DE102009034347A1 - Adjustment element for setting the pinhole position - Google Patents

Abstract

Laser Scanning Microscope (LSM) with an adjustment element for setting the pinhole position, consisting of a mirror which can be introduced into the beam path for reflecting the illumination light in the direction of the microscope detection,
characterized in that
the mirror is mounted in a lens mount which can be introduced into the objective turret of the LSM, and at least one lens for focusing the illumination light onto the mirror is arranged in front of the mirror in the illumination direction.

Description

State of the art

In 1 is a schematic extract from the analog representation of the structure of a laser scanning microscope (LSM) in DE 19702753 A1 . US 6563632 shown.

On the picture description given there is referred to below.

For the present invention is important that the sample by means of a scanning module scanned via a microscope beam path and the light originating from the sample (for example fluorescent light) through the lens and a tube lens back towards the detection via a scan lens and the scanner.

In Direction of detection are for confocal detection in front of the detectors Pinholes provided in their size and position are adjustable (see again, for example, the above publication).

It is important that the pinhole opening optimally for incident light is positioned to be an unnecessary Avoid vignetting of the sample light.

The anyway, weak sample light in some fluorescence phenomena should not be unnecessarily reduced in its luminous efficacy.

to correct setting of the pinhole to the detection beam path is the pinhole, for example, in a direction perpendicular to the optical Axis moves.

Out DE 10 2005 020 542 A1 Various optical arrangements for adjusting the Pinholelage are known.

In the non-prepublished patent application DE 10 2008 007 178 a calibration device which can be introduced into the beam path is provided in an objective revolver which contains a test structure which may have reflective grating elements.

The The invention will be described below with reference to the schematic drawings explained in more detail.

2 shows an invention on a Objektivanschraubfläche 4 optical adjustment element to be fastened, which can be unscrewed into the objective revolver (not shown) at a free point.

It has a mirror on its underside 7 , Preferably, a plane mirror, which reflects the light of the LSM light source.

The mirror can also have a curvature in order to adapt the image position, for example, to wavelengths used for which the transmitted (intermediate) image is color-corrected. For each other wavelength then an individual curvature (focusing / defocusing) is necessary. The mirror must not necessarily be in an image plane with a suitable curvature. The arrangement of lens (s) ( 6 ) and mirrors ( 7 ) can also be designed achromatic, so that an adjustment of the pinhole over a wide spectral range is possible.

If the pinhole calibration is to take place for only one wavelength, the use of the lens (s) ( 6 ) are waived. For the mirror ( 7 ) is then to choose a suitable for the wavelength of the illumination light curvature. Likewise, the use of a plane mirror is possible. Then the adjustment can be made optimally for the wavelength for which the entire optical system of the microscope is designed.

The illumination light is on the mirror 7 via a lens arrangement 6 focused and arrives on his way back, here via a reflector 3 in the direction of detection via the tube lens 2 of the microscope (one or more lenses are possible) in an intermediate image plane 1 in front of a scan lens, which is not shown here (auf 1 and the related description in DE 19702753 A1 is referred). A possible position of the pupil of the optics 6 is with 5 shown schematically.

Advantageous are the mirror 7 and the lenses 6 firmly integrated in a lens mount, which schematically as a sleeve 8th in 2 is shown.

On This way, the adjustment can be comfortable screw in and out and it is conceivable that the application optimized additional adjusting elements are introduced into the nosepiece can.

With a displacement of the pinhole in front of the detector perpendicular to the optical axis can now on the detector passing through the pinhole reflection signal of the mirror 7 be detected and a displacement of the pinhole as in DE done until the detection signal is maximized or corresponds to a predetermined value, for example, a lower threshold for the detection sensitivity.

Then, by adjusting the objective turret, the adjusting element swung out and the actual microscope objective is swiveled in.

The Invention has the advantage that all imaging elements of the microscope and the scan module in the optimization process of Pinholelage can be included.

Furthermore, by the additional optical elements 6 which can be broadly corrected spectrally, a broad spectral range are covered, ie the different light sources of LSM with different wavelengths can be adjusted with one and the same adjustment element with respect to the Pinholelage and the optimal Pinholestellung can for the individual wavelengths (as described in DE) stored and assigned during operation.

A further adjustment of the beam position to the pinhole under the possible use of the invention Adjustment element will be described below.

Here the pinhole can be fixed in its position to the optical axis or additionally also be displaceable.

3 shows the beam path of the ZEISS LSM 710
( http://www.zeiss.de/C1256CFB00332E16/0/F7BE630BECC94A08C12573F3005483F4/$file/60-1-0001_d.pdf )
with the inventive concept with two main color splitters HFT2 for the substantially visible region and HFT1 for the substantially invisible region and an additional tunable laser.

The light of two laser or laser groups LQ1 and LQ2 passes through main color divider HFT1 and HFT2 for the separation of illumination and detection beam path, which can be configured as switchable dichroic filter wheels and can also be interchangeable to make the selection of wavelengths flexible, and reflect the illumination light first via a scanner, preferably consisting of two independent galvanometric scanning mirrors for the X and Y deflection, in the direction of a scanning optics SL and over this and the microscope objective in a conventional manner (see 1 and related explanation) to the sample. The sample light passes in the reverse direction through the dividers HFT1, HFT2 in the direction of detection D.

Here the detection light first passes over a pinhole PH a Pinholeoptik PHO upstream and downstream of the pinhole and a Filter arrangement F for narrow-band filtering unwanted Radiation components, consisting for example of notch filters, and passes through a beam splitter BS, which optionally with appropriate circuit over a transmissive component a decoupling to external detection modules allows a mirror M as well as other deflections a grating G for the spectral splitting of the detection radiation.

The divergent spectral components split by the grating G are determined by means of of an imaging mirror IM collides and gets in direction a detector assembly consisting of individual PMT1, PMT2 in the edge region and a centrally located multi-channel detector MPMT.

Instead of of the multichannel detector can also be another single detector used become.

In front a lens L1 are two perpendicular to the optical axis displaceable prisms P1, P2 in the edge region; which is part of the Unite spectral components, which on the lens L1 on the individual PMT1 and 2 are focused. The remainder of the detection radiation becomes after passing through the plane of PMT1 and 2 over a second lens L2 is collimated and spectrally separated to the individual Detection channels of the MPMT directed.

By Displacement of the prisms P1, P2 can be set in a flexible manner which part of the sample light is spectral over the MPMT is detected separated and which part via the prisms P1, P2 summarized by PMT1 and 2 is detected.

4 shows the operation of an adjustable coupling mirror M from 1 , It is schematically shown the design of the mirror as a solid-state articulation mirror for tilting the mirror in one direction (eg x-direction).

Of the shown lever is usually operated by a motor an adjustment of the mirror angle around the pivot point shown to realize.

The Use of solid-state joints allows an extremely Precise backlash adjustment small deflection angle of the Laser light. By combination with another solid-body joint can be an additional tilt of the mirror realize in y-direction.

By adjusting the mirror M, the position of the illumination beams LQ1 and LQ2 to the optical axis and thus to the pinhole PH is slightly shifted and pivoted-adjusting according to 2 the adjustment described can be done.

Of the Focus of the illumination light does not have to be compelling are exactly in the Pinholeebene, but this is preferred because the adjustment is then best possible.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• DE 19702753 A1 [0001, 0015]
• US 6563632 [0001]
• DE 102005020542 A1 [0008]
• - DE 102008007178 [0009]

Cited non-patent literature

• - http://www.zeiss.de/C1256CFB00332E16/0/F7BE630BECC94A08C12573F3005483F4/$file/60-1-0001_d.pdf [0024]

Claims (12)

Laser scanning microscope (LSM) with an adjusting element for adjusting the pinhole position, consisting of an insertable into the beam path mirror for reflecting the illumination light in the direction of the Mikrokopdetektion, characterized in that the mirror is mounted in a einbringbaren in the nosepiece of the LSM lens mount and at least one lens for focusing the illumination light on the mirror is arranged in front of the mirror in the illumination direction. Laser scanning microscope according to claim 1, wherein the mirrored in the illumination direction last lens surface is trained. Laser scanning microscope according to claim 1 or 2, wherein the mirror surface via the microscope arrangement Intermediate image levels is focused in the Pinholeebene. Laser scanning microscope according to one of the preceding Claims, wherein the mirror surface plane, spherical or aspherical. Laser scanning microscope according to one of the preceding Claims, wherein in the illumination direction a lens surface a microscope objective in a spectral range not used for observation is formed mirrored. Laser scanning microscope according to one of the preceding Claims, wherein a microscope objective as an adjustment serves. Adjustment element for setting the pinhole position an LSM consisting of a mirror, the at least one lens is arranged upstream of the focusing of the illumination light. Adjustment element according to claim 7, wherein the in the illumination direction last lens surface is formed mirrored. Adjusting element according to claim 7 or 8, wherein the Mirror surface designed plane, spherical or aspherical is. Adjusting element according to claim 7, 8 or 9, wherein in the illumination direction, a lens surface of a microscope objective mirrored in a non-observed spectral range is trained. Adjusting element according to one of claims 7-10, wherein a microscope objective is the adjusting element. Method for adjusting a pinhole in one Laser scanning microscope with an adjustment of the pinhole and / or a displacement of the optical axis of the detection beam path takes place, except for at least one detector of the LSM a maximum Signal is set, characterized in that one optical adjusting element is pivoted into the beam path that of a mirror and an upstream in imaging direction Lens arrangement exists and reflected from the mirror illumination light is detected, wherein for one or more wavelengths the adjustment of the pinhole or displacement of the optical axis he follows and upon reaching a maximum or predetermined Detection signal the Pinholestellung and / or position of the optical Axis is stored.

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