DE19758748C2 - Laser Scanning Microscope - Google Patents

Abstract

Laser scanning microscope, DOLLAR A with a UV laser and a light guide connected downstream of the UV laser for transmitting the UV laser light to a sample to be examined, the UV light to be kept away from the light guide (14.1) being steerable into a light trap and used as a modulation means AOTF (AcoustoOpticalTunableFilter) (32) is provided, which deflects the UV light to be kept away from the light guide (14.1) into the light trap and controls the UV light to be fed into the light guide (14.1) to a constant intensity.

Description

State of the art

In the Handbook of Biological Confocal Microscopy, Second Edition (James B. Pawley), Plenum Press New York and London 1995 on page 519, Fig. 6 a fiber coupling optics is described.

On page 595, Fig. 14, a telecentric system for multiple detection beam paths is described.

US 5283433 shows a coupling optics for Detection beam paths.

DE 43 23 129 A1 describes in column 6 centerable confocal diaphragms which can be varied in terms of their diameter. US 5444528, US 5377003, US 5317379, US 5216484 describe the mode of action of an AOTF.

US 5081350, EP 283256 A2, WO 90/00754 A1 describe one Fiber connection between laser and scanning unit.

EP 283256 A2 describes a microscope with scanning fiber described in which an optic on the output side of the fiber is attached to the light in a point of light converge. To monitor the laser power is a Monitor diode provided.

In a laser scanning microscope, the illumination side irradiated several wavelengths and also several Wavelength bands detected.

DE 44 46 185 A1 describes the coupling of a UV Using laser beam into an optical fiber magneto-optical, mechanical or electro-optical Closure means.

The object of the invention is to couple a UV Simplify laser beam in a laser scanning microscope and at the same time to ensure the stability of the excitation.  

The task is performed using a laser scanning microscope Preamble of claim 1 by the characterizing Features resolved.

Representation of the mode of operation and advantages of the solution according to the invention on the basis of the exemplary embodiments according to the schematic representations FIGS. 1-6:

Show it:

Fig. 1 shows a modular arrangement of microscope M, scan head S and laser unit

Fig. 2 is an illustration of the beam path in the scan head S

Fig. 3, the optical effect of the displaceable collimating optics 16

Fig. 4 shows the optical effect of the pinholes displaceable in the direction of the optical axis

Figure 5 shows the optical effect of the vertically displaceable to the optical axis pinholes at different reflecting beam splitters.

Fig. 6 scan head S. microscope M and a fiber behind the pinhole in the detection beam path

• 1. A microscope unit M and a scan head S are shown schematically in FIG. 1, which have a common optical interface via an intermediate image Z according to FIG. 2.
  The scan head S can be connected both to the phototube of an upright microscope and advantageously also to a side outlet of an inverted microscope. In Fig. 1 a switchable between Auflichtscan and transmitted light scanning mitttels a pivotable mirror 14 microscopic beam path is shown, with the light source 1, the illumination optics 2, beam splitter 3, lens 4, sample 5, the condenser 5, the light source 7, receiver arrangement 8, a first tube lens 9, an observation beam path with a second tube lens 10 and an eyepiece 11 and a beam splitter for coupling the scanning 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 optical fibers 14.1 , 14.2 .
  The optical fibers 14.1 , 14.2 are coupled in by means of displaceable collimation optics 16 , which will be discussed in more detail below, 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.
  The actual scanning unit consists of scanning objective 22 , scanner 23 , main beam splitter 24 and a common imaging optics 25 for detection channels 26.1-26.4 .
  A deflecting 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 filter 30 and suitable receiver elements 31 (PMT, photomultiplier) are arranged downstream.
  The beam splitters 27 , 28 can advantageously, as shown schematically in FIG. 5, be designed as a dividing wheel with a plurality of positions, which can be switched over by stepper motors.
• 2. Advantageously, UV radiation is coupled into glass fiber 14.1 , preferably a single-mode glass fiber using an AOTF Acousto Optical Tunable Filter, as a beam deflector, ie if the radiation is not supposed to fall on the fiber input, it is transmitted from the fiber input by means of the AOTF , for example in the direction of a light trap, not shown.
  The coupling optics 33 for coupling the laser radiation have lens systems, not shown, for coupling, the focal length of which is determined by the beam cross section of the laser and the numerical aperture required for the optimum coupling.
  In the laser module 13.2 , single and multi-wavelength lasers are provided, which are coupled individually or together via an AOTF into one or more fibers.
  Furthermore, the coupling can also take place simultaneously via a plurality of fibers, the radiation of which is mixed on the microscope side after passing through a matching lens by color combiners.
  Mixing of the radiation from different lasers at the fiber input is also possible and can be carried out using the schematically illustrated, replaceable and switchable splitter mirror 39 .
• 3. The laser radiation emerging from the fiber end of the fibers 14.1 , 2 at the scanning unit S in FIGS . 2 and 3 is collimated to an infinity beam by means of the collimation optics 16 .
  This is advantageously done with a single lens, which has a focusing function by displacement along the optical axis by means of a control unit 37 which can be controlled via a central control unit 34 , in that its distance from the end of the optical fiber 14.1 , 2 on the scanning unit can be changed according to the invention.
  The effect of the displacement of the collimation optics 16 is shown schematically in FIGS . 3a and 3b.
  In Fig. 3a, the beam path for two different wavelengths, λ1, λ2 shown. Since for a polychromatic light source by means of a fixed imaging optics in an image plane is only imaged for a medium wavelength of the spectral range, the distance between the fiber end and the collimation optics is changed by means of the control unit 37 . The lens positions S1, S2 result for the two illustrated wavelengths in order to ensure the same focus position for both wavelengths.
  This advantageously has the effect that, in the case of fluorescence microscopy, the fluorescence radiation arises in the focus of the objective 4 set to infinity and the excitation radiation is focused in the same plane. Several fibers and fiber collimators can also be used to set different chromatic compensations for different excitation wavelengths.
  Furthermore, a chromatic correction of the optics used, in particular of the microscope objectives, can take place in this way. Different chromatic compensations can be set independently using several coupling fibers and collimation optics for different wavelengths.
  The variable collimation by displacement of the lens 16 can also be used for realizing a z-scans by the z-direction is displaced by means of the displaceable collimator lens 16, the focus in the specimen in an optical section and after the other is detected. This is shown in FIG. 3b for a wavelength λ, the positions S1, S2 corresponding to the focus positions F1, F2.
• 4. In FIG. 2, a monitor diode 19 (which may also have a front focusing lens, not shown here) is used in conjunction with a line- or area-selective filter wheel or filter slide 21 , controlled by a control unit 36 , for permanent monitoring of the scan module Coupled laser radiation, in particular to control the power in a certain laser line in isolation and, if necessary, to stabilize it by means of a control signal from the control unit 34. The detection by means of the monitor diode 19 detects the laser noise and variations due to the mechanical-optical transmission system.
  An error signal can be derived from the detected instantaneous laser power, which has an on-line effect directly on the laser or an intensity modulator (ASOM, AOTF, EOM, shutter) connected downstream of the laser for the purpose of stabilizing the laser power radiated into the scan module.
  By activating the filter unit 21 , wavelength stabilization of the intensity and laser power control can thus take place.
  A connection to the detection 31 (PMT) and in each case to the central control unit can be used to reduce the noise by forming signal quotients / or signal subtraction of the detection signal and the monitor signal of the diode 19 , in that the corresponding sensor signal of a detection channel is pixel-by-pixel as pixel image information on the signal of the Monitor diode is normalized (e.g. division) in order to reduce fluctuations in intensity in the image.
• 5. In FIG. 1, pinholes 29 in the detection channels 26.1-26.4 are shown schematically in various ways. In particular, they can be arranged to be displaceable perpendicular to the optical axis or in the direction of the optical axis, and their diameter can be changed in a known manner, for example by means of a scissor mechanism or a cat's eye. The adjustment of the pinhole diameter allows them to be adapted to the diameter of the Airy discs at different observation wavelengths.
  In Figs. 4 and 5 drive means 38 are shown schematically for the adjustment or shifting of the individual pinholes, the data lines have to the central control unit 34th
  The controllable displaceability of the pinholes in the direction of the optical axis is shown schematically in FIG. 4. It is advantageous for the compensation of optical errors, in particular chromatic longitudinal aberrations. These errors can occur with the scan lens 22 , but also, for example, with the imaging optics 25 common to the detection channels.
  For different wavelengths λ1, λ2, chromatic longitudinal deviations result in different focus positions, which correspond to different pinhole positions P1, P2. When imaging optics are replaced, for example the microscope objective, if the chromatic longitudinal error of the optics used is known, the pinholes can be automatically displaced along the optical axis via the control unit 34 and control and displacement means 38 . An exact setting can be made to the excitation wavelength used.
  By means of a common imaging optics 25 for all detection channels, which advantageously consists of only one optical element, the image generated by the scanning objective 22 and lying in infinity is imaged in the pinhole plane. The common imaging optics 25 bring about an improved transmission efficiency compared to known solutions. In cooperation with the imaging optics with individually adjustable pinholes in the individual detection channels, an exact adjustment can nevertheless be carried out.
• 6. Different dichroic beam splitters 28 can be used in the beam path, depending on the wavelength used, in order to block only these and feed them to a detection beam path. Partial revolvers or part wheels (not shown) are therefore provided in different beam paths for pivoting in different, as small as possible dividers, in particular parting wheels, whose wheel axis is inclined at 45 degrees to the optical axis, so that the dividers are only ever shifted in the reflection plane. Since the dividers 28 attached to the divider wheels cannot be adjusted exactly the same or fluctuations within their adjustment or standard wedge tolerances can cause different beam deflection angles, as shown in FIG. 5, the respective pinhole is shifted via control unit 38 perpendicular to the optical axis in accordance with FIG beam deflection. Here, two different positions of dividers 28.1 , 28.2 are shown schematically on a divider wheel, not shown, driven by a control unit 36 , which cause focus positions in the plane of the pinholes 29 that are displaced perpendicular to the optical axis.
  Here, by means of the control unit 34 via the control units 36, 38 coupling the position of the pinhole 29 be made with the Teilerradstellung for the divider 28, that is for all different configurations divider divider turret is stored optimum Pinholeposiition and retrievable.
  This affects not only the position of a certain divider wheel, but also the position of several divider wheels, so that the optimal pinhole position is always set automatically.
• 7. FIG. 6 schematically shows how an optical fiber 40 can be attached to the pinhole 29 , at the exit to the PMT behind the pinhole, in order to guide the radiation through the pinhole of the detection channel to an external sensor 31 .
  This is advantageously done without additional coupling optics close behind the pinhole with the aid of the optical fiber 38 .
  Since the pinhole opening is adjustable, the exchange of fibers with different core diameters is greatly simplified by adapting the pinhole size to the core diameter.

List of the reference numerals used

M microscope
S scan head

1

light source

2

illumination optics

3

beamsplitter

4

lens

5

sample

6

condenser

7

light source

8th

receiver

9

tube lens

10

tube lens

11

eyepiece

12

beamsplitter

13.1

.

13.2

laser

14

optical fibers

15

swiveling mirror

16

collimating optics

17

beam deflection

18

semi-transparent mirror

19

monitor diode

20

neutral density filters

21

line filter

22

scanning objective

23

scanner

24

Main beam splitter

25

imaging optics

26.1-26.4

detection channels

27

deflecting prism

28

.

28.1

.

28.2

dichroic beam splitter

29

adjustable pinholes

30

emission filter

31

PMT (photomultiplier)

32

AOTF (Acousto Optical Tunable Filter)

33

coupling optics

34

central control unit

35

.

36

.

37

.

38

local control units for diode

19

. filter changer

21

. collimator optics

16

, adjustable pinholes

29

39

beamsplitter

40

optical fiber
S1, S2, F1, F2 focus positions
P1, P2 pinhole positions

Claims (1)

Laser scanning microscope,
with a UV laser and a light guide downstream of the UV laser for transmitting the UW laser light to a sample to be examined
and with modulation means in front of the light guide, which couple the UW laser light into the light guide only for a desired period of time and keep it away from the light guide outside this period,
characterized by
that the UV laser light to be kept away from the light guide (14: 1) can be steered into a light trap
and that the modulation means are designed as AOTF (Acousto Optical Tunable Filters) ( 32 ) which deflects the UW laser light to be kept away from the light guide ( 14.1 ) into the light trap
and controls the UV laser light to be fed into the light guide ( 14.1 ) to a constant intensity.