is expediently movable, preferably mounted rotatable and displaceable. In this way, all areas of the sample can be made accessible to manipulation, in particular also to spatially strictly localized manipulation.
• the control unit is expediently designed to control both manipulation optics.
• the arrangement expediently has an evaluation unit which converts the light detected pixel-wise, for example on a planar CCD detector, into data, ie digital signals, and also evaluates the signal, wherein the signal conversion is often also carried out in the detection means itself.
• FIG. 1 shows an optical arrangement for photomanipulation of a sample 1.
• the sample 1 is picked up by a sample holder 2.
• the sample 1 may be embedded in a gel cylinder made of agarose which is fixed in the sample holder 2.
• the sample holder 2 is rotatably mounted, which here by the Arrow is indicated.
• the sample holder 2 is also slidably mounted, that is movable in all three spatial directions, so that all areas of the sample 1 can be illuminated and detected.
• the sample 1 can also be movably mounted and the sample holder 2 can be firmly constructed, so that the movements of sample 1 and sample holder 2 are decoupled.
• the sample 1 or light which comes from the sample 1 is imaged at least partially on the detection device, ie the CCD camera 6, by imaging optics with an imaging objective 7 which is located in an imaging beam path.
• the light sheet for illumination is essentially flat in the focus of the imaging objective 7.
• the optical axis of the imaging lens 7 also intersects the plane of the sheet of light at a non-zero angle, preferably perpendicular, as shown in FIG.
• the optical arrangement also has a control unit 8, which in the example has an optional nal evaluation unit 9 is combined. In the evaluation unit 9, the detected light is converted into data and evaluated. The direction in which light is detected is indicated by the arrow marked "D" between imaging objective 7 and CCD camera 6.
• the optical arrangement furthermore has means for photomanipulation of the sample 1.
• These means for photomanipulation comprise a first manipulation optics, by means of which light of a first manipulation light source 10 is coupled into the illumination beam path for forming a substantially planar manipulation light sheet.
• the direction in which manipulation light is directed to the sample is indicated by the arrow marked "M" between the lens 5 and the sample holder 2.
• the second manipulation optics can be designed as a laser scanning microscope. This allows the confocal illumination of the sample via the imaging beam path. If the deflecting mirrors 12 are configured not as fully reflective, but as partially transmissive mirrors, light from the first manipulation light source 10 can also be coupled into the imaging beam path, as indicated by the dashed line. With a corresponding configuration of the coupling element 13, even light from the illumination source 3 can be coupled into the imaging beam path and thus enables normal observation in incident light.
• a combination of the second Manupulationsoptik as shown in Figure 3, with an arrangement according to Figure 2 is possible. Another variant is shown in FIG. 4, here the first manipulation light source 10 and the second manipulation light source 16 are identical.
• Another application example is a sample that shows no response at all below a power density threshold, but only when it is excited above that threshold. If the power density of the light of the two manipulation light sources when impinging on the sample is in each case less than the threshold value, in the sum but above this, so a locally very narrow area can be selected in this way. If the sample has an absolute melting point in the range of the added power densities of the manipulation light sources, the sample 1 can be broken up at this point, for example by the superimposition of the two beams. This can be used, for example, for microdissection. The light source can be the same in the two cases just described.
• irradiating a sample labeled with Dronpa 3 dyes does not cause exposure to light of 405nm wavelength nor to irradiation of 488nm wavelength light. Only with a simultaneous combination of both excitation wavelengths, bright emissions become apparent. However, in the sample 1, these emissions occur only in the region in which the two beams overlap or intersect, that is to say in a spatially very restricted area, even if the manipulation optics in each case illuminate a larger area in each case.

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• Physics & Mathematics (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Optics & Photonics (AREA)
• Microscoopes, Condenser (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

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• 2007
  • 2007-09-28 DE DE102007047464.6A patent/DE102007047464B4/de active Active
• 2008
  • 2008-09-16 EP EP08802224A patent/EP2208101A1/de not_active Withdrawn
  • 2008-09-16 WO PCT/EP2008/007690 patent/WO2009043473A1/de active Application Filing
  • 2008-09-16 US US12/680,165 patent/US8547634B2/en active Active
  • 2008-09-16 JP JP2010526193A patent/JP5636280B2/ja active Active

Non-Patent Citations (1)

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