Classifications

• • 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
  • G02B21/0004—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
• • 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
• • 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

Definitions

• the invention relates to an optical arrangement in the beam path of a confocal fluorescence microscope, with at least one laser light source, a device arranged in the illumination / detection beam path for separating the excitation light reflected on the object from the fluorescent light emitted by the object, a lens arranged between the device and the object and one Detector downstream of the device in the detection beam path
• dichroic beam splitters to separate the excitation light reflected on the object from the fluorescent light emitted by the object. In the case of simultaneous multi-color application, multiple dichroic beam splitters are used accordingly.
• the fluorescent light freed from the reflected excitation light is used - after separation by means of a beam splitter - detected by means of a special detector
• the beam splitters used in practice are generally expensive These beam splitters are not very suitable for quantitative comparative measurements of high precision and high measurement dynamics, especially since these beam splitters are also temperature-dependent.
• dichroic beam splitters have transmission losses of around 10% for detection
• the entire numerical aperture is used for illumination or for scanning. This leads to an illumination focus that is very small in the lateral direction, so that longer ones
• the present invention is based on the object of designing and developing an optical arrangement in the beam path of a confocal fluorescence microscope in such a way that an increase in the fluorescence yield can be achieved with a simple design compared to the generic arrangement with a conventional dichroic beam splitter
• the optical arrangement according to the invention in the beam path of a confocal fluorescence microscope achieves the above object by the features of patent claim 1.
• the optical arrangement mentioned at the outset is characterized in that the device comprises a mirror and that the mirror in the illumination / detection beam path is arranged and dimensioned in such a way that that it does not expand the dark field illumination of the object coming from the laser light source Reflected excitation beam in the lens and the fluorescent light coming from the object with a full numerical aperture - reduced by the effective cross-section of the mirror in the detection beam path - in the direction of the detector.
• the Device comprises a mirror and that the mirror in the illumination / detection beam path is arranged and dimensioned such that it reflects the unexpanded excitation beam coming from the laser light source into the lens for dark field illumination of the object and reduces the fluorescent light coming from the object with a full numerical aperture around the effective cross section of the mirror in the detection beam path - in the direction of the detector
• the device for separating the excitation light reflected on the object from the fluorescent light emitted by the object - instead of a conventional dichroic beam splitter - can be a mirror which is arranged in the illumination / detection beam path.
• This mirror is here to be dimensioned in such a way - sufficiently small - that it reflects the unexpanded excitation beam coming from the laser light source into the objective and illuminates the fluorescent light coming from the object with a full numerical aperture in the direction of the detector, with the fluorescent light around the im Detection beam path effective cross-section of the mirror is reduced.
• the main reflection of the excitation light reflected by the object is advantageously reflected out of the detection beam path at the mirror
• the mirror is dimensioned or made small in such a way that it causes a loss of approximately 1% for the fluorescent light to be detected in the detection beam path. This enables efficient detection with a particularly high dynamic range
• the mirror used here could be designed as an independent component in the context of a particularly simple embodiment, the mirror in turn being carried by a holder. In the context of such a configuration, the use of a conventional beam splitter is completely eliminated, since here only the small one is in the beam path at the appropriate point Mirror is arranged
• the mirror could be designed as a preferably integral mirrored area of an - otherwise conventional - beam splitter, the mirror or the mirrored area being at least largely arranged or formed in the center of the beam splitter. This small - integral - mirror could Its shape is approximately round or elliptical or oval.
• this is a mirrored area in the middle of a beam splitter which, like an isolated mirror, reflects the unexpanded excitation beam into the lens
• the lighting takes place with an extremely small numerical aperture to favor the dynamics
• the non-reflecting area of the beam splitter could have approximately 10% reflection and 90% transmission in the direction of the detector.
• this could be an anti-reflection (AR) coating on the side of the beam splitter facing the detector.
• This beam splitter does not Fluorescence light in accordance with the usual - infinite - beam path with the full numerical aperture - reduced by the fluorescence light striking the mirrored area - passing in the direction of the detector of the mirror
• losses of only 1% can be realized, which leads to the already mentioned high dynamic
• the non-reflecting area of the beam splitter could be designed and possibly coated in such a way that the optical properties of the non-reflecting area of the beam splitter are at least largely independent of temperature. The reproducibility of the measurement is thereby favored
• the object could be arranged at an angle. This means that the object itself is not orthogonal to the optical one Axis is arranged, so that there is a separate beam path for the back-reflected excitation light, which is at least slightly - spatially - offset from the illuminating beam path.
• This special beam path allows conventional light traps to be arranged without any problems in order to effectively mask out the back-reflected excitation light
• the object can be moved in its plane in such a way that the illuminated object area always has the has the same distance from the lens.
• the light reflected and / or scattered on the object does not return to the laser light source in accordance with the above explanations, but instead into a light trap via the special beam path insulated component - have an absorbing area for absorbing the excitation light scattered and / or reflected by the object.
• the provision of special light traps is no longer necessary, which results in a spatial reduction in the arrangement.
• the mirror - in the context of a round or elliptical or oval configuration - Have a specular or reflective area and an absorbent area
• the beam splitter has, in addition to the mirrored area, an absorbing area for absorbing the excitation light scattered and / or reflected by the object.
• the provision of a special light trap can also be omitted, so that this embodiment also has the advantage involves being able to build the arrangement with the smallest space requirement
• Laser light sources an at least approximately the same - axial - focus position is possible.This on the one hand provides the necessary lighting tolerance in the object position along the optical axis and on the other hand ensures lighting with a high intrascene dynamic range.To avoid repetition, reference is made to the previous statements to avoid repetition
• FIG. 1 shows a schematic representation of the basic structure of an optical arrangement according to the invention in the beam path of a confocal Fluorescence microscope
• FIG. 2 shows a schematic representation of a beam splitter with a central mirror
• FIG. 3 shows a schematic representation of a mirror that can be used instead of the beam splitter as a separate component
• FIG. 4 shows a schematic illustration of a mirror with an integral reflecting and absorbing area
• FIG. 1 shows a schematic representation of an exemplary embodiment of an optical arrangement in the beam path of a confocal
• the device 6 comprises a mirror 13, the mirror 13 being arranged and dimensioned in the illumination / detection beam path 3, 4, 5 such that it is used for dark field illumination of the object 7, the unexpanded excitation beam coming from the laser light sources 1, 2 or that Excitation light 8 is reflected into the objective 10 and the fluorescent light 9 coming from the object 7 with a full numerical aperture - reduced by the cross section of the mirror 13 effective in the detection beam path 4, 5 in the direction of the detector 11.
• the device 6 is designed as a beam splitter, the mirror 13
• FIG. 3 shows an alternative embodiment of the device 6 in such a way that it is simply and simply designed as a singular mirror 13, the mirror 13 being an independent component.
• the mirror 13 could have an overall mirrored surface as shown in FIG. 3
• the mirror 13 is likewise designed as a separate component, the surface of the mirror 13 being subdivided into a reflecting mirror surface 17 and an absorbing surface 18. This measure makes the provision of a separate light trap for reflecting back unnecessary. it suggests 19
• the jerk reflexes or the jerk-reflected excitation peak 19 can be made "harmless" by the sample or the object 7 is skewed, that is to say is not arranged orthogonally to the optical axis 23.
• the reflected reflection excitation light 19 is displaced slightly to the illumination beam path 20 or to the beam path of the excitation light 8, so that the reflected reflection excitation light 19 is reflected back along its own beam path 21

Landscapes

• Physics & Mathematics (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Optics & Photonics (AREA)
• Health & Medical Sciences (AREA)
• Computer Vision & Pattern Recognition (AREA)
• General Engineering & Computer Science (AREA)
• Engineering & Computer Science (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Microscoopes, Condenser (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (1)

Family

ID=7880995

Family Applications (1)

Country Status (5)

Cited By (5)

Families Citing this family (16)

Citations (5)

Family Cites Families (15)

• 1998
  • 1998-09-15 DE DE19842153A patent/DE19842153C2/de not_active Revoked
• 1999
  • 1999-09-13 DE DE59913082T patent/DE59913082D1/de not_active Expired - Lifetime
  • 1999-09-13 US US09/554,083 patent/US6785302B1/en not_active Expired - Lifetime
  • 1999-09-13 EP EP99955690A patent/EP1031055B1/de not_active Revoked
  • 1999-09-13 WO PCT/DE1999/002910 patent/WO2000016149A1/de not_active Ceased
  • 1999-09-13 JP JP2000570627A patent/JP4197395B2/ja not_active Expired - Lifetime

Patent Citations (5)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events