DE102006046111A1 - Laser scanning microscope for measuring fluorescence anisotropy of sample, has evaluation module deriving fluorescence anisotropy from detected fluorescent radiation in consideration of direction of stimulated radiation - Google Patents

Abstract

The microscope has a lambda/4 compensator (2) emitting stimulated radiation of a laser (1) as linear polarized stimulated radiation (AS). Polarization direction of the radiation is turned with a predetermined angular speed. A scanner (7) guides the polarized stimulated radiation over a sample (P) in order to stimulate a fluorescent radiation (FS), and a detection module (10) detects the stimulated fluorescent radiation. An evaluation module (14) derives fluorescence anisotropy from the detected fluorescent radiation in consideration of the direction of the stimulated radiation. An independent claim is also included for a laser scanning microscopic method for measuring fluorescence anisotropy of a sample.

Description

The The invention relates to a laser scanning microscope and a laser scanning microscopy method for measuring the fluorescence anisotropy of a sample.

at Previously known method, the sample is time sequentially with linearly polarized excitation light of a first direction and then a second direction perpendicular to the first direction, applied and sequentially fluorescence radiation measured. From these measurements, it is, for example, at Difficult to macromolecules to extract all factors that influence the fluorescence anisotropy exercise, like e.g. the size, shape, Orientation and / or flexibility of the macromolecule.

Further it is known to excite the sample with unpolarized light and to provide two detection channels, in which the fluorescence radiation of a first polarization state and the fluorescence radiation of a second polarization state simultaneously to eat. However, this is very expensive, since two detection channels with corresponding optics, Provide detectors and evaluation is provided.

outgoing It is the object of the invention to provide a laser scanning microscope to measure the fluorescence anisotropy of a sample provide, with the difficulties described above can be. Furthermore, a laser scanning microscopy method for measuring the fluorescence anisotropy of a sample become.

The Task is solved through a laser scanning microscope to measure the fluorescence anisotropy of a Sample, with an excitation module containing a laser, the excitation radiation emits, a polarization unit, the supplied excitation radiation of the Lasers emits as linearly polarized excitation radiation whose Polarization direction at a predetermined angular velocity rotates, and has a scanner, that of the polarization unit emitted polarized excitation radiation over the sample leads to To excite fluorescence radiation, a detection module that stimulates the excited Fluorescence detected, and an evaluation module, the the detected fluorescence radiation taking into account the polarization direction the excitation radiation derives the fluorescence anisotropy.

By is the rotating polarization direction of the excitation radiation it is possible to detect the same portion of the sample several times, with each time the polarization direction of the excitation radiation is rotated Has. From the measured values can then even for each detected point of the sample section fluorescence anisotropy be determined quantitatively.

Under Fluorescence anisotropy here becomes especially the dependence the intensity the fluorescence radiation from the polarization direction of the linear understood polarized excitation radiation.

The Angular velocity is preferably constant. She can, however not be constant. In particular, the angular velocity changed or to be set to certain predetermined values.

The Excitation module focuses the linearly polarized excitation radiation preferably linear on or in the sample and leads the focused excitation radiation across the linear extension over the Sample. This is a fast line scanner to realize with a sample section can be detected quickly. For example, image acquisition rates easily possible from 50 to 100 frames per second.

Especially the microscope is designed so that the depth of focus in the sample variable is so that optical Cuts can be performed in different sample depths. To the fluorescence radiation is preferably detected confocally to an excellent depth resolution to reach.

The Angular velocity of rotation of polarization direction of Excitation radiation can be selected be that pro Rotation of the polarization direction by 180 ° at least three times (preferably five to ten times) the same portion of the sample is detected. This can be then excellent dependence the fluorescence from the polarization direction of the excitation radiation capture quantitatively.

at The laser can linearly polarize the excitation radiation of the microscope and the polarization unit can rotate a λ / 2 plate for the wavelength have the excitation radiation through which the excitation radiation goes through, causing the rotation of the polarization direction is effected. That's it in a simple way, the desired To realize rotation.

Especially is the axis of rotation of the λ / 2 plate in addition to the cross section of the excitation radiation, so that the to be provided Rotary axis no shading effect or other influences on the Excitation radiation exerts.

The polarization unit can be a dre In this case, the excitation radiation passes as a circularly polarized excitation radiation through the rotating λ / 4 plate, whereby the desired rotations of the polarization direction is effected. Also in this case it is preferred that the axis of rotation of the λ / 4 plate is adjacent to the cross section of the excitation radiation (the axis of rotation is laterally offset from the center axis of the excitation radiation, for example).

Especially the laser can emit the excitation radiation linearly polarized and the polarization unit has a second λ / 4 plate which is fixed and so oriented that they the linearly polarized excitation radiation of the laser in circular converted polarized excitation radiation, which then on the rotating λ / 4 plate is steered.

The Task is further solved by a laser scanning microscopy method for measuring fluorescence anisotropy a sample in which generates linearly polarized excitation radiation whose polarization direction is at a predetermined angular velocity turns and over one Tested is to excite fluorescence radiation, in which the excited fluorescence radiation is detected and at which from the detected fluorescence radiation considering the direction of polarization, the excitation radiation the fluorescence anisotropy is derived.

With this method can be quantitatively determine fluorescence anisotropy very well.

Especially the linearly polarized excitation radiation is linear or focused into the sample and transverse to the linear expansion over the Tested. This can quickly get the desired Sample section scanned and detected.

The Angular velocity can be chosen in particular so that per rotation the polarization direction by 180 ° at least three times the same section of the sample is detected. It is preferred the same section five up to ten times per 180 ° conversion detected.

at The microscopy method can be used to generate the excitation radiation first generated linearly polarized laser radiation and by a rotating λ / 2 plate for the wavelength led the laser radiation be, causing the rotation of the polarization direction and thus the excitation radiation is generated.

Prefers the axis of rotation of the λ / 2 plate is next the laser radiation.

at The microscopy method can be used to generate the excitation radiation first generates circularly polarized laser radiation and by a rotating λ / 4 plate guided , causing the rotation of the polarization direction and thus the excitation radiation is generated.

The Axis of rotation of the λ / 4 plate may be adjacent to the cross section of the laser radiation.

at The microscopy method can also be used to generate the excitation radiation first generated linearly polarized laser radiation and through a second λ / 4 plate guided which is fixed and oriented so that the linearly polarized laser radiation converted into the circularly polarized laser radiation, which then through the rotating λ / 4 plate guided becomes.

The The invention will be described by way of example with reference to the accompanying drawings explained in more detail. It demonstrate:

1 a schematic view of a first embodiment of the laser scanning microscope;

2 a front view of the rotating analyzer 5 from 2 ;

3 a front view of the rotating analyzer 5 from 1 in a different rotational position than in 2 ;

4 schematically the intensity of the fluorescence radiation for a point as a function of the angle of rotation of the polarization direction of the excitation radiation, and

5 another embodiment of the laser scanning microscope.

At the in 1 In the embodiment shown, the laser scanning microscope for measuring the fluorescence anisotropy of a sample comprises a laser 1 , in this order a λ / 4 compensator 2 , a beam shape optics 3 , a deflecting mirror 4 , a rotatable analyzer 5 , a beam splitter 6 , a scanner 7 as well as a lens 8th are subordinate. These elements form together with the laser 1 an excitation module 9 of the laser scanning microscope.

Furthermore, the laser scanning microscope comprises a detection module 10 that the beam splitter 6 is downstream and a detection optics 11 and a detector 12 includes.

Furthermore, there is a control module 13 provided that the laser 1 , the analyzer 5 , the scanner 7 and the detector 12 controls and also at the same time the signals of the detector 12 receives and to an evaluation module 14 forwards.

During operation, the laser generates 1 Excitation radiation AS of a predetermined wavelength λ is to be excited with the fluorescence radiation FS in the sample P to be examined. The laser 1 generated excitation radiation AS is linearly polarized here, the polarization direction is in the plane of the drawing. The linearly polarized excitation radiation AS impinges on the λ / 4 compensator 2 , which is aligned so that the λ / 4 compensator 2 emitted excitation radiation is circularly polarized. The circularly polarized excitation radiation then strikes the beamform optics 3 which changes the beam cross section of the circularly polarized excitation radiation from the originally substantially circular cross section into a line-shaped cross section.

The circularly polarized excitation radiation with a line-shaped cross section becomes over the deflecting mirror 4 on the analyzer 5 steered and passes through the analyzer 5 , The analyzer 5 generates in turn linearly polarized excitation radiation (still with a line-shaped cross-section), the direction of the linear polarization due to the fact that the analyzer 5 turns, is changed continuously.

In 2 is a front view of the analyzer 5 shown in the schematic, the polarization direction, the transmitted excitation radiation AS after passing through the analyzer 5 indicated by the arrows P1.

As 2 can also be taken, is the axis of rotation 15 to which the analyzer 5 turns, outside the beam cross section of the excitation radiation AS, so that no shading or disturbance of the excitation radiation due to the mechanical axis of rotation to be provided 15 given is.

Because the analyzer 5 around the axis of rotation 15 is rotated at a predetermined speed (arrow P2) is at a second time after the in 2 time shown the analyzer 5 in another rotational position, the in 3 is shown schematically. At this time, therefore, the polarization direction of the linearly polarized excitation radiation AS after passing through the analyzer 5 rotated by about 45 °, as can be seen from the schematic representation of the arrows P1.

The linearly polarized excitation radiation AS with the time-varying or rotating polarization direction impinges on the beam splitter 6 and turns it into a scanner 7 reflected, the excitation radiation AS to the lens 8th which focuses the excitation radiation onto or into the sample P. The scanner serves to guide or scan the excitation radiation AS transversely to its longitudinal direction over the sample P, so that a predetermined image field of the sample P is subjected to the excitation radiation.

In the sample, fluorescence radiation is generated due to the excitation radiation, which is transmitted via the objective 8th on the scanner 7 meets, which acts for the fluorescent radiation FS descending, so that the fluorescence radiation FS to the beam splitter 6 is reflected from the beam splitter 6 to the detection optics 11 and onto the linear, spatially resolving detector 12 meets. It is thus possible to simultaneously detect the fluorescence radiation which is generated in the line-shaped region at a time which is impinged by the linear excitation radiation, in which case the detection is confocal, so that an excellent depth resolution takes place (in the propagation direction of the excitation radiation AS). By scanning transversely to the longitudinal direction of the linear excitation radiation, an entire image or an optical section of the predetermined sample regions can be detected in a depth of the sample P quickly.

The rotational speed of the analyzer 5 is now chosen so that per rotation of the polarization direction by a total of 180 °, the entire sample area is scanned and detected at least three times. For example, at an image acquisition rate of 100 images per second, the rotation frequency of the analyzer 5 be chosen so that it is 5 Hz. This means that one image per 18 ° rotation angle or 10 images per rotation angle range of 180 ° is / are recorded.

In 4 is schematically shown for a point of the recorded image, the measured intensity in 10 ms steps, which corresponds to a change in the rotation angle of the linear polarization of the excitation radiation of 18 °. The solid line is that by means of the evaluation module 14 derived therefrom intensity curve IK, which shows the fluorescence anisotropy as a function of the polarization direction of the excitation radiation. From this intensity curve IK for a point, for example, a fluorescence anisotropy value can be derived which is defined as (I (parallel) - I (perpendicular)) / (I (parallel) + I (perpendicular)).

I (parallel) is the measured intensity when the polarization of the Excitation radiation parallel to the anisotropic preferred direction of is to be measured sample. I (vertical) is the fluorescence intensity when the polarization direction of the excitation radiation perpendicular to the anisotropic Preferred direction is.

With the described method is thus measured the fluorescence radiation of each point of the sample area, which was generated by the excitation radiation, wherein the polarization direction of the excitation radiation is always rotated by 18 ° between two measurements of the fluorescence radiation of the same point.

The However, it also means that if the sample area is scanned from left to right and whole left the first measurement is performed with the polarization direction 0 °, the measurement in the middle of the range then with a rotation of the polarization direction of 9 ° is executed. At the next Passage then becomes the measurement of the left area with one turn the polarization direction of 18 ° executed and the measurement of the center is performed with a rotation of 27 °. The difference of the rotation However, the direction of polarization between two measurements is for each Point in the example described here 18 °.

Of the absolute difference of the rotational position of the polarization direction the excitation radiation for different Scanning direction dots (e.g., left area and center of scan area) is therefore constant and can therefore be taken into account mathematically become.

The rotational speed of the analyzer 5 is preferably constant and can be varied, so that always the rotational speed in dependence on the measuring speed (ie the number of frames per second) can be set, which provides the desired number of measured values (nodes) per rotation of the polarization direction by 180 °.

The described measurement can for different optical sections (ie in different sample depths) carried out so that one Three-dimensional image of the fluorescence anisotropy of the sample can be determined can.

In 5 is a modification of the laser scanning microscope of 1 shown, wherein like elements are designated by like reference numerals and reference is made to the description of the above statements.

The embodiment of 5 is different from that of 1 in that instead of the λ / 4 compensator 2 and the analyzer 5 a λ / 2 plate 16 between the deflecting mirror 4 and the beam splitter 6 is arranged. As a result, the excitation radiation AS, which is on the λ / 2 plate 16 meets, linearly polarized, since the laser emits linearly polarized laser radiation. Thus, upon rotation of the λ / 2 plate 16 the linear polarization direction through the λ / 2 plate 16 rotated through the excitation radiation AS. Therefore, in the same way as in connection with 1 described embodiment, the sample with the linearly polarized excitation radiation whose polarization direction is rotated, are applied.

The beam splitter 6 is in the embodiments of 1 and 5 formed as a linear mirror, which is just so large that it the excitation radiation AS with a linear cross-section to the scanner 7 hinreflektiert. However, the fluorescence radiation has, as a rule, a larger cross section than the linear cross section of the excitation radiation, so that only the part of the fluorescence radiation incident on the linear mirror 6 meets, not to the detection optics 11 arrives. Thus, however, the majority of the fluorescence radiation reaches the detection optics 11 and can by means of the detector 12 be detected.

Around To produce three-dimensional images of the sample is known in the art Make the excitation radiation at different depths in the Sample focused and then across the scanner at this depth across to the linear Distraction of the excitation radiation deflected.

With this microscope it is possible the orientation of a labeled with fluorescent dye molecule precisely determine. With that you can for example in connection with distance-sensitive experiments precise and novel information about molecular interactions of cell structures and cellular compartments to be delivered. There are also statements about the rotation correlation times of proteins or degradation processes. So can also fibril angles of fibers (e.g., wood, textile polymer) are determined become. The microviscosity of cell membranes can be determine. It is also possible To capture quantifications of association reactions between biomolecules. In particular, the location of the molecule dipole (e.g., for measurement the fibril angle in cellulose fibers) can be precisely analyzed.

Claims (16)

Laser scanning microscope for measuring the fluorescence anisotropy of a sample (P), with an excitation module ( 9 ), which has a laser ( 1 ) emitting excitation radiation (AS), a polarization unit ( 2 . 5 ; 16 ), which supply the supplied excitation radiation (AS) of the laser ( 1 ) emits as linearly polarized excitation radiation whose polarization direction rotates at a predetermined angular velocity, and a scanner ( 7 ), which corresponds to that of the polarization unit ( 2 . 5 ; 16 ) emitted polarized excitation radiation over the sample (P) leads to excite fluorescence radiation (FS), a detection module ( 10 ), which detects the excited fluorescence radiation (FS), and an evaluation module ( 14 ), which from the detek fluorescence radiation. taking into account the polarization direction of the excitation radiation, the fluorescence anisotropy is derived. Microscope according to Claim 1, in which the excitation module ( 9 ) linearly excitation radiation linearly focused on or in the sample (P) and transverse to the linear expansion over the sample (P) leads. Microscope according to one of the above claims, at the angular velocity is chosen so that per rotation of the polarization direction at least 180 ° three times the same portion of the sample (P) is detected. Microscope according to one of the preceding claims, in which the laser ( 1 ) emits the excitation radiation linearly polarized and the polarization unit ( 2 . 5 ; 16 ) a rotating λ / 2 plate ( 16 ) for the wavelength of the excitation radiation (AS) through which the excitation radiation (AS) passes, whereby the rotation of the polarization direction is effected. Microscope according to Claim 4, in which the axis of rotation of the λ / 2 plate ( 16 ) is adjacent to the cross section of the excitation radiation (AS). Microscope according to one of Claims 1 to 3, in which the polarization unit comprises a rotating λ / 4 plate ( 5 ) for the wavelength of the excitation radiation and the excitation radiation as circularly polarized excitation radiation through the rotating λ / 4 plate ( 5 ), thereby causing the rotation of the polarization direction. Microscope according to claim 6, wherein the axis of rotation of the λ / 4-plate ( 5 ) is adjacent to the cross section of the excitation radiation. Microscope according to Claim 6 or 7, in which the laser emits the excitation radiation in a linearly polarized manner and the polarization unit ( 2 . 5 ) a second λ / 4 plate ( 2 ), which is fixed and oriented so that it the linearly polarized excitation radiation of the laser ( 1 ) converts into circularly polarized excitation radiation. Laser scanning microscopy method for measurement the fluorescence anisotropy of a sample (P), at the linear polarized excitation radiation is generated, the polarization direction rotates at a predetermined angular velocity and the over a Sample (P) guided is used to stimulate fluorescence radiation (FS), where the excited Fluorescence radiation (FS) is detected and at the time of the detected fluorescence radiation taking into account the polarization direction the excitation radiation, the fluorescence anisotropy is derived. A microscopy method according to claim 9, wherein the linearly polarized excitation radiation linearly onto or into the sample (P) focused and transverse to the linear expansion over the Sample (P) guided becomes. A microscopy method according to claim 9 or 10, wherein the angular velocity is chosen so that per rotation of the polarization direction at least 180 ° three times the same portion of the sample (P) is detected. Microscopy method according to one of claims 9 to 11, in which initially generated for generating the excitation radiation linearly polarized laser radiation and by a rotating λ / 2 plate ( 16 ) is guided for the wavelength of the laser radiation (AS), whereby the rotation of the polarization direction causes and thus the excitation radiation is generated. Microscopy method according to Claim 12, in which the axis of rotation of the λ / 2 plate ( 16 ) is adjacent to the cross section of the laser radiation (AS). Microscopy method according to one of claims 9 to 11, in which initially generated for generating the excitation radiation circularly polarized laser radiation and by a rotating λ / 4-plate ( 5 ) is guided, whereby the rotation of the polarization direction causes and thus the excitation radiation is generated. Microscopy method according to claim 14, wherein the axis of rotation of the λ / 4-plate ( 5 ) is adjacent to the cross section of the laser radiation. Microscopy method according to claim 14 or 15, in which the laser radiation is first generated in a linearly polarized manner and transmitted through a second λ / 4 plate ( 2 ), which is fixed and oriented so that it converts the linearly polarized laser radiation into the circularly polarized laser radiation.