DE19829954A1 - Beam splitter for use in a laser scanning microscope - Google Patents

Abstract

Beam splitter to divide excitation and emission radiation in a laser scanning microscope consists of colored glass plates (FT) arranged on a rotating splitter (TR).

Description

In FIG. 4 is a microscope unit M and a scan head S are schematically shown, having a common optical interface via an intermediate image and form a LSM.

The scan head S can be attached to the phototube of an upright microscope as well as attached to a side exit of an inverted microscope become.

A microscopic beam path that can be switched between incident light scan and transmitted light scan by means of a pivotable mirror 14 is shown, with light source 1 , illumination optics 2 , beam splitter 3 , objective 4 , sample table 5 , condenser 6 , light source 7 , receiver arrangement 8 , a first tube lens 9 , an observation beam path a second tube lens 10 and an eyepiece 11 and a beam splitter for coupling the scan beam. A laser module 13.1 , 13.2 receives the lasers and is connected to the laser coupling unit of the scan head S via single-mode optical fibers 14.1 , 14.2 .

The optical fibers 14.1 , 14.2 are coupled in by means of displaceable collimation optics and beam deflection elements 17.1 , 17.2 .

By means of a partially transparent mirror 18 , a monitoring beam path in the direction of a monitor diode 19 , which is advantageously arranged upstream of a line filter 21 and a neutral filter 20 , which is advantageously arranged on a rotatable filter wheel (not shown), is suppressed.

The actual scanning unit consists of scanning objective 22 , X / Y scanner 23 , main beam splitter 24 and a common imaging optics 25 for detection channels 26.1-26.4 .

A deflection prism 27 behind the imaging optics 25 reflects the radiation coming from the object 5 in the direction of the dichroic beam guide 28 in the convergent beam path of the imaging optics 25 , the pinholes 29 which can be adjusted in the direction and perpendicular to the optical axis and whose diameter can be changed individually for each detection channel and emission filters 30 and suitable receiver elements 31 (PMT) are arranged downstream.

A control unit / computer unit 34 is provided, which is connected, among other things, to the table 5 and the scanners 23 and controls them.

The divider turret TR according to the invention and adjacent components are shown in FIG. 2.

It can advantageously be used in the beam path in FIG. 4 at the location of the beam splitter 24 for separating excitation and fluorescence or the dichroic beam splitters 28 , the dichroic beam splitters each transmitting a certain upper radiation component and reflecting the lower one in the direction of the next detection.

The motor of the divider turret can be controlled via the control unit 34 in FIG. 4.

The respective position of the Divider revolver and thus the pivoted beam splitter can be detected.

The representation is shown in FIG. 2 in the direction of view of the transmitted beam, a reflected beam Sr running as indicated in the representation.

FIG. 1 shows a side view from the direction A in FIG. 2.

Fig. 3 shows a longitudinal section through the divider turret.

The TR divider turret is inclined at 45 degrees to the vertical, thus radial Deviations in position of the revolver no deviation in the angular position of the contained divider causes.

It is at fixed bearing points OF each via a ball bearing KL, preferably resilient on one side, that is to say without play, supported and in corresponding conical or spherical recesses on the Drive axle A and articulated at the bearing points OF.

The actual part wheel, which is attached to an axis A, points Recesses in which the color splitter glasses FT preferably exchangeable are arranged.

A likewise stationary motor M drives the dividing wheel TR via a pinion R and one provided on the divider gear Z to between different dividers switch.

The motor M is controlled by a control unit.

Colored glasses are advantageous as a substrate for the exchangeable beam splitters  used, for example for single and multiphoton fluorescence.

The filter sets for classic fluorescence microscopy consist of three Components an excitation filter, a beam splitter and a blocking filter. The excitation filter can also be activated by controlling an AOTF or AOM will be realized.

The beam splitter separates the excitation radiation from the emission radiation. The excitation radiation can be reflected in the sample and the emission radiation can be transmitted for detection. (The opposite way is also possible, transmission of the excitation radiation, reflection of the emission radiation.)
As a rule, dichroic beam splitters are used here.

The emission filter blocks the excitation radiation with a high optical density and transmits the fluorescence emission.

According to the invention, the emission filter is replaced by the fact that as a substrate for a suitable colored glass is used for the dichroic beam splitter.

Above the specified wavelength, these are transmissive and reflective below, so that only the fluorescent radiation to be detected arrives at the detector.

When excited with a bandpass BP 546/12, for example, a Color splitter FT 580 (substrate OG 590 from Schott) used.

When excited with a bandpass BP 4361/2 For example, an FT 460 color splitter (substrate GG 475) is used.

The colored glasses with long-pass characteristics are particularly suitable for conventional fluorescence (1-photons). B.
WG 280, 295,. . ., 360,
GG 375, 385,. . ., 495
Floor 515,. . ., 590
RG 610,. . ., RG 850

Multiphoton fluorescence

For multiphoton fluorescence, the excitation is long-wave and Emission short-wave.

The long-wave colored glasses are particularly suitable here Excitation radiation block z. B. the BG 39 absorption glass.

A color splitter KP 685 (on size 39) is used for TiSa laser excitation. Emission filter not necessary, but possible e.g. B. as an LP filter.

Claims (1)

Beam splitter to separate excitation and emission radiation in one Laser scanning microscope, which consist of colored glasses and preferably replaceable, are arranged on a rotatable divider revolver.