GB2381690A

GB2381690A - Beam shut-off in a laser scanning microscope

Info

Publication number: GB2381690A
Application number: GB0222444A
Authority: GB
Inventors: Guenter Schoeppe; Sebastian Tille; Ulrich Meisel; Gunter Moehler
Original assignee: VEB Carl Zeiss Jena GmbH
Current assignee: Jenoptik AG
Priority date: 2001-10-02
Filing date: 2002-09-27
Publication date: 2003-05-07
Anticipated expiration: 2022-09-27
Also published as: GB0222444D0; JP2003195178A; GB2381690B; DE10148679A1

Abstract

A laser scanning microscope 100 contains moving mirrors 3,4 to enable a laser beam to scan an object 10 in a raster fashion. In order to prevent the beam from irradiating the target when not required the beam is directed onto a beam blocking device or 'stop' 6 which prevents the light from reaching the target scanning area. The stop may be arranged in a frame shape which surrounds the scanning area to enable the beam to be easily deflected for shut-off at any point during the scan. The use of the stop is a cheap alternative to more expensive methods employing acousto-optical modulators.

Description

238N 690

Device for beam shut-off in a laser scanning microscope The invention relates to a device for beam shut-off in a laser scanning microscope comprising a deflecting device, which guides a laser beam over an image field in a raster

5 scanning manner, as well as to a method of operating a laser scanning microscope, wherein beam strut-off of a laser beam takes place.

In laser scanning microscopy, an area of an object is raster-scanned by a laser beam point by point. In doing so, a parallel laser beam, which is few millimetres in diameter, is 10 deflected according to a desired pattern, in a raster-like manner - e. g. in a similar manner as an electron beam in a Braun tube - in most cases by using a deflecting device. This deflected laser beam is focused by an optical system - called a scanning objective - in an intermediate image plane of the laser scanning microscope and imaged by an objective of the microscope onto or into the object. By said beam deflection, an object area of 15 adjustable size is raster-scanned which area has a predetermined maximum size for optical reasons. The laser beam sweeps a field, while raster-scanning the object. The expansion of

the laser beam varies according to the distance from the intermediate image plane.

The focused laser beam interacts with the object and may return as reflected radiation or 20 fluorescence radiation along the same path taken by the illumination radiation.

Between the laser and the deflecting device, there is provided, in most cases, a beam splitter, which guides the radiation taken up by the object' in an application-dependent manner, more or less completely into a detector beam path.

2s In the detector beam path, there is arranged, in all cases, at least one imaging system, which focuses the radiation in a farther image plane. In said plane, there is at least one small stop, called a pinhole, and, behind said stop, a detector. Depending on the particular application, there may also be further elements arranged in the detector beam path.

Pl00973GB 2 Since the reduction of the laser diameter at a negatively infinite point, the object and the pinhole lie in optically conjugated planes, such arrangements are called confocal. They exhibit special, advantageous imaging properties.

s A main field of application of confocal laser scanning microscopy is for fluorescence

tests, since this method enables resolution of a very thin layer of an object. Radiation originating from other planes is very strongly attenuated. The situation is similar with regard to reflective objects. Moreover, scattered radiation originating from interfaces and mounts of the optical components used is strongly attenuated.

Therefore, objects showing fluorescence properties upon irradiation are particularly useful in this known type of microscopy. However, flourescent objects, in particular, are light-sensitive, i.e. their fluorescence decreases with increasing irradiation time, so that it is desired to avoid unnecessary irradiation thereof if no image is recorded. If the laser beam were constantly incident on the object during positioning of the object and adjustment of Me microscope, this would "burn" into the object holes or traces no longer fluorescent. This is the case, for example, if the position of the focal plane is adjusted to a new section.

so Therefore it is common to switch-off the laser beam in laser scanning microscopes if no image is to be recorded. This is referred to as "beam shut-off''.

In principle, the laser could be switched off to this end. However, this approach is not convenient, since it leads to unstable operating conditions and a shorter life of the lasers 25 conventionally used. Shutoff by means of mechanical shutters is too slow.

For beam strut-off of the laser, in case its radiation might be incident on areas of the object which are not intended to be imaged, it is known to shut off the beam using acousto-

optical modulators (AOM) or acousto-optical tunable filters (AOTF). These components 30 are usually arranged in the parallel part of a non-expanded laser beam and allow the laser .,...,,,,,....,,._1.._ 1.._1 1. 51 _11' 1 1111_' '' _11 1 - 1151'! '1_ 1151 1 1111111 1 51 111 118 11- 1 1 1181111111 1115 1 - 1111

P 1 00973GB 3

beam to be switched on and off with an extremely short time of response. However, such acousto-optical components are relatively expensive parts.

Therefore, the problem underlying the invention is to provide a device for beam shut-off 5 in a laser scanning microscope and a method of operating a laser scanning microscope using beam shut-off, wherein expensive acousto-optical modulators can be dispensed with. According to a first aspect of the present invention, a device for beam shut-off in a laser 0 scanning microscope comprising a deflecting device, which guides a laser beam over an object in a raster-scanrung manner, said laser beam being guided within a field, comprises

a stop which is arranged following the deflecting device and adjacent said field and to

which stop the deflecting device directs the laser beam if the latter is not intended to be incident on the object.

Thus, the invention renounces the previous principle of having a controllable element, which is constantly arranged in the beam path, switch off the laser beam in such a manner that the laser beam no longer exits from this element. Instead, the laser beam is directed to a stop on which it is absorbed. To do so, the deflecting device, which is an essential part of 20 a laser scanning microscope for raster-scanning deflection of the laser beam anyway, is suitably controlled. Thus, the deflecting device is used to prevent illumination of the object by the laser beam. Thus, there is no longer a shut-off in the conventional sense. The laser beam is guided to a stop, which lies outside the field swept by the laser beam during

raster-scanning of the object, and is absorbed there. This prevents the laser beam from 25 reaching the object, and thus no damage can be done to the object. In continuation of the terminology introduced, this is also referred to, in connection with this invention, as beam shut-off.

Pl00973GB 4 The speed at which this beam shut-off takes place is given by the deflecting device, which usually works very fast in a laser scanning microscope, because this is required for other reasons anyway (time for recording an image).

s The stop to which the laser beam is directed for beam shut-off, may principally be arranged at any location following the deflecting device in the optical path. The closer the stop is arranged to the intermediate image plane, the smaller the beam diameter of the laser beam will be in the stop plane and the quicker the transition will be made, during beam shut-off, from hill illumination of the object to full beam shut-off. At the lo intermediate image plane of the optical system, the cross-section of the laser beam has the size of the spot diameter, i. e. its strongest reduction. When it is guided, in a raster-

scanrung manner, to the edge of a stop lying in the intermediate image plane, the laser beam is then shut-off very quickly and, in extreme cases, from one image point to the next. It is therefore advantageous to arrange the stop as closely as structurally possible to the intermediate image plane of the microscope, or within said plane.

This embodiment has not only the advantage that shut-off is effected particularly quickly, because the laser beam may be guided very quickly at a given deflection rate from the image field to the stop with its entire beam cross-section, but also that the stop may be

20 kept very small, being merely required, in particular, to have the she of the laser beam diameter, since the laser beam is of nearly minimal diameter when it is incident on the stop. Further, it is possible to protect the object from the laser beam during the phases of reversal of the deflecting device, which are not represented in the image anyway, or during the scaring pauses.

2s The stop merely needs to be designed such that the entire laser beam can be placed thereon. It is to be designed such that it covers at least that area to which the deflecting device may permanently direct a beam. Thus it is sufficient, in principle, to have a circular stop of suitable size, which is arranged near the field such that the deflecting device may

30 direct the laser beam thereto. Advantageously, a small circular stop is arranged at the _,__1 _1_114_11 1 151_1 11 1, B; 1 15 111 111 1_ 110 111115 1 1ll15111 1 1111 1

P I 00973GB 5

location of the so-called parking position of the laser beam, to which the laser beam is directed in the scan pauses, i. e. at a location outside the field to be imaged.

For beam shut-off, the invention advantageously uses an already existing deflecting 5 device. Said device may be substantially of any design as long as it is capable of directing the laser beam to the stop lying adjacent the field to be scanned. Commonly, the deflecting

device causes biaxial deflection. In laser scanning microscopy, two tilted mirrors are conventionally used, since they provide maximum freedom in guiding the laser beam over the image field being raster- scanned on the object. In such a deflecting device, which is

lo able to permanently direct the laser beam to one point, a simple circular stop is also sufficient for beam shut-off.

In a deflecting device comprising a tilted mirror and a rotating polygon mirror, however, a linear or line-shaped stop is required.

A conventional scanning movement normally starts in one corner of the image field being

raster-scanned on the object and leads to the diagonally opposite corner. The next scanning operation then starts again at the starting point of the previous' scanning operation. In order to protect the object from undesired irradiation between two scans, it is 20 preferable to provide the stop with an L-shaped design and having two edges thereof arranged adjacent the field, so that the inner edges of the stop are located at the edge of the

field to be imaged, since the laser beam may then be returned from the end point of a

scanning operation to the starting point of the next scanning operation on the L-shaped stop, so as to be on the stop near the starting point of this scan, before the scanning 25 operation is started. In doing so, the laser beam is guided, first parallel to one, then parallel to the other edge of the stop outside the image field, to the starting position. Upon shut-off

of the laser beam, it is possible to activate it repeatedly by simply guiding the laser beam from the stop to the starting point. This may be effected very quickly.

P 1 00973GB 6

In a further embodiment, the stop is provided as a window surrounding the field, because

then, the shortest possible distance is present from any point of the raster-scanned image field to the stop. Thus, a particularly quick shutoff may be achieved, or the beam may be

shut off during the phases of reversal.

When operating a laser scanning microscope, it may be required, for technical reasons associated with an examination, to reduce the size of the raster-scanned field

(optoelectronic zoom) and/or to move the centre of a currently scanned area within the maximum possible field (offset). To do so, the stop may be adapted with fixed dimensions

0 to the maximum size of the filed to be raster-scanned. Particularly advantageously, however, it may be provided adjustable in location and size, so as to be adaptable as a function of the selected deflection pattern (within the maximum possible size of the field).

For this purpose, it is advantageously provided that the edges of said window be formed by adjustable larnellae.

According to a second aspect of the present invention a method of operating a laser scanning microscope, wherein an object is rasterscanned by a laser beam, comprises deflecting the laser beam to a stop arrangement adjacent the field in order to shut off the

laser beam from unintentional incidence on the target. This method has the advantages zo described above in cormection with We device and allows fast laser beam shut-off.

Further, said shut-off merely requires a change in the control of the deflecting device, obviating separate control of a shut-off element.

In a method, wherein the light beam is guided from a starting point on the object to an end Is point during a scan, it is convenient to guide the image beam to the end point of the stop and to return it on the stop to a point near the starting point. Thus, on the one hand, a very quick shut-off is achieved upon completion of a scanning operation. On the other hand, it is possible to quickly activate the laser beam again at Me beginning of a scanning operation. !-1 _ 1 _ t -11 111 111141 Illllleall_nallll ll lllllllll lll Ill la_I-11111111

P100973GB 7

In principle, the laser beam may be guided in any manner in order to scan the object.

However, a particularly convenient embodiment with quick raster-scanning of a rectangular area is obtained if the laser beam is deflected across the object in a reciprocating manner in a first direction and, orthogonal thereto, in a second direction.

s The invention is explained in more detail below with reference to the Figures wherein: Fig. 1 shows a schematic representation of a laser scanning microscope; Fig. 2a-e show different embodiments of the stop of the invention; lo Fig. 3 shows the required shape of the stop of 2c for arrangement outside the intermediate image layer; Fig. 4 shows the basic design of the stop of 2c taking into account the zoom and offset during the scanning operation, and Fig. 5 shows a schematic representation of a scanning operation with the path of the laser 15 beam relative to the stop and the represented image field.

Figure 1 shows a schematic representation of a laser scanning microscope 100. A parallel laser beam 1 exiting from a laser or from a fibre (not shown) is reflected at a main beam splitter 2 in the direction of a biaxial deflecting device comprising scanner mirrors (tilted 20 mirrors) 3 and 4 (x and y axes) and, in the resting position of both mirrors, is directed along the optical axis to a scanning objective 5. By this light beam represented at 7b, a laser spot is generated at a centre of an intermediate image 8.

A broken line 8a in the plane of the intermediate image 8 represents the size of a Is maximum representable field, depending on optical conditions and associated with the

maximum size of an object to be raster-scanned. The path of the light beam 7b is further represented in the direction of an objective 9. By this light bundle, finally a small laser spot is generated on or in an object 1O.

Pl00973GB 8 In the plane of the intermediate image 8, there is arranged an L-shaped stop 6 (see Fig. 2c) according to the invention, whose function shall be explained later. A light bundle 7a schematically represents the beam path for the starting point of a scanning operation, while a light bundle 7c shows the position of the laser beam upon completion of a s scanning operation.

The radiation returning from the object 10 passes through elements 9, 5, 4, 3 in the opposite direction and is then finally transmitted, in an application-dependent manner, partially or nearly completely from the main beam splitter 2 in a detection beam path to an lo optical system 11 (pinhole objective) and is focused by said optical system 11 in the plane of a stop 12 (pinhole).

The light passed through the pinhole is finally converted to electrical signals by a detector 13. The stop 6 serves to quickly shut off the laser beam. For this purpose, the laser beam is directed to the stop. The term "shut-off' is to be understood from the perspective of the object 10, i.e. the laser beam 1 does, in fact, illuminate the scanner mirrors 3, 4 during shut-off, but is then absorbed on the stop 6 so that it is not incident on the object 10.

Fig. 1 schematically shows three different deflections of the laser beam 1. The laser beam referred to as 7a is incident on a point of the image field to be scanned, which point is the

starting point of a scanning operation. The laser beam referred to as 7b is in the midst of a scaring operation. The laser beam referred to as 7c is directed to stop 6 and is thus not 25 incident on the object 10. Thus, a shut-off of the laser beam 1 is achieved wherein the object is not illuminated by the laser beam 1.

By way of example, Figures 2a - c show different embodiments of the stop: _,,,_tl li,' jI IIe i II-IICI: 111101l'l1l '-ellill l 1 Aim-,lil 1 -!111

P 1 00973GB 9

Fig. 2a shows a circular stop 14, which is located near the starting position of the laser beam. This prevents the laser beam from reaching the object during the adjustment procedures or the scanning pauses. For this purpose, the scanner mirrors are controlled such that the laser beam is incident on a point slightly to the left, in a diagonally upward 5 direction, outside the maximum representable field, i.e. the so-called parking position 20

in Fig. 5, where the beam is incident on the small circular stop 14 of Fig. 2a.

Fig. 2b shows the L-shaped stop 6 as also used in the embodiment of Fig. 1, which stop limits the magnum representable field at two edges. This prevents the laser beam from

lo being incident on the object when returning to the starting point from the end point 23 in Fig. 5 of the scanned field.

During a scanning operation with meander-shaped scanning of a scan field (the scaling

beam reciprocating in one direction and slowly progressing in the direction orthogonal 15 thereto), the scanner mirrors must be reversed at the line beginnings and ends 22 from one direction of movement to the other, as is evident from Fig. 5. In these phases, distortions would appear in the represented image. Therefore, one guides the scanners a little beyond the maximum representable field and does not represent the signals generated thereby. In

order to prevent the object 10 from being irradiated during these phases of reversal, the 20 stop shown 15 in Fig. 2c takes the form of a frame bordering the image field and

providing a window as a square opening, so that the laser beam may pass this stop 15 only for those areas of the scanned image field which are actually imaged sufficiently free from

distortion. 25 When scanning the objects, it is common to reduce the scanned field for enlarged

representation of small details, while representing the image of this smaller field in the

size of the full image (opto-electronic zoom). such case, and as shown in Figure 4, it is convenient, in order to prevent irradiation of object areas which are not represented, to make the stop 15 from movable, straight lamellae 16-19, each of said lamellae 16-19

P 00973GB 10

forming one edge of the field left blank by the stop 15. Said lamellae are then adjusted

such that only the area 8b, which is to be represented in the image field, is left blank.

It is also possible that a detail outside the centre of the field might be of interest after

s adjustment of the opto-electronic zoom. By suitable adjustment of the lamellae 16-19, a smaller area of the field, eccentric relative to the centre of the field, may be selected.

Using the above-described design of stop 15, a smaller object area 8b lying symmetrically to the new field centre 8c may be readily irradiated with a laser beam.

lo The possibilities of adjusting the larnellae 16-19 to fit precisely for these cases are numerous. The parameters for their required location may be obtained from the control parameters of the scanners using known procedures.

Due to apparatus restrictions, it is not usually possible in each case to arrange the stop 6, Is 14, 15 exactly in the intermediate image plane 8. If the stop has to be arranged in a different plane, the laser beam 7a, 7b, 7c will not have attained its strongest reduction yet.

In this case, in order to illuminate the edge portions of the imaged object field at full

intensity, the stop must be made larger around the diameter of the light bundle at the location of the stop than the image field to be imaged, and the corners of the image may be

20 rounded off with the radius of the light bundle in said plane. These relationships are illustrated in Fig. 3 with respect to the stop shape 15 according to Fig. 2c.

Fig. 5 schematically shows the scanning of a rectangular field.

2s The laser beam is guided to the parking position 20 by corresponding control of the scanner mirrors before the scanning operation. When scanning starts, the laser beam is guided to position 21 at the first point of the field 8 to be imaged and it very quickly

reaches the point of reversal 22, from which it is guided to the other point of reversal 22 in the opposite direction. These points of reversal are outside the field to be imaged. During

__ _ _._.. _.... __..,., all,-.,,-,-,,,,._,,,,, A! 1 Bl 81 at_ Ill l -_

P 1 00973GB

the process of reversal of the scanner mirrors, the laser beam is incident on the stop 15, so that the object 10 is not irradiated then.

Once the laser beam has reached the final point of reversal 23 at the bottom edge of the s image, it is returned, on the stop, along a path shown at 24, outside the field to be imaged,

to the parking position 20.

Claims

P 00973GB 12 Claims

1. A device for beam shut-off in a laser scanning microscope comprising a deflecting device which guides a laser beam over an object in a rasterscanning manner, said laser 5 beam being gruded within a field, and a stop, which is arranged following the deflecting

device and adjacent said field and to which stop the deflecting device directs the laser

beam if the latter is not intended to be incident on the object.

2. The device as claimed in Claim 1, characterized in that an objective comprising an lo intermediate image plane is arranged following the deflecting device and that the stop lies near said intermediate image plane.

3. The device as claimed in any one of Claims I or 2, characterised in that the stop is L-shaped and has two edges thereof arranged adjacent the field.

4. The device as claimed in any one of Claims 1 or 2, characterised by a circular stop.

5. The device as clanned in any one of Claims 1 or 2, characterised in that said stop is provided as a window frame surrounding the field.

6. The device as claimed in Claim 5, in which the frame is formed by adjustable larnellae forming the edges of the window.

7. A method of operating a laser scanning microscope, wherein a laser beam is 2s guided over a field so as to be incident on an object, comprises deflecting the laser beam

to a stop arrangement adjacent the field in order to shut off the laser beam from

unintentional incidence on the target.

8. The method as claimed in Claim 7, wherein the laser beam, starting from a starting 30 point located at the edge of the field, is guided to an end point, also located at the edge of

' i lll_ IR,lIl8: I 1 1 Ill 11:1 1l"

Pl00973GB 13 the field, and is then guided back to the stop and onto the stop to a point located near the

starting point.

9. The method as claimed in Claim 8, wherein the laser beam is deflected, in a 5 reciprocating manner, in a first direction and, perpendicular thereto, in a second direction, scanning the image field.

10. A method of operating a laser scanning microscope on an object substantially as herein described with reference to the accompanying drawings.

11. A device for beam shut off in a scanning microscope comprising a deflecting device which guides a laser beam over an object in a rasterscanning manner, said laser beam being guided within a field, substantially as herein described with reference to, and

as shown in, the accompanying drawings.