DE20221635U1 - Optical system for stimulating, measuring fluorescence on/in specimens treated with fluorescent paint has geometric axis at least partly identical with optic axis between mirror and detector - Google Patents

Abstract

The system has a laser, mirror, deflection element, lens, specimen table, perforated stop, spectral filter, detector and a constant geometric axis that does not pass through the specimen and on which an optical arrangement, second focal point, perforated stop, spectral filter and detector are arranged with the mirror and deflection element. The geometric axis is at least partly identical with the optical axis between the mirror and the detector. The system has a laser (1), mirror (4), deflection element (7), lens (8), movable specimen table (13), perforated stop (18), spectral filter (19), detector (20) and a constant geometric axis (G) that does not pass through the specimen and on which an optical arrangement, a second focal point, perforated stop, spectral filter and detector are arranged together with the mirror and deflection element. The geometric axis is at least partly identical with the optical axis (5) in the region between the mirror and the detector. Independent claims are also included for the following: a method of stimulating and measuring fluorescence on or in specimens treated with fluorescent paints.

Description

The The invention relates to an optical system for exciting and measuring of fluorescence on or in treated with fluorescent dyes Samples, according to the generic term of the independent Claim 1. Such optical systems are e.g. as raster-light microscopes known.

• • eine Lichtquelle, die einen Lichtstrahl produziert;
• • einen Probenträger zum Halten der Probe;
• • eine Optik zum Bilden eines ersten Brennpunktes auf der Probe;
• • eine optische Anordnung zum Abbilden eines zweiten Brennpunktes mit dem Licht, welches die Probe durchstrahlte bzw. von der Probe reflektiert wurde bzw. welches die an oder in der Probe ausgelöste Fluoreszenz darstellt;
• • einen Photodetektor zum Messen der Intensität des zweiten Brennpunkts; und
• • einen Rastermechanismus zum gegenseitigen Bewegen von Probe und erstem Brennpunkt.

Scanning light microscopes have been known for several decades. Their functional principle is based on the fact that a light beam is concentrated to a small point of light (the so-called first focal point) on a sample. The sample and this point of light are mutually moved so that a certain area of the sample is swept (scanned) by the light spot. The light which penetrates or is reflected by the sample during the scanning or the fluorescence triggered on or in the sample is referred to below as "sample-originating light" and measured by one or more photodetectors. An enlargement image is created by assigning a specific area on an image of the sample to an original measurement point. Basically, therefore, such a scanning light microscope comprises:

• • a light source that produces a beam of light;
• A sample carrier for holding the sample;
• An optic for forming a first focus on the sample;
• An optical arrangement for imaging a second focal point with the light which was irradiated or reflected by the sample or which represents the fluorescence triggered on or in the sample;
• A photodetector for measuring the intensity of the second focus; and
• A raster mechanism for moving sample and first focus.

In a conventional scanning light microscope becomes the light beam for illuminating the sample in the direction of the two spatial axes X and Y distracted. This approach has the disadvantage that the angle of incidence of the refracted by the projectile lens light on the sample varies and image distortion in the image of the sample light through the objective lens generated. This image corruption (aberration) can be corrected by a corresponding construction of the objective lens become. However, such an expensive and the optics disadvantageous effect simultaneously in terms of light collection efficiency and working distance limiting.

According to US 5,081,350 This problem has been solved by the disclosure of a device with which the sample is scanned by a light beam. In this case, the device for illuminating the sample and the device for measuring the signal coming from the sample are mounted on a reciprocating unit. The sample is mounted on a sample stage which is movable perpendicularly to this oscillation, so that the sample can be scanned at a constant angle of incidence of the illumination. Because, especially for the application of a quick grid method, the light source should preferably be arranged outside of the moving part of the scanning light microscope, there is proposed the use of fiber optic light guides, which optically connect the light source with the projective. However, there is a risk that these fiber optic cables will be damaged by the frequent and rapid back and forth moving.

Out US 5,260,569 An improved, generic device is known, which solves the above-described problems of the prior art by proposing a raster-light microscope, which as a light source, a laser, a mirror, for deflecting the coming out of the laser and parallel to a optical axis to the mirror incident light toward a sample, a deflecting element for deflecting this light on this mirror, optics for forming a first focal point, a mirror and the optics comprehensive, linearly along the optical axis back and forth arranged movable A unit in which the mirror and optics are arranged immovably relative to one another, an oscillating linear drive operatively connected to this unit, a sample table movable at least in the direction of the X and Z spatial axes for aligning the sample with respect to the first focal point, an optical arrangement for imaging a second Focal point with sample-derived light, an i a second aperture focal aperture for masking sample originating light which is farther than a certain distance from the focal point of said aperture, a spectral filter for selecting a portion of the sample passing through the aperture, and a detector for Measuring the intensity of the portion of sample-derived light transmitted by the aperture and selected by the spectral filter. The optic also acts as a collimator for the light originating from the sample, and the mirror is also designed to deflect this collimated light counter to the direction of incidence of the light from the laser and parallel to the optical axis.

In addition to all features of the preamble of claim 1 disclosed US 5,260,569 In addition, a raster-light microscope in which the light emitted by a light source is directed in parallel by means of a collimating lens acting as part of the projecting lens. The collimated light propagates in the direction parallel to the scanning direction of the microscope. That is why the collimated light beam falls - regardless of the current position of the back and forth movable unit - always from the identical direction to the mirror, which is fixed in the reciprocating unit. As a result, the light beam from the mirror to the sample is always reflected in the same direction and in collimated form. This koilimierte light is focused after a 90 ° reflection at the mirror by means of another projectile lens, which is also fixed in the reciprocating unit, in a first focal point. Therefore, the sample can be scanned by the reciprocating unit and by the light of a light source attached to the reciprocating unit. However, it is preferable to mount the light source and photodetector outside the reciprocating unit to make this unit simpler and easier to enable rapid scanning.

task The present invention is an alternative optical system or to propose an alternative optical method, the simpler, more flexible system design or system usage additional options essentially reveals the advantages of the state of the art having.

These Task becomes - according to one first aspect - with a system according to the feature combination of the independent claim 1 solved, in addition to the nearest Prior art known features of the preamble proposed It will make the optical system steady and not through the sample extending geometric axis G includes, on which the optical Arrangement, the second focal point, the pinhole, the spectral filter and the detector - together with the mirror and the deflection - are arranged, these geometric axis G in the area between the mirror and the detector at least partially identical to the optical axis. Further inventive features arise from the dependent ones Claims.

These Task becomes - according to one second aspect - with a method according to the feature combination of independent claim 25 solved, in addition to the nearest Prior art known features of the preamble proposed is that an optical system with a steady and not through the sample extending geometric axis G is provided, on which the optical arrangement, the second focal point, the pinhole, the spectral filter and the detector - together with the mirror and the deflection - arranged are, with this geometric axis G in the area between the mirror and the detector at least partially identical to the optical axis is. Further inventive Features emerge from the dependent claims.

Based of schematic drawings, which are merely preferred examples of embodiments represent and scope of the disclosure in relation to the present Do not limit the invention, this invention will now be closer explained. Showing:

1 a quasi-spatial scheme of an optical system according to a first embodiment with two lasers;

2 a vertical partial section of an optical system according to a second embodiment with at least one laser;

3 a vertical partial section of an optical system according to a third embodiment with at least one laser.

1 shows a quasi-spatial scheme of a reflection-type optical system according to a first embodiment. Preferably, this optical system is suitable for exciting and measuring fluorescence on or in samples treated with fluorescent dyes and comprises a first laser 1 , This sends - to excite the first fluorescent dyes - light of a first wavelength 2 out, so that these first fluorescent dyes light a second wavelength 3 emit. A mirror 4 directs this from the first laser 1 coming and parallel to an optical axis 5 on the mirror 4 incident light of the first wavelength 2 in the direction of a sample 6 um (cf. 2 and 3 ). A deflecting element 7 directs the light 2 from the first laser 1 on this mirror 4 around. An optic 8th forms a first focal point 9 for that from the first laser 1 incident and through the mirror 4 redirected light of the first wavelength 2 , One unity 10 includes the mirror 4 and the optics 8th , This mirror 4 and the optics 8th are in unity 10 immovably arranged against each other.

This unit 10 is linear along the optical axis 5 arranged back and forth movable and with an oscillating linear drive 11 operatively connected. The oscillating linear drive 11 can eg as a stack of piezo elements; be formed as a mechanical, for example, a connecting rod comprehensive drive or as excited by ultrasonic waves, naturally oscillating solid. However, preferred is the formation of an oscillating linear drive 11 as a "voice coil" as in US 5,260,569 and in particular in US 5,880,465 is described. In this case, a device for generating the reciprocating movement is used, which essentially corresponds to a loudspeaker, wherein the membrane movements by means of an operative connection to the unit 10 be transmitted.

This look 8th is suitably designed such that it also acts as a collimator for the second wavelength light emitted by the first fluorescent dyes 3 ("sample-derived light"). The mirror 4 also directs this collimated light 12 opposite to the direction of incidence of the first wavelength light 2 and parallel to the optical axis 5 around.

This optical system also includes a movable at least in the direction of the X and Z spatial axes table 13 for samples treated with at least one first fluorescent dye 6 and for aligning the sample 6 opposite the first focus 9 , Preferably, the table is 13 also movable in the direction of the Y-space axis, wherein the X-axis and the Y-axis at least approximately parallel to the surface 15 a sample carrier 14 run.

The X, Y and Z spatial axes are all at least approximately at right angles to each other, with the Y axis parallel to a horizontally extending part of the optical axis 5 and extends to the likewise horizontal geometric axis G.

Preferably, the table is 13 built in several parts and includes a in the direction of the X-axis over a smaller distance (thin arrow) moving part 13 ' , an in the direction of the X-axis over a greater distance (thick double arrow) moving part 13 '' and a movable in the direction of the Y-axis over a greater distance (thick double arrow) part 13 ''' , The whole table 13 or at least the part 13 ' is movable in the direction of the Z-axis. To move the parts of the table 13 are preferably used electric motors.

As a sample carrier 14 For example, glass slides are suitable, as they have long been known from light microscopy. Other or similar sample carriers may be made of plastic. Still other, substantially flat, eg also suitable for scanning tunneling microscopy sample carrier may be made of silicon or pyrolytic graphite and the like, or comprise these substances. It can also be provided that sample carriers are used which have a defined graduation on the surface. This division may consist of a regular groove-shaped subdivision; but it can also have an array of pits. Examples of such an array of wells are silicon or glass plates having a plurality of etched depressions. Further examples are standard microtiter plates ^TM (trademark of Beckman Coulter, Inc., 4300 N. Harbor Blvd., PO Box 3100 Fullerton, CA, USA 92834) or "microplates" which have 96, 384, 1536 or more wells in the mold of potty or so-called "wells" include.

While dry or at least dried or immobilized samples are preferably prepared for examination on microscope slides, sample carriers 14 with wells or wells liquid or in a liquid samples 6 take up. Has proved to be particularly suitable for use in the inventive optical system, a frame 31 which essentially has the outer dimensions of a microplate. Therefore, this frame fits in or on the same sample table 13 like a microplate and can be placed there according to a microplate automatically, ie with a robotic arm or taken away. This frame 31 is for receiving and holding a plurality of slides, in particular plastic, glass, Sili zium, pyrolytic graphite and the like, formed. The arrangement of 4 glass slides on such a frame has proven to be particularly suitable. This will be the slides 14 for example as in 1 shown or in a row, longitudinal edge on the longitudinal edge - each pushed together or slightly spaced from each other (see. 1 ) - arranged. The frame 31 is preferably made of plastic and injection molding, whereby the production price can be kept low and the dimensional accuracy high. This dimensional accuracy significantly facilitates the use of such frames 31 in all kinds of devices for the automated handling of microplates. Of course, the frames 31 also by hand with the slides 14 equipped and used in the inventive optical system. With slides 14 stocked frame 31 Furthermore, it facilitates the further handling of these secured slides, which no longer have to be touched directly between the process steps to be carried out in devices for automated handling.

The optical system also includes an optical arrangement 16 for imaging a second focal point 17 with the through the optics emitted by the first fluorescent dyes 8th collimated and the mirror 4 deflected light of the second wavelength 3 ("sample-derived light"). One in the second focus 17 arranged pinhole 18 serves to hide light of the second wavelength 3 which is farther away than at a certain distance from the focal point 17 on this pinhole 18 incident. Preferably, this pinhole 18 formed exchangeable, so that the sharpness and the brightness of the second focal point can be increased or decreased as required by means of different diameters of this Konfokalaperturblende. Especially in extremely low light conditions, a larger aperture, which reduces the sharpness of image will be of benefit. A replaceable pinhole 18 allows a choice of different hole diameters to match the image in the second focus 17 to the sample volume or to the desired penetration depth of the excitation beam in the sample as well as the selection of the corresponding depth of field of the detector arrangement.

The optical system also includes a first spectral filter 19 for selecting a portion of the through the pinhole 18 continuous light of the second wavelength 3 and a first detector 20 for measuring the intensity of the pinhole 18 transmitted and from the first spectral filter 19 selected part of the light of the second wavelength 3 ("sample-derived light").

The optical system according to the invention comprises a continuous and not through the sample extending, geometric axis G, on which the optical arrangement 16 , the second focal point 17 , the pinhole 18 , the first spectral filter 19 and the first detector 20 - together with the mirror 4 and the deflecting element 7 Are arranged, said geometric axis G in the region between the mirror 4 and the first detector 20 at least partially with the optical axis 5 is identical.

This common arrangement on the geometric axis G has the advantage that the scanning of the sample by means of a very simply built back and forth movable unit 10 can be done at high speed. In fact, this unit includes 10 only the mirror 4 and the optics 8th , The oscillation frequency is preferably 20 Hz, and the oscillation amplitude D may be up to 25 mm or more.

The light of the away from this unit 10 placed laser 1 and also from the sample, in parallel opposite to that of the laser 1 extending light represents a purely optical connection between the raster portion of the system and the excitation and measuring part of the same, so that no vibrations are transmitted from the raster portion of the measuring part of the system. In addition, this arrangement allows the bundled light directly 2 a laser 1 to feed into the optical system. The mirror 4 directs the bundled laser beam 2 on the optics 8th which in turn causes the resulting focus point always at the same location on the optical axis 5 directs. This optical axis 5 runs between the mirror 4 and the sample 6 preferably perpendicular. This optical system based on the coupling of a collimated laser beam suffers less light losses than those which feed and deflect a collimated light beam to obtain a first focal point.

In the system according to the invention, the optical axis is 5 along a preferably horizontally extending part of the geometric axis G identical. In a direction parallel to these axes 5 , G is a collimated laser beam for exciting the sample and in the opposite direction, a collimated beam with light originating from the sample is used. As a result, the deflecting element 7 which the laser beam 2 in the direction of the mirror 4 which is supposed to be permeable to the collimated light beam, should be a simple glass pane. Typically, about 4% of a light beam is reflected at an interface glass / air and about 96% is transmitted. A coating (antireflection coating) increases the transmission in case of need and reduces the reflection of such a glass pane. This simple glass pane replaces the one US 5,260,569 known and more expensive polarization beam splitter (see there 1 , Reference number 13 ) and quarter wave plates (see there 1 , Reference number 18 ), which are necessary in the prior art, because in both directions, a collimated light beam, which requires the entire surface of the mirror and filter, is used.

Another advantage of this inventive system is that the laser beam 2 for exciting the sample fluorescence probably parallel to, but not in the center of the optical axis 5 and not in the center of the geometric axis G must pass. This is primarily for determining a mutually optimal Z position of sample 6 and first focus 9 advantageous. The laser beam then preferably runs at a distance (A) from the optical axis identical to the geometric axis G. 5 , So it is an "acentric illumination" of the sample possible, as in the example 2 and 3 is shown.

Furthermore, in the system according to the invention there is the possibility of a preferred deflecting element 7 to use, which for deflecting the light 2 from the first laser 1 in one to the optical axis 5 parallel direction on this mirror 4 one for the laser beam 2 certain highly reflective area 21 covers and in its remaining areas both for the light of the first 2 as well as the second wavelength 3 is essentially permeable. This results in the use of such a "pin mirror" relative to the deflection element 7 Simple glass plate used a greatly improved yield for the collimated laser light to excite the fluorescence of the fluorescent dyes in or on the samples 6 , The loss of about 5% of the originating from the specimen and the high specular area 21 deflected light does not matter here.

To make the device more compact, it can be provided that the optical system a simple mirror for mirroring between the deflector 7 and the mirror 4 extending light rays includes (not shown). This simple mirror is then also arranged in the geometric axis (G). However, in this case, the optical arrangement 16 , the second focal point 17 , the pinhole 18 , the first spectral filter 19 and the first detector 20 - According to the deflection by the simple mirror - arranged on a different geometric G G '.

This in 1 illustrated optical system with two preferably monochromatic lasers of different wavelength also includes a second laser 1' , which - for exciting second fluorescent dyes - to the light of the first wavelength 2 parallel light of a third wavelength 2 ' emits so that these fluorescent dyes light of a fourth wavelength 3 ' emit. A second spectral filter 19 ' selects a portion of the light of the fourth wavelength 3 ' off and a second detector 20 ' measures the intensity of the second spectral filter 19 ' selected portion of the light of the fourth wavelength 3 ' , In addition, a beam splitter element 26 provided, which for the emitted by the first fluorescent dyes and the optics 8th collimated light 12 the second wavelength 3 is at least partially transmissive and which for mirroring or deflecting the light of the fourth wavelength 3 ' from the geometric axis G in the direction of a second detector 20 ' is trained. This beam splitter element 26 may be designed as a dichroic mirror or, for example, as a 50% beam splitter.

An object lighting 30 eg in the form of a simple lamp, is preferably also removed from the unit 10 provided so that the objects to be scanned can be viewed or imaged, if necessary, optically. The light used is by means of one on the axes 5 G arranged dichroic mirror gimmed. The spectral filters 19 . 19 ' are designed to filter out this light. A separate detector (not shown) may be provided to view the visual image of the samples 6 hold. Alternatively, the detector can also be used 20 be used to scan the sample, for example, in fluorescence mode.

2 shows a vertical partial section of an optical system, for exciting and measuring fluorescence on or in samples treated with fluorescent dyes, according to a second embodiment. This optical system comprises all the features of claim 1. For this second, somewhat simpler embodiment is only a monochromatic laser 1 intended. The table 13 is also movable in the direction of the Y-space axis, wherein X- and Y-axis at least approximately parallel to the surface 15 a sample carrier 14 run. The under the 1 All discussed sample carriers can all be used in this embodiment as well.

The deflecting element 7 for redirecting the light 2 from the first laser 1 in one to the optical axis 5 parallel direction on this mirror 4 includes one for the laser beam 2 certain highly reflective area 21 , In its other areas is the deflecting element 7 both for the light of the first 2 as well as the second wavelength 3 essentially permeable. The high-reflectance area 21 of the deflecting element 7 is at a distance A from the optical axis identical to the geometric axis G. 5 arranged so that the beam path for the out of the laser 1 coming light of the first wavelength 2 parallel and at the distance A to these axes 5 , G runs.

An opaque aperture 22 is used to temporarily hide one used for focusing and stimulation and the sample 6 reflected laser beam between the deflecting element 7 and the optical arrangement 16 arranged. In the state shown, this aperture 22 pushed into the beam path.

The laser 1 coming, bundled light 2 becomes at the highly reflective area 21 of the deflecting element 7 in the direction of - preferably inclined to the horizontal by 45 ° mirror 4 , parallel to the axes 5 , G and deflected at a distance A to these axes. The mirror 4 directs this bundled laser beam 2 parallel and at a distance A to the now at least approximately perpendicular optical axis 5 around, whereupon the laser beam in the optical axis 5 lying focal point 9 is steered. The course of the laser beam described so far 2 is used for focusing, ie for setting an optimal working distance between optics 8th and sample 6 used. This will (as described later) the mutual position of focus 9 and sample 6 determined and adjusted. The focus 9 concentrated focusing beam 25 has a spot diameter between the diffraction limit and 2 mm. A preferred spot diameter is less than 15 μm. The focusing beam 25 is reflected from the sample and reaches at a distance from the optical axis 5 again the mirror 4 , which reflects this reflection beam in a direction parallel to the horizontal axes 5 , G distracts.

To determine an exact working distance - which is preferably up to 7 mm, but can also be more or less - this acentric illumination is particularly well suited because the laser beam impinges on the sample at an angle which is smaller than 90 °. It is obvious that with decreasing angle of incidence the accuracy or the resolution increases in the direction of the Z-axis, while an angle of incidence of 90 ° allows the lowest resolution in the direction of the Z-axis, because, for example, it is not possible to determine the depth at which the focal point 9 within a sample 6 located. From the above it also follows that the resolution of the optical system in the direction of the Z-axis increases with increasing distance A. The parallel to the axes 5 G running reflection beam is through an optical arrangement 16 in the second focus 17 directed where he pinhole 18 passes. The spectral filter is now preferably pulled out of the beam path or replaced by a neutral density filter (gray filter), so that the reflection beam on the detector 20 impinges and from this the intensity of the reflection beam can be measured.

If the optimal working distance is not with the reflection, but with the intensity of the fluorescence excited in or on the samples or on the basis of in or on the sample 6 be created, diffused scattered light, so the aperture 22 pushed into the beam path (see. 2 ) and the spectral filter 19 or a neutral density filter (gray filter) in front of the detector 20 placed. The bundled laser beam 1 then becomes an excitation beam 24 used, which in the same way as the focusing beam 25 in the first focus 9 is steered. Part of it is essentially dome-shaped from the sample 6 Expansive fluorescence is reflected in optics 8th collimated (ie, collimated) and from the mirror 4 in the direction of the optical arrangement 16 distracted. Despite a small loss, essentially from the area of the highly reflective area 21 of the deflecting element 7 and the aperture 22 is determined, this collimated fluorescent light is incident on the optical arrangement 16 and becomes the second focal point 17 focused. There passes - depending on the selected diameter of the pinhole 18 - A certain part of the focused fluorescent light this pinhole 18 and the spectral filter 19 and is from the detector 20 detected. For scanning the sample, the same beam path, as just described, is used.

The first spectral filter 19 is between the pinhole 18 and the first detector 20 arranged. It can also act as a window in the detector 20 be used. However, it is preferred to form the first spectral filter 19 as a filter slide with, for example, five different, automatically interchangeable filters, this spectral filter 19 manually interchangeable with another filter pack with another filter set.

This optical system preferably comprises a computer or microprocessor for recording and processing by the detector 20 recorded measurement signals and for outputting of these signals corresponding data. This computer or microprocessor is also preferably for controlling the movements of the table 13 educated. The movable in the directions of the X and / or Y and / or Z-axis table 13 is preferably also formed tiltable about the X and / or Y axis.

According to the method according to the invention, such an optical system is provided with a geometrical axis G which is continuous and does not extend through the sample. On this geometric axis G, the optical arrangement 16 , the second focal point 17 , the pinhole 18 , the first spectral filter 19 and the first detector 20 - together with the mirror 4 and the deflecting element 7 - arranged. In this case, this geometric axis G is in the region between the mirror 4 and the first detector 20 at least in part with the optical axis 5 identical.

While stimulating and measuring the of the samples 6 emitted fluorescence becomes the unit 10 this optical system fixed according to a first use. The sample table 13 is simultaneously moved in the direction of the X and / or Y axis, with the Y axis parallel to the axes 5 , G runs. With this table movement, which eg through the parts 13 ' (X-movement) and 13 '' '(Y-motion) can be performed, with continuous excitation and measurement, a line scan, at point excitation and point measurement, a point scan, on or in the samples 6 allows.

While stimulating and measuring the of the samples 6 emitted fluorescence becomes the unit 10 this optical system according to a second mode of use in the direction of the Y-axis back and forth moves. The sample table 13 is simultaneously fixed or moved in the direction of the X-axis. With these movements of unity 10 alone, with continuous excitation and measurement becomes a line scan, with simultaneous movement of unit 10 (Y-movement) and table 13 (Movement of the part 13 ' results in X-motion), with continuous excitation and measurement, a surface scan is performed on or in the samples 6 allows. The Y-axis runs parallel to the axes 5 , G. The oscillating linear drive 11 for the unit 10 is preferably designed as a "voice coil".

In front the effective scanning of the samples becomes the Z position the sample carrier set.

As an alternative light source for the excitation of fluorescence laser pulses or lamp flashes can be used. Then be preferred discrete, single laser pulses or discrete small series of a few laser pulses which fluoresce at one point in or on samples 6 stimulates. Specifically, when measuring the fluorescence in samples presented, for example, in the 1536 wells of a high density microplate, such spot measurements are preferably made. Between or during the measurements become unity 10 and sample carriers 14 shifted against each other, especially the shift of the unit 10 can happen extremely fast in the direction of the Y-axis. Measuring the fluorescence in these 1536 wells, which takes about 1 minute in the area scan with continuous excitation and measuring with the currently fastest devices, can thus be shortened to about 10 seconds. For microplates with even more wells or even smaller grid dimensions, even greater time savings are achieved.

For example on essentially flat sample carriers 14 immobilized samples 6 is used to define an optimal Z position of the movable table 13 or a sample 6 - Between the deflection 7 and the optical arrangement 16 arranged, opaque aperture 22 pulled out of the beam path. On it becomes one hand during a Z movement of the table 13 through the first detector 20 generated and recorded in a computer or microprocessor series of Messnsnalen - calculates the maximum of these measurement signals corresponding Z position of the table and the table 13 moved to this Z-position. To set the maximum of these measurement signals, the geometric center is preferably taken between the inflection points of the rising and falling edge of the measurement signal. This method is preferably performed on at least three locations of a slide and the table 13 - according to the defined maxima for these three places - as far as necessary in the directions of the X, Y and Z axis shifted and tilted about the X and Y axis. It is advantageous if this definition of an optimal Z-position is performed by means of a computer or microprocessor, which is the one from the detector 20 acquired measuring signals and processed, which outputs data corresponding to these signals and also the movements of the table 13 controls.

3 shows a vertical partial section of an optical system, for exciting and measuring fluorescence on or in samples treated with fluorescent dyes, according to a third Auführungsform. This optical system comprises all features of claim 1. For this third embodiment, one or more preferably monochromatic lasers 1 . 1' be provided. The table 13 is preferably movable in the direction of the X, Y and Z spatial axes, wherein the X and Y axes are at least approximately parallel to the surface 15 a sample carrier 14 run. In addition, the table 13 preferably tiltable about the X and / or Y axis. The under the 1 and 2 All discussed sample carriers can all be used in this embodiment as well.

The deflecting element 7 for redirecting the light 2 from the first laser 1 in one to the optical axis 5 parallel direction on this mirror 4 includes one for the laser beam 2 certain highly reflective area 21 , In its other areas is the deflecting element 7 both for the light of the first 2 as well as the second wavelength 3 essentially permeable. The high-reflectance area 21 of the deflecting element 7 is in the center of the optical axis identical to the geometric axis G. 5 arranged so that the beam path for the out of the laser 1 coming light of the first wavelength 2 parallel and in the center of these axes 5 , G runs.

This optical system comprises a separating element 23 for separating the from the laser 1 coming light of the first wavelength 2 in an excitation beam 24 for the fluorescence and one to this excitation beam 24 parallel focusing beam 25 , An opaque aperture 22 is for temporarily masking a used for exciting the fluorescence laser beam between the deflecting element 7 and the separator 23 arranged. This separator 23 if only one laser can 1 should be used as a simple, plane-parallel glass plate with back Vollverspiegelung be formed.

The bundled light 2 from the laser 1 is about 4% of the uncoated first surface of the separator 23 in the direction of the deflecting element 7 reflected. There, this laser beam strikes a non-mirrored area and becomes again approximately 4% and at a distance A parallel to the axes 5 , G in the direction of the mirror 4 distracted. The laser 1 coming, bundled light 2 will also be on the back surface of the separator 23 mirrored. In the case of a mirrored glass panel at the rear, approx. 96% of the laser light is used 2 on optical axis 5 on the deflector 7 directed where the light 2 on its highly reflective area 21 meets. The separating element 23 thus fulfills the task of the laser beam 2 in one on the optical axis 5 extending excitation beam 24 and a focusing beam parallel thereto at a distance A 25 separate.

As an alternative separating element can also be a simple, plane-parallel glass plate, which is obliquely placed in the beam path can be used. The position of this plane-parallel glass plate should be between the laser 1 and the deflecting element 7 are located; Preferably, this is plane-parallel glass but plate between the laser 1 and the separator 23 arranged (not shown). Most of the laser beam passes through the glass plate with a slight offset. About 4% are reflected back into the glass plate at the rear glass / air interface. Again, a proportion of about 4% of the reflected beam then undergoes a renewed reflection at the front interface. This results in that a small portion of the laser light parallel to the main beam in the direction of the deflecting element 7 is steered. In this case, the separating element 23 designed as a front surface mirror and does not serve to separate the beam.

If two or more monochromatic lasers of different wavelengths are to be used, is used as a separator 23 used a plane-parallel formed as a dichroic mirror plate. Each laser can then - according to the wavelength of its light - its own separation element 23 . 23 ' be assigned, which is durchstrahlbar by the other lasers. These separating elements are then preferably arranged on a common optical axis and direct the laser beams in the direction of a single deflecting element 7 like this 1 is apparent.

That of the lasers 1 . 1' coming, bundled light 2 . 2 ' becomes at the highly reflective area 21 of the deflecting element 7 in the direction of - preferably inclined to the horizontal by 45 ° - mirror 4 distracted.

Preferably, the brighter of the two by the separating element 23 generated rays thus hits the high-reflectance area 21 of the deflecting element 7 on where he is in the direction of the optical axis 5 and the geometrical axis G identical thereto, on the mirror 4 is steered. This excitation beam 24 , which focuses by means of inserted aperture 22 is preferably hidden by the mirror 4 in the direction of the at least approximately perpendicular optical axis 5 distracted, pierces the optics 8th and hits in the optical axis 5 to the test 6 on. The at least approximately perpendicular to the sample 6 incident excitation beam 24 is especially suitable for fluorescence in samples 6 which are immersed in a liquid or in liquid form. Such samples are preferably presented in the wells of microplates for examination. Even if high-density microplates are to be used with not only 96 but 384, 1536 or more wells, the at least approximately perpendicular excitation beam always reaches the samples 6 ,

In contrast to the use of known and expensive, so-called "f (θ) optics" to achieve at least almost perpendicular to a sample 6 incident excitation beam, the present invention allows the use of much lower cost optical elements for the mirror 4 and the optics 8th ,

The second, preferably weaker light beam thus strikes a non-mirrored region of the deflection element 7 where he (about 4%) parallel to the direction of the axes 5 , G and at the distance A running on the mirror 4 is steered. This focusing beam 25 is preferably only with inserted aperture 22 active and is from the mirror 4 parallel and at a distance A to the direction of the at least approximately perpendicular optical axis 5 distracted. The optics 8th directs the focusing beam on the in the at least approximately vertical optical axis 5 lying focal point 9 ,

The above for the use of the focusing beam (see second embodiment, 2 ) and based on the reflection reflections on the focus, ie to set an optimal working distance between optics 8th and sample 6 apply mutatis mutandis here. The same applies to the focusing by measuring the fluorescence or by hand in or on the sample 6 produced diffused scattered light, wherein using the third embodiment described herein and when measuring the fluorescence, the aperture 22 retracted from the beam path remains.

Part of it is essentially dome-shaped from the sample 6 Expansive fluorescence is reflected in optics 8th collimated (ie, collimated) and from the mirror 4 in the direction of the optical arrangement 16 distracted. Despite a small loss, essentially from the area of the highly reflective area 21 of the deflecting element 7 is determined, this collimated fluorescent light is incident on the optical arrangement 16 and becomes the second focal point 17 focused. There passes - depending on the selected diameter of the pinhole 18 - A certain part of the focused fluorescent light this pinhole 18 and the spectral filter 19 and is from the detector 20 detected. For scanning the sample, the same beam path, as just described, is used.

This one always in the optical axis 5 extending excitation beam is from the sample 6 reflects and runs in the opposite direction towards the laser 1 . 1' ,

When using two monochromatic lasers 1 . 1' is preferably a beam splitter element 26 between the pinhole 18 and the first detector 20 (see. 3 ) arranged. A first collection list 28 serves to collect the through the pinhole 18 continuous light of the second Wel lenlänge 3 , The beam splitter element 26 becomes a second condenser lens 28 ' downstream, with which through the pinhole 18 continuous light of the fourth wavelength 3 ' collected and the second detector 20 ' with the second spectral filter 19 ' is forwarded.

Optionally, further optical elements (lenses, mirrors, diaphragms) for optimized beam guidance with regard to signal efficiency and filtering effect between the pinhole 18 and the detector 20 - Preferably between the spectral filter 19 and the detector 20 - to be placed.

The Reference numerals in the various figures indicate the respective same characteristics. Any combinations of the shown or described embodiments belong to the scope of the present invention.

• • der Einsatz von Probenträgern 14 in der Grösse einer Mikroplatte oder von noch grösseren Probenträgern wird ermöglicht;
• • der Arbeitsabstand kann bis zu 7 mm oder mehr betragen;
• • die numerische Apertur des Objektivs liegt über einem Wert von 0.4 und beträgt bis zu 0.6 oder mehr;
• • insbesondere dank dem Einsatz eines Gegenschwingers 29 kann die Oszillationsamplitude D bis zu 25 mm oder mehr betragen;
• • die zumindest näherungsweise senkrechte Anregung der Proben wird mit einer kostengünstigen und weitgehend aberrationsfreien Optik erreicht;
• • das Umlenkelement 7 kann als einfache Glasscheibe oder als Pinspiegel ausgebildet sein und ermöglicht als einfaches optisches Element das Trennen von Anregungsstrahl und Fluoreszenz;
• • die Verwendung eines Umlenkelements 7 in der Form eines Pinspiegels ermöglicht den gleichzeitigen Einsatz von mehreren monochromatischen Lasern unterschiedlicher Wellenlänge und die Vermessung einer nahezu unbegrenzten Zahl verschiedener Fluoreszenzfarbstoffe unterschiedlicher Emissionswellenlänge;
• • der Austausch von zwei monochromatischen Lasern unterschiedlicher Wellenlänge bedingt keine weiteren Umbauten am optischen System;
• • die Anwendung der offenbarten unterschiedlichen Fokussiermodi in Kombination mit einer Lochblende unterschiedlichen Durchmessers erlaubt das in den Fokus bringen, Anregen und Vermessen der Fluoreszenz verschiedenster Proben und Probenformate.

Among the advantages of the present invention different from the prior art include:

• • the use of sample carriers 14 in the size of a microplate or even larger sample carriers is enabled;
• • the working distance can be up to 7 mm or more;
• • The numerical aperture of the lens is above a value of 0.4 and is up to 0.6 or more;
• • especially thanks to the use of a counter-oscillator 29 the oscillation amplitude D can be up to 25 mm or more;
• • The at least approximately vertical excitation of the samples is achieved with a cost-effective and largely aberration-free optics;
• • the deflection element 7 can be designed as a simple glass plate or as a pin mirror and allows the separation of the excitation beam and fluorescence as a simple optical element;
• • the use of a deflecting element 7 in the form of a pin mirror allows the simultaneous use of several monochromatic lasers of different wavelengths and the measurement of a virtually unlimited number of different fluorescent dyes of different emission wavelength;
• • the replacement of two monochromatic lasers of different wavelengths requires no further modifications to the optical system;
• The use of the disclosed different focusing modes in combination with a pinhole of different diameters allows to focus, excite and measure the fluorescence of a wide variety of samples and sample formats.

Claims (29)

Optical system for exciting and measuring fluorescence on or in samples treated with fluorescent dyes, comprising: a) at least one first laser ( 1 ), which is used to excite first fluorescent dyes light of a first wavelength ( 2 ), so that these first fluorescent dyes light a second wavelength ( 3 ) emit; b) a deflecting element ( 7 ), which serves as an optical element for separating an excitation beam ( 24 ) of the first wavelength ( 2 ) of the fluorescent light of the second wavelength ( 3 ) is trained; c) a mirror ( 4 ) for redirecting the from the first laser ( 1 ), on the mirror ( 4 ) incident, focused light of the first wavelength ( 2 ) in the direction of a sample ( 6 ) or for deflecting the from the direction of the sample ( 6 ) and on the mirror ( 4 ) incident light of the second wavelength ( 3 ) towards a detector ( 20 ); d) an optic ( 8th ), to form a first focal point ( 9 ) on or in the sample ( 6 ) for the first laser ( 1 ) incident and through the mirror ( 4 ) deflected, focused light of the first wavelength ( 2 ); e) a unit ( 10 ), which the mirror ( 4 ) and the optics ( 8th ), wherein mirror and optics in the unit are arranged immovably against each other and this unit ( 10 ) linearly reciprocally arranged and with an oscillating linear drive ( 11 ), the optics ( 8th ) as a collimator for that of the first fluorescent dyes on or in the sample ( 6 ) emitted light of the second wavelength ( 3 ) and the mirror ( 4 ) also to divert this collimated light ( 12 ) opposite to the direction of incidence of the focused light of the first wavelength ( 2 ) are formed; f) a table ( 13 ) for receiving sample carriers ( 14 ) for samples treated with at least one first fluorescent dye ( 6 ); g) an optical arrangement ( 16 ) for imaging a second focal point ( 17 ) with the first fluorescent dyes on or in the pro ( 6 ) emitted by the optics ( 8th ) collimated and the mirror ( 4 ) deflected light of the second wavelength ( 3 ); h) one in the second focal point ( 17 ) arranged aperture ( 18 ) for masking light of the second wavelength ( 3 ) which is farther away than at a certain distance from the focal point ( 17 ) on this pinhole ( 18 ) impinges; i) a first spectral filter ( 19 ) to select through the aperture ( 18 ) transmitted light of the second wavelength ( 3 ); and j) a first detector ( 20 ) for measuring the intensity of the pinhole ( 18 ) and from the first spectral filter ( 19 ) selected light of the second wavelength ( 3 ), one of the laser ( 1 ) emitted and at the deflecting element ( 7 ) in the direction of the mirror ( 4 ) deflected focusing beam ( 25 ) of light ( 2 ) azent and at a distance (A) to an optical axis ( 5 ) of this optical system is arranged to extend. Optical system for exciting and measuring fluorescence on or in samples treated with fluorescent dyes, comprising: a) at least one first laser ( 1 ), which is used to excite first fluorescent dyes light of a first wavelength ( 2 ), so that these first fluorescent dyes light a second wavelength ( 3 ) emit; b) a deflecting element ( 7 ), which serves as an optical element for separating an excitation beam ( 24 ) of the first wavelength ( 2 ) of the fluorescent light of the second wavelength ( 3 ) is trained; c) a mirror ( 4 ), for deflecting the from the first laser ( 1 ) coming to the mirror ( 4 ) incident, focused light of the first wavelength ( 2 ) in the direction of a sample ( 6 ) or for deflecting the from the direction of the sample ( 6 ) and on the mirror ( 4 ) incident light of the second wavelength ( 3 ) towards a detector ( 20 ); d) an optic ( 8th ), to form a first focal point ( 9 ) on or in the sample ( 6 ) for the first laser ( 1 ) incident and through the mirror ( 4 ) deflected, focused light of the first wavelength ( 2 ); e) a unit ( 10 ), which the mirror ( 4 ) and the optics ( 8th ), wherein mirror and optics in the unit are arranged immovably against each other and this unit ( 10 ) linearly reciprocally arranged and with an oscillating linear drive ( 11 ), the optics ( 8th ) as a collimator for that of the first fluorescence dyes on or in the sample ( 6 ) emitted light of the second wavelength ( 3 ) and the mirror ( 4 ) also to divert this collimated light ( 12 ) opposite to the direction of incidence of the focused light of the first wavelength ( 2 ) are formed; f) a table ( 13 ) for receiving sample carriers ( 14 ) for samples treated with at least one first fluorescent dye ( 6 ); g) an optical arrangement ( 16 ) for imaging a second focal point ( 17 ) with that by the first fluorescent dyes on or in the sample ( 6 ) emitted by the optics ( 8th ) collimated and the mirror ( 4 ) deflected light of the second wavelength ( 3 ); h) one in the second focal point ( 17 ) arranged aperture ( 18 ) for masking light of the second wavelength ( 3 ) which is farther away than at a certain distance from the focal point ( 17 ) on this pinhole ( 18 ) impinges; i) a first spectral filter ( 19 ) to select through the aperture ( 18 ) transmitted light of the second wavelength ( 3 ); and j) a first detector ( 20 ) for measuring the intensity of the pinhole ( 18 ) and from the first spectral filter ( 19 ) selected light of the second wavelength ( 3 ), wherein the oscillating linear drive ( 11 ) for moving the unit back and forth linearly ( 10 ) comprises a connecting rod. Optical system according to claim 2, characterized in that one of the lasers ( 1 ) emitted and at the deflecting element ( 7 ) in the direction of the mirror ( 4 ) deflected focusing beam ( 25 ) of light ( 2 azentrisch and at a distance (A) to an optical axis ( 5 ) of this optical system is arranged to extend. Optical system according to claim 1 or 3, characterized in that in addition one of the laser ( 1 ) emitted and at the deflecting element ( 7 ) in the direction of the mirror ( 4 ) deflected excitation beam ( 24 ) of light ( 2 ) parallel to the focusing beam ( 25 ) and acentric at a distance (A) from the optical axis (FIG. 5 ) is arranged running. Optical system according to claim 1 or 3, characterized in that in addition one of the laser ( 1 ) emitted and at the deflecting element ( 7 ) in the direction of the mirror ( 4 ) deflected excitation beam ( 24 ) of light ( 2 ) identical to the focusing beam ( 25 ) is arranged running. Optical system according to claim 1 or 3, characterized in that in addition one of the laser ( 1 ) emitted and at the deflecting element ( 7 ) in the direction of the mirror ( 4 ) deflected excitation beam ( 24 ) of light ( 2 ) and centric to the optical axis ( 5 ) is arranged running. Optical system according to one of the preceding claims, characterized in that the deflecting element ( 7 ) for redirecting the bundled light ( 2 ) from the first laser ( 1 ) in the direction of this mirror ( 4 ) one for the collimated laser beam ( 2 ), high-reflectance area ( 21 ) and in its other areas, both for the light of the first ( 2 ) as well as the second wavelength ( 3 ) is substantially permeable. Optical system according to claim 7, characterized in that the high-specular region ( 21 ) of the deflecting element ( 7 ) at a distance (A) to an optical axis ( 5 ) is arranged so that the beam path for the out of the laser ( 1 ), bundled light of the first wavelength ( 2 ) at a distance (A) to this optical axis ( 5 ) runs. Optical system according to one of the preceding claims, characterized in that the table ( 13 ) for aligning the sample ( 6 ) opposite the first focal point ( 9 at least effectively in Direction of the X and Z spatial axes of a substantially rectangular coordinate system is movable. Optical system according to claim 9, characterized in that the table ( 13 ) for aligning the sample ( 6 ) opposite the first focal point ( 9 ) is formed in the direction of the Z-space axis about the X and / or Y-axis tiltable or linearly displaceable. Optical system according to claim 9 or 10, characterized in that the table ( 13 ) is also movable in the direction of the Y-space axis, wherein X- and Y-axis at least approximately parallel to the surface ( 15 ) of a sample carrier ( 14 ) lie. Optical system according to one of claims 4 to 6, characterized in that it has an opaque aperture ( 22 ) for temporary masking of the excitation beam ( 24 ) and / or the focusing beam ( 25 ) or the corresponding one of the sample ( 6 ) reflected laser beam. Optical system according to one of the preceding claims, characterized in that it also has a separating element ( 23 ) for separating the from the laser ( 1 ) coming light of the first wavelength ( 2 ) into an excitation beam ( 24 ) for the fluorescence and one to this excitation beam ( 24 ) parallel focusing beam ( 25 ). Optical system according to claim 13, characterized in that the separating element ( 23 ) is selected from a group comprising a dichroic mirror, a plane-parallel glass plate with back full mirroring and a simple, plane-parallel glass plate obliquely placed in the beam path. Optical system according to one of the preceding claims, characterized in that it comprises an offset element which is arranged between the laser ( 1 ) and the mirror ( 4 ) is arranged and designed as a simple, obliquely placed in the beam path, plane-parallel glass plate. Optical system according to one of the preceding claims, characterized in that the first spectral filter ( 19 ) between the pinhole ( 18 ) and the first detector ( 20 ) is arranged. Optical system according to one of the preceding claims, comprising: a) a second laser ( 1' ), which is used to excite second fluorescent dyes light of a third wavelength ( 2 ' ) so that these fluorescent dyes light a fourth wavelength ( 3 ' ) emit; b) a second spectral filter ( 19 ' ) for selecting the light of the fourth wavelength ( 3 ' ); and c) a second detector ( 20 ' ) for measuring the intensity of the second spectral filter ( 19 ' ) selected light of the fourth wavelength ( 3 ' ), characterized in that it also has a beam splitter element ( 26 ) which is emitted for the light emitted by the first fluorescent dyes and by the optics ( 8th ) collimated light ( 12 ) of the second wavelength ( 3 ) is at least partially transmissive and which for mirroring or deflecting the light of the fourth wavelength ( 3 ' ) in the direction of a second detector ( 20 ' ) is trained. Optical system according to claim 1 or 17, characterized in that it additionally comprises at least one converging lens ( 28 . 28 ' ) for collecting the through the aperture ( 18 ) continuous light of the second ( 3 ) or fourth wavelength ( 3 ' ). Optical system according to claim 17 or 18, characterized in that the beam splitter element ( 26 ) between the pinhole ( 18 ) and the detector ( 20 ) is arranged. Optical system according to one of the preceding claims, characterized in that the from the sample table ( 13 ) recordable sample carriers ( 14 ) is designed as a slide, in particular made of plastic, glass, or silicon. Optical system according to one of claims 1 to 19, characterized in that the from the sample table ( 13 ) recordable sample carriers ( 14 ) is designed as a microplate, in particular with 96, 384, 1536 or more wells. Optical system according to one of claims 1 to 19, characterized in that the from the sample table ( 13 ) recordable sample carriers ( 14 ) as a framework ( 31 ) is designed for receiving and holding a plurality of microscope slides, in particular made of plastic, glass, silicon and the like, and has the outer mass of a microplate. Optical system according to one of the preceding claims, characterized in that the sample table ( 13 ), for stimulating and measuring the samples ( 6 ) emitted fluorescence, is designed to be movable in the direction of the X-axis, and that the unit ( 10 ) is simultaneously reciprocable in the direction of the Y-axis, wherein the Y-axis is parallel to the optical axis ( 5 ) runs. Optical system according to one of claims 1 or 23, characterized in that the oscillating linear drive ( 11 ) is designed as a "voice coil". Optical system according to one of claims 1, 2, 23 or 24, characterized in that the unit ( 10 ) also a counter-oscillator ( 29 ), which, for absorbing vibrations of the unit, in push-pull to the linear drive ( 11 ) is movable in the direction of the Y-axis. Optical system according to one of the preceding claims, characterized in that the pinhole ( 18 ) is designed to be interchangeable for different hole sizes. Optical system according to one of the preceding claims, characterized in that the first spectral filter ( 19 ) is designed as a filter slide with at least two different, mutually automatically interchangeable filters and this filter slide is manually designed to be interchangeable with another filter slide. Optical system according to one of the preceding claims, characterized in that it comprises a computer or microprocessor for recording and processing the data by means of detectors ( 20 . 20 ' ) comprises detected measurement signals and for outputting data corresponding to these signals. An optical system according to claim 28, characterized in that the computer or microprocessor is also used to control the movements of the table ( 13 ) is formed, wherein the table ( 13 ) Is designed to be displaceable in the directions of the X and / or Y and / or Z-axis and tiltable about the X and / or Y-axis.