JP2003195178A - Device for beam shut-off in laser scanning microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device for beam shut-off in a laser scanning microscope and a method for scanning the laser scanning microscope by using beam shut-off in order to dispense with an expensive acoustooptical modulator. <P>SOLUTION: In the laser scanning microscope scanning an image field 4, the laser beam is deflected to a stop 6 arranged near the image field 4 in order to shut off the laser beam 1 if the laser beam 1 is not intended to be incident on the image field 4. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a beam blocking device in a laser scanning microscope provided with a deflection device for guiding a laser beam onto an image forming area by a raster scanning method,
It also relates to a method for operating a laser scanning microscope in which a beam interception of the laser beam is performed.

[0002]

2. Description of the Related Art In a laser scanning microscope, a predetermined area of an object is raster-scanned by a laser beam.
In that implementation, a collimated laser beam of a few millimeters in diameter is deflected according to a desired raster-like pattern, most often by using a deflection device, like an electron beam in a cathode ray tube, for example. The deflected laser beam is focused on an intermediate image plane of a laser scanning microscope by an optical system called a scanning objective lens, and is focused on or into an object by the objective lens of the microscope. Is imaged. The beam deflection causes a raster scan of a target area of adjustable size and for optical reasons a predetermined maximum size. The laser beam sweeps a predetermined area while raster-scanning the object. The divergence of the laser beam varies according to the distance from the intermediate image plane.

The focused laser beam interacts with the object and can return as reflected or fluorescent radiation along the same path as the illuminating radiation. In many cases a beam splitter is provided between the laser and the deflection device, which beam guides the radiation received by the object into the detector beam path in a sensible manner.

In all cases, the detector beam path is provided with at least one imaging system for focusing the radiation in a further imaging plane. At least one small aperture (Bl) called a pinhole is provided in the plane.
end) and there is a detector behind the diaphragm.
Depending on the particular application, additional elements may be placed in the detector beam path.

Since the reduction portion of the laser diameter at the point of negative infinity, the object, and the pinhole exist in each plane optically conjugated, such an arrangement is confocal (ko).
nforkal). The arrangement exhibits particularly favorable image characteristics.

The main field of application of confocal laser scanning microscopy technology is the fluorescence test method, since it allows the resolution of very thin layers of an object. is there. Radiation emanating from other planes is very strongly attenuated. This situation is similar to the situation for reflective objects. Furthermore, scattered radiation originating from the interfaces and attachments of the optical components used is strongly attenuated.

Thus, in this known type of microscope use, objects that exhibit fluorescent properties upon irradiation are particularly useful. However, especially fluorescent objects are light-sensitive, i.e. the fluorescence of the objects decreases as the irradiation time increases, so it is desirable to avoid unnecessary irradiation of the objects when no image is recorded. When the laser beam is constantly incident on the object during positioning of the object and adjustment of the microscope, the fluorescent traces or holes in the object are no longer “brenned”.
do not do. This is the case, for example, when the position of the focal plane is adjusted to a new section, so in laser scanning microscopes it is common to turn off the laser beam when no image is recorded. This is called "beam interception".

In principle, the laser can be turned off for this purpose. However, this method is not convenient because the operating conditions of the conventionally used laser are unstable and the life thereof is shortened. Also, the blocking with a mechanical shutter is too late.

An acousto-optic modulator (AOM) or an acousto-optic tunable filter (AOTF) for blocking the laser beam when it can impinge on the area of the object for which imaging is not intended. It is known to interrupt the beam with. These components are typically located in the parallel portion of the unexpanded laser beam, and
Allows the laser beam to be switched on and off with a very short response time. However, such acousto-optic components are relatively expensive parts.

[0010]

The problem underlying the present invention is therefore to eliminate the need for expensive acousto-optic modulators.
A beam blocking device in a laser scanning microscope and a method of operating a laser scanning microscope using beam blocking.

[0011]

According to the invention, a beam blocking device in a laser scanning microscope comprising a deflection device for guiding a laser beam guided in an area in a raster scanning manner onto an object is provided. The problem is solved by a diaphragm provided close to the area. The deflecting device directs the laser beam to this diaphragm if the beam is not intended to be incident on the object.

The present invention thus does not use the previous principle of providing a controllable element that is permanently placed in the beam path and cutting the laser beam so that it no longer exits the element. . Instead, the laser beam is directed to and absorbed on the diaphragm. To do this, the deflection device, which is an essential part of the laser scanning microscope for the raster scanning deflection of the laser beam, is optionally controlled appropriately. The deflection device is thus used to prevent irradiation of the object by the laser beam. Therefore, blocking in the conventional sense no longer exists. The laser beam is guided during a raster scan of the object towards an aperture which lies outside the area swept by the laser beam and is absorbed there. This prevents the laser beam from reaching the target, so that damage to the target cannot occur. Following the terminology introduced, this is also referred to as beam blocking in the context of the present invention.

The speed at which this beam interception occurs is provided by the deflection device. The deflection device usually operates very fast in laser scanning microscopes because it is needed for other reasons (time to record the image) anyway.

The diaphragm, to which the laser beam is directed for beam interception, is in principle located anywhere in the optical path following the deflection device. The closer the diaphragm is to the intermediate image plane, the smaller the beam diameter of the laser beam in the diaphragm plane, and between complete irradiation and complete beam interception of the object during beam interception. The transition to is done more quickly. The cross section of the laser beam at the intermediate imaging plane of the optical system has the spot diameter, ie the most reduced size. When the laser beam is guided by the raster scanning method to the edge of the diaphragm located in the intermediate image plane, the laser beam is extremely extreme from one image point to the next image point. Quickly shut off. Therefore, the diaphragm is preferably arranged as close as structurally possible to the intermediate image plane of the microscope or in this plane.

This embodiment has the advantage that the interruption is particularly rapid because the laser beam can be guided very quickly over its beam cross section from the imaging area to the diaphragm at a given deflection speed. Not only that, since the diameter of the laser beam when it impinges on the diaphragm is almost minimal, the diaphragm need only have the size of the laser beam diameter, and thus be kept very small. It also has the advantage of gaining. Furthermore, the object may be protected from the laser beam during the reversal phase of the deflection device, which is not imaged in any way, or during a scan pause.

The diaphragm only needs to be designed such that the entire laser beam can rest on it. The diaphragm should be designed such that it covers at least the area in which the deflection device permanently directs the beam. Therefore, in principle, it is sufficient to provide a circular diaphragm of suitable size, which is arranged close to said area so that the deflection device can direct the laser beam to said circular diaphragm. It A small circular diaphragm is preferably arranged at the so-called standby position of the laser beam to which the laser beam is directed when the scan is interrupted, i.e. at a position outside the area to be imaged. It

For beam blocking, the present invention preferably uses existing deflection devices. The device may be of virtually any design so long as it is capable of directing a laser beam to the diaphragm located close to the area to be scanned. Generally, the deflection device causes biaxial deflection. Two tilting mirrors are conventionally used in laser scanning microscopes because they provide the greatest degree of freedom in guiding the laser beam onto the imaged area that is being raster scanned over the object. Because it does. In a deflection device, such as one that can permanently direct the laser beam to a single point, a simple circular diaphragm is sufficient to provide beam blocking.

However, in a deflecting device with tilting mirrors and rotating polygon mirrors, a linear or line-shaped stop is required. Conventional scanning movements typically start at one corner of the imaging area being raster scanned with respect to the object and result in diagonally opposite corners. Then, the next scanning operation starts again at the starting point of the previous scanning operation. In order to protect the object from undesired irradiation between the two scans, it has an L-shaped design so that the inner edge of the diaphragm is located at the edge of the area to be imaged and said It is preferred to deploy a diaphragm having two edges arranged close to the area,
In this case, the laser beam returns from the end point of the scanning operation to the start point of the next scanning operation on the L-shaped diaphragm so that the laser beam is scanned before the scanning operation is started. This is because it can be located on the diaphragm in the vicinity of the start point of. In doing so, the laser beam is guided to the starting position outside the imaging area, parallel to one edge of the diaphragm and then parallel to the other edge. After interruption of the laser beam, the laser beam may be repeatedly activated by simply guiding the laser beam from the stop to the starting point. This can be done very quickly.

In a further embodiment the aperture is arranged as a window surrounding the area. In that case, there is the shortest possible distance from any point of the raster scan image towards the aperture. Thus, a particularly rapid shutoff can be achieved, i.e. the beam can be shut off during the phase of inversion.

Reducing the size of the raster scan area (optoelectronic zoom) and / or the area currently being scanned for technical reasons related to inspection when operating a laser scanning microscope. It may be necessary to move the center of the in the largest possible area (deflection). To do this, the diaphragm can be fitted with a fixed size to the maximum size of the area to be raster scanned. However, the diaphragm may be arranged positionably and size-adjustably so that it can be adapted particularly preferably (within the maximum possible size of the region) as a function of the selected deflection pattern. For this purpose, the diaphragm is advantageously arranged such that each edge of the window is formed by an adjustable slice.

In the operation method of a laser scanning microscope in which an object is raster-scanned by a laser beam, the above-mentioned problem is solved in order to cut off the laser beam when it is not intended to reach the object.
The solution is that the laser beam is deflected to an aperture located close to the swept area during the raster scan of the object. This method has the advantages described above with respect to the device and enables fast laser beam blocking. Furthermore, since the blocking only requires a change in the control of the deflection device, a separate control of the blocking element is avoided.

In the method in which the light beam is guided from the start point to the end point on the object during scanning,
It is expedient to guide the image beam to the end point of the stop and return it on the stop to a point near the start point. Therefore, on the one hand, a very rapid shut-off is achieved at the completion of the scanning operation. On the other hand, the laser beam can be acted upon again quickly at the beginning of the scanning operation.

In principle, the laser beam can be guided in any manner to scan the object. However, laser
A particularly preferred embodiment with a rapid raster scan of a rectangular area is provided where the beam is deflected across the object in a first direction and back and forth and in a second direction orthogonal to the first direction. can get.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. FIG. 1 shows a schematic view of a laser scanning microscope. A collimated laser beam 1 emanating from a laser or fiber is reflected by the main beam splitter 2 in the direction of a biaxial deflection device comprising scanner mirrors 3 and 4 (x and y axes). Further, the parallel laser beam 1 is directed along the optical axis with respect to the scanning objective lens 5 at the seating positions of both mirrors. This light beam, represented by the reference 7b, produces a laser spot in the center of the intermediate image 8.

A broken line 8a on the plane of the intermediate image 8 represents the maximum drawing area. The area depends on the optical conditions and is related to the maximum size of the object to be raster scanned. The path of the light beam 7b is further represented in the direction of the objective lens 9. This light flux eventually produces a small laser spot on or in the object 10.

In the plane of the intermediate image 8, an L-shaped diaphragm 6 according to the invention (see FIG. 2c) is arranged. The function of the L-shaped diaphragm 6 will be described below. The light beam 7a schematically shows the beam path with respect to the starting point of the scanning operation, while the light beam 7c
Indicates the position of the laser beam at the completion of the scanning operation.

The radiation returning from the object 10 is reflected by the elements 9,
5, 4 and 3 in the reverse direction, and finally from the main beam splitter 2 to the optical system 11 (pinhole objective lens) depending on the application from the main beam splitter 2 in the detection beam path. The light is transmitted and focused by the optical system 11 in the plane of the diaphragm 12 (pinhole).

The light passing through the pinhole is finally
It is converted into an electric signal by the detector 13. The aperture 6 is
It serves to quickly shut off the laser beam. For this purpose the laser beam is directed to the diaphragm.
The phrase “Abschalten” should be understood from the perspective of the object 10. That is, the laser beam 1 actually illuminates the scanner mirrors 3, 4 during the interruption, but is not incident on the object 10 because it is absorbed on the diaphragm 6 thereafter.

FIG. 1 shows three different deflections of the laser beam 1. A laser beam, designated 7a, impinges on a predetermined point of the imaging area 4 to be scanned. This predetermined point is the starting point of the scanning operation. The laser beam, designated 7b, is in the middle of a scanning operation. The laser beam 7c is directed to the diaphragm 6 and therefore does not enter the object 10. Thus, the interruption of the laser beam 1 is achieved in that the object is not illuminated by the laser beam 1.

For example, FIGS. 2A to 2C show various embodiments of the diaphragm. FIG. 2A shows the circular diaphragm 14. The circular diaphragm 14 is arranged close to the starting position of the laser beam. This prevents the laser beam from reaching the object during the conditioning procedure or the scan pause. To this end, each scanner mirror is located outside the maximum representable area and slightly diagonally to the left on the left side, namely at the so-called standby position 20 shown in FIG. The beam is controlled to be incident. At the standby position 20, the beam is incident on the small circular diaphragm 14 shown in FIG.

FIG. 2b shows an L-shaped diaphragm 6, which is also used in the embodiment of FIG. 1, which limits the maximum representable area on two sides. This allows
The laser beam is the end point 2 of the scanning area in FIG.
Incident on the object is prevented when returning from 3 to the starting point.

During the scanning operation by meandering scanning (that the scanning beam reciprocates in the first direction and travels slowly in the second direction orthogonal to the first direction) with respect to the scanning region, FIG. As will be seen, each said scanner mirror must be reversed at the beginning and end 22 of the line from movement in one direction to movement in the other. At these stages, distortion appears in the rendered image. Therefore, the scanner is guided slightly beyond the maximum representable area and the signal produced thereby is not represented. In order to prevent the object 10 from being illuminated during these inversion steps, the laser beam squeezes the aperture 15 only in the area of the scanning imaging area that is actually imaged with little distortion. A diaphragm 15 shown with a square aperture in FIG. 2c is suitable so that it can pass through.

When scanning an object, it is general to narrow the scan area to enlarge and display small details, while expressing the image of the small area with the maximum image size. (Photoelectric zoom). In such a case, the linear movable slices 16 to 1 are arranged to prevent the irradiation of the target region which is not expressed.
It is convenient to create the diaphragm 15 at 9. The slice 16
19 to 19 form one side of the area left as an opening by the diaphragm 15. Each of the slices is then adjusted so that only the area 8b to be represented in the imaging area is left as an aperture (see FIG. 4).

After adjusting the opto-electrical zoom, it is possible to be interested in details outside the center of the area. A small area of the area eccentric from the center of the area is a thin piece 16
Through 19 suitable adjustments. By using the design of the diaphragm 15 described above, the small target area 8b that exists symmetrically with respect to the new area center 8c can be easily illuminated by the laser beam.

To fit these cases exactly,
The possibilities for adjusting the flakes 16 to 19 are diverse. The required position parameters for the slices 16 to 19 can be determined from the scanner control parameters. This procedure is known from the prior art.

Due to the limitations of the device, it is not possible in each case to arrange the diaphragms 6, 14, 15 exactly in the intermediate image plane 8. If the diaphragm has to be arranged in another plane, the laser beams 7a, 7b,
7c has not yet achieved its ultimate reduction. In this case, in order to illuminate the edge of the imaged target area with the highest intensity, the diaphragm is arranged around the diameter of the light beam at the position of the diaphragm more than the imaging area to be imaged. It must be oversized and each corner of the image can be rounded with the radius of the light beam in said plane. These relationships are shown in FIG. 3 for the diaphragm shape 15 according to FIG.

FIG. 5 schematically shows scanning of a rectangular area. The laser beam is guided to the waiting position by corresponding control of each scanner mirror before the scanning operation. When the scanning is started, the laser beam is guided to a position 21 at the first point of the area 8 to be imaged, reaching the reversal point 22 very quickly and from there to the other reversal point 22. You will be guided in the direction. These reversal points are outside the imaging area to be imaged. During the process of reversing each scanner mirror the laser
Since the beam is incident on the diaphragm 15, the object 10 is not irradiated thereafter.

When the laser beam reaches the final reversal point 23 at the bottom of the image, it follows the path indicated by the reference numeral 24 on the diaphragm outside the area to be imaged to the waiting position 10. Is returned.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a laser scanning microscope.

2A is a diagram showing various embodiments of the diaphragm of the present invention; FIG. 2B is a diagram showing various embodiments of the diaphragm of the present invention;
(C) The figure which shows various embodiment of the diaphragm of this invention. (E)
FIG. 4 is a view showing various embodiments of the diaphragm of the present invention.

FIG. 3 FIG. 2 for a placement outside the intermediate image layer.
The figure which shows the required shape of the diaphragm of (c).

FIG. 4 is a diagram showing a basic design mode of the diaphragm of FIG. 2 (c) in consideration of zooming and deviation during a scanning operation.

FIG. 5 is a schematic diagram of a scanning operation illustrated by the path of a laser beam with respect to an aperture and an imaging area.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Sebastian Tire             Federal Republic of Germany D-077743 Jena             Boats Strasse 1 (72) Inventor Ulrich Meisel             Federal Republic of Germany D-077743 Jena             Jahnstrasse 21 (72) Inventor Gunter Mailer             Federal Republic of Germany D-077743 Jena             Orchidee Enveke 3 F-term (reference) 2H052 AA07 AC15 AC27 AC34

Claims (9)

[Claims] 1. Beam interception in a laser scanning microscope with a deflection device (3, 4) for guiding a laser beam (1) guided in an area (8) onto an object (10) in a raster scanning manner. A device (6, 14, 15) arranged in proximity to the area (8) following the deflection device (3, 4) and a laser beam (6) for the object (10). If the incidence of 1) is not intended, the deflection device (2,3) is directed to the diaphragm (6,14,15) by the laser beam (1).
A device characterized by: 2. Following said deflection device (3, 4),
Objective according to claim 1, characterized in that an objective lens (5) with an intermediate image plane (8) is arranged and the stop (6, 14, 15) is located in the vicinity of the intermediate image plane. Device. 3. The diaphragm (6) is L-shaped and has two edges arranged close to the region (8). The device described in. 4. The circular diaphragm (14) is characterized in that
Or the device according to any one of 2. 5. Device according to claim 1 or 2, characterized in that the diaphragm is provided as a window (15) surrounding the area (8). 6. A device according to claim 5, characterized by adjustable flakes (16-19) forming each edge of the window (15). 7. A method of operating a laser scanning microscope in which a laser beam (1) is guided onto an area (8) so as to be incident on an object (10), the laser beam ( If the incidence of 1) is not intended, the beam is directed towards an aperture (6, 14, 15) arranged close to the region (8) in order to block the laser beam (1). Operation method characterized by being deflected. 8. The laser beam starting from a starting point located at the edge of the region (8) is
Was guided to an end point (10), which is likewise arranged on the edge of the, and then placed on the stop (14, 15) as well as on the stop and close to the start point. The method of claim 7, wherein the method is guided to a point. 9. The laser beam (1) is deflected so as to reciprocate in a first direction that scans the imaging area (4) and is deflected in a second direction orthogonal to the first direction. The method of claim 8, wherein the method is performed.