DE102014110341A1 - Method and apparatus for microscopically examining a sample - Google Patents

Abstract

The invention relates to a method for microscopically examining a sample (5). The method includes the step of contacting the sample (5) with an optically transparent medium (12) having a higher refractive index than the sample (5), generating an illuminating light beam (13) and directing the illuminating light beam (13) by an illumination lens (1), which focuses the illumination light beam. Subsequently, the deflecting of the illumination light beam (13) in the direction of the sample to be examined (5) with a deflection on a detection lens (2) arranged deflection such that the illumination light beam on an interface (10) between the optically transparent medium (12) and the Sample (5) meets and there is totally reflected to the evanescent illumination of the sample (5). Finally, the detection of the fluorescent light emanating from the sample (5) and passing through the detection objective (2) takes place.

Description

The invention relates to a method for microscopically examining a sample.

The invention also relates to a device for carrying out such a method.

In microscopic fluorescence investigations of samples, these are usually illuminated directly with an illuminating light beam in order to optically excite the sample in the illuminated area and subsequently to detect the fluorescent light emanating from the sample. In scanning microscopy, the focus of an illumination light bundle is usually meander-shaped over or through the sample with the aid of a controllable beam deflection device, which may for example contain one or more tilt mirrors, and the sample is thus scanned sample by sample point.

As an alternative to direct sample illumination, it is also possible to illuminate the sample evanescently. In this case, the excitation light is totally reflected at an interface to the sample, wherein the excitation of the sample is carried out by evanescent electromagnetic field decaying with the penetration depth. For this type of sample analysis, the term TIRFM (total internal reflection fluorescence microscopy) has become common.

Out DE 103 44 410 A1 is a scanning microscope with evanescent sample illumination known. The scanning microscope includes a light source, whose light is coupled into a cover glass, so that it can spread in space by total internal reflection and can illuminate a sample arranged on the cover glass over a large area evanescently. In addition, the scanning microscope has a point detector which receives detection light emanating from a grid point of the sample, and a beam deflecting device arranged in the beam path of the detection light for displacing the position of the grid point in the sample. However, this device has the disadvantage that the coupling of the illumination light in the cover glass is not very efficient and therefore only a reduced amount of light for evanescent sample illumination is available.

In addition, the methods and devices known from the prior art with regard to the evanescent sample illumination often have the disadvantage that the exact location of the sample illumination can not be adjusted flexibly and with sufficient precision.

In addition, the methods and devices known from the prior art have the disadvantage that the samples before microscopic examination with evanescent lighting consuming in special sample chambers or between coverslips must be arranged and prepared before the sample chamber or the arrangements of coverslips between them the sample is mechanically clamped, can be placed on the stage of a microscope. Often just the edge regions of the sample, which abut the interface at which the total reflection should take place, are damaged by the continuous pressure load, so that the subsequent microscopic examination is at least adversely affected or even made completely impossible.

It is the object of the present invention to specify a method for the microscopic examination of a sample which can be used flexibly for different samples and arrangements of samples and can be carried out in particular in a manner that protects the sample.

• a. In-Kontakt-bringen der Probe mit einem optisch transparenten Medium, das einen höheren Brechungsindex aufweist als die Probe,
• b. Erzeugen eines Beleuchtungslichtbündels,
• c. Lenken des Beleuchtungslichtbündels durch ein Beleuchtungsobjektiv, das das Beleuchtungslichtbündel fokussiert,
• d. Umlenken des Beleuchtungslichtbündels, das das Beleuchtungsobjektiv durchlaufen hat, in Richtung auf die zu untersuchende Probe mit einem an einem Detektionsobjektiv angeordneten Umlenkmittel, derart dass das Beleuchtungslichtbündel auf eine Grenzfläche zwischen dem optisch transparenten Medium und der Probe trifft und dort zur evaneszenten Beleuchtung der Probe totalreflektiert wird,
• e. Detektieren des von der Probe ausgehenden und durch das Detektionsobjektiv verlaufenden Fluoreszenzlichtes. Beispielsweise mit einem Detektor, der ein zur Lichtleistung des Fluoreszenzlichts proportionales elektrisches Signal erzeugt.

The problem is solved by a method which includes the following steps:

• a. Contacting the sample with an optically transparent medium having a higher refractive index than the sample,
• b. Generating an illumination light beam,
• c. Directing the illumination light beam through an illumination objective that focuses the illumination light beam,
• d. Redirecting the illumination light bundle, which has passed through the illumination objective, in the direction of the sample to be examined with a deflecting means arranged on a detection objective, such that the illumination light beam strikes an interface between the optically transparent medium and the sample and is totally reflected there to evanescent illumination of the sample .
• e. Detecting the emanating from the sample and passing through the detection lens fluorescent light. For example, with a detector that generates an electrical signal proportional to the light output of the fluorescent light.

It is a further object of the present invention to provide a device which makes it possible to image a microscopic sample according to the above-mentioned method with evanescent illumination.

This object is achieved by a device which has an illumination objective and a detection objective, on which a deflection means for deflecting an illumination light beam is arranged on an interface between an optically transparent medium and a sample.

The invention has the advantage that an evanescent sample illumination with particularly large Efficiency is possible. This is particularly due to the fact that the illumination light beam due to the deflection with the deflection means, which takes place after passing through the illumination lens, can be easily and reliably directed at the required angle of incidence to effect a total internal reflection on the interface between the optically transparent medium and the sample ,

In addition, the invention has the further, particular advantage that the impact on the interface between the optically transparent medium and the sample can be changed easily and without the risk that the critical angle of total reflection is passed through unintentionally, which will be explained in more detail below is.

In addition, the method according to the invention can advantageously also be carried out in such a way that a sample can be brought into an examination position completely without pressure loading of the edge surface to be examined and only brought into contact with the optically transparent medium immediately before the generation of a microscopic image, so that the microscopic examination of a largely unloaded sample is possible. This is further explained below in particular in connection with some embodiments.

In order to be able to reliably align the illuminating light bundle with the angle of incidence required for total internal reflection on the interface between the specimen and the optically transparent medium, the boundary surface is preferably aligned at an angle other than zero to the optical axis of the illuminating objective and / or the detection objective. Particularly flexible and versatile is an embodiment in which the interface between the sample and the optically transparent medium is aligned perpendicular to the optical axis of the illumination objective and / or the detection objective. In particular, such an alignment makes it possible to illuminate the sample and in particular one and the same sample area of the sample from different directions and in particular in each case with the same angle of incidence. This is done, for example, to investigate the fluorescence properties of the sample as a function of the polarization of the illumination light, in particular as a function of the orientation of the plane of the linear polarization of the illumination light.

In particular, for this purpose, it can be advantageously provided that the illumination light bundle after deflection extends in a plane which has an angle different from zero degrees to the optical axis of the illumination objective. For example, it can be provided that the illumination light beam is deflected such that it strikes the interface between the sample and the optically transparent medium at an angle of incidence in the range of 55 to 70 degrees, in particular in the range of 60 to 64 degrees. Within these areas, evanescent sample illumination of most biological samples is possible with sufficient penetration depth.

In a particularly advantageous embodiment of the method according to the invention, the impact location and / or the angle of incidence and / or the direction of incidence of the illumination light beam are changed to the interface between the sample and the optically transparent medium during the microscopic examination.

Changing the location of incidence makes it possible, for example, to sequentially illuminate and examine different sample areas by supplying the fluorescent light emanating from these sample areas to an area detector for detecting a two-dimensional image, such as a CCD detector, and to generate an image of the respective sample area. In particular, it is possible, for example, to scan the peripheral surface of the sample which is in contact with the optically transparent medium systematically along a scanning path and to assign a surface image to each sample region which has been swept thereby. From the obtained image information for the individual sample areas, an overall image of the sample can then be reconstructed. For example, the point of incidence of the illumination light beam on the interface between the sample and the optically transparent medium can be changed continuously along a, in particular meandering, scan path. In this way, the fact can be taken into account that the illuminated area is usually smaller than the field of view of the detection objective. By juxtaposing several images of different sample areas, the entire field of view of the detection objective can be utilized.

However, such a procedure is probably reserved for special applications, while usually only a sufficiently large illumination focus is generated in order to be able to evanescently illuminate the entire sample area of interest simultaneously and simultaneously with an area detector for acquiring a two-dimensional image, such as a CCD detector, for example. to create an image of the sample area.

In order to get the largest possible illumination spot, the focus of the Lighting beam in the illumination light propagation direction should be as long as possible and have the largest possible focus diameter. A long focus in the illumination light propagation direction is important because the illumination spot on the interface between the optically transparent medium and the sample is elongated due to the large angle of incidence at which the illumination light beam strikes the interface. In the case of an illumination light bundle which is circular in cross section perpendicular to the direction of propagation, the incident surface on the interface between the optically transparent medium and the sample is of course elliptical. In that regard, the focal length should preferably be greater than twice the large semiaxis of the ellipse of the impact surface. A large focus diameter has an effect especially in the direction of the small half-axes.

In order to be able to generate the largest possible focus diameter and a focal length that is as long as possible in the propagation direction, the illumination objective preferably has a low numerical aperture of, for example, 0.05. With such an objective, an incident surface of approximately 100 m by 10 m could be illuminated quite roughly and depending on other parameters, such as the chosen angle of incidence to the interface between the optically transparent medium and the sample.

In order to achieve an enlargement of the incident surface, in particular in the direction of the small semiaxes, instead of a circular illuminating light bundle, a correspondingly oriented light stripe, that is to say a lighting bundle that is oblong in cross section perpendicular to the propagation direction, or a quasi-stripe of light can be coupled into the illumination objective, which will be described in more detail below is explained.

In a particular embodiment, as an alternative or in addition to a change in the location of incidence, the angle of incidence of the illuminating light beam is changed to the interface between the sample and the optically transparent medium. In this way it is possible, for example, to vary the penetration depth of the evanescent field into the sample and / or the size of the spot illuminated by the illuminating light bundle.

As an alternative or in addition to a change in the point of incidence and / or a change in the angle of incidence, the direction of incidence of the illuminating light bundle can also be changed to the interface between the sample and the optically transparent medium. This can be done in particular in order to simultaneously rotate the direction of incidence of the linear polarization direction of the light striking the interface, if, for example, properties of the sample which depend on the linear polarization direction are to be investigated.

In order to be able to change the point of incidence and / or the angle of incidence and / or the direction of incidence of the illuminating light bundle to the interface between the specimen and the optically transparent medium, it is possible in particular to use a beam deflecting device which can be adjusted with respect to the deflection angle. This can include, for example, a gimbal-mounted tilting mirror or two tilting mirrors that can be rotated about different axes of rotation. In particular, it can be provided that the illumination light beam is moved with the beam deflection device relative to the illumination objective and / or relative to the deflection means.

As an alternative or in addition to the use of an adjustable beam deflection device, it is also possible that the incidence and / or the angle of incidence and / or the direction of incidence of the illumination light beam on the interface between the sample and the optically transparent medium by moving the sample relative to the illumination objective and / or is changed relative to the illumination light beam. For this purpose, in particular, a sample table, which can preferably be displaced in three spatial directions, can be used.

In addition to the possibilities mentioned for changing the point of incidence, the angle of incidence and / or the direction of incidence, it is also possible to change these parameters by moving the deflecting means relative to the sample. This can be done alternatively or in addition to the use of a beam deflecting device and / or to move the sample. In a particular embodiment, the deflection means is immovably attached to the detection lens. In particular, however, in order to be able to change the impact location and / or the angle of incidence and / or the direction of incidence, the deflecting means can advantageously be movably arranged on the detection objective, in particular fastened thereto.

In a particular embodiment, the deflection means is arranged in the front region and / or in the vicinity of the front lens.

In a particular embodiment, the deflection means is designed as a mirror with several facets. In particular, such an embodiment makes it possible to change the point of incidence and / or the angle of incidence and / or the direction of incidence of the illumination light beam on the interface between the sample and the optically transparent medium in that - for example by means of an adjustable beam deflector or by moving the deflection - successively different facets are illuminated. Here can be provided in particular that the facets are spatially differently aligned in order to effect each a reflection in another spatial direction. Alternatively, it is also possible, for example, for the deflection means to have a frustoconical mirror surface. Such an embodiment makes it possible to rotate the illuminating light bundle continuously and descriptively around a cone surface around an axis running through the point of impact.

As explained, the method according to the invention offers a whole range of possibilities for setting the point of incidence and / or the angle of incidence and / or the direction of incidence, which has the very special advantage that the user can adapt the method individually to the particular sample-specific or examination-specific requirements ,

In the event that the impact location is changed, in particular even if a scanning of the interface between the optically transparent medium and the sample takes place, an assignment of the respective impact location to a respective sample area can be carried out to produce an overall image of the sample composed of several images.

In particular, it can be provided that for each impact location, taking into account the angle of incidence and / or the refractive index of the sample and / or the refractive index of the optically transparent medium and / or the wavelength of the illumination light beam and / or the diameter of the impact location, an associated sample area is determined. In such a determination, errors, in particular system-related errors, are preferably corrected or compensated. In particular, a compensation or correction of spatial deviations of the actual course of the illumination light beam relative to a to be expected according to the principles of geometric optics beam path, such as by the Goos-chicken effect, take place. Preferably, a compensation or a correction of the errors, which would be caused by a deviation of the thickness of the optically transparent medium, in particular a cover glass, from a desired thickness. For example, it is not uncommon for the actual thickness of a cover glass to deviate significantly from the standard thickness of, for the most part, 170 m. Such compensations and the consideration of such correction factors have the advantage that a particularly good resolution of the image of the sample to be examined can be achieved.

In an advantageous embodiment, each impact location and / or each assigned sample area is assigned at least one specific, in particular two-dimensional, image which was obtained by detecting the detection light emanating from the sample during the illumination of the respective impact location.

The term image is understood in particular to mean a reproduction of the sample or a part of the sample in the form of data that can be displayed, for example by means of a PC, and / or in the form of an image visible to the naked eye.

In particular, the detector may be designed such that it generates an electrical signal proportional to the light output of the received detection light for each pixel. In particular, it can also be provided that the detector - simultaneously or sequentially - specifically receives fluorescent light of different wavelengths and respectively generates corresponding electrical detection signals. For example, the detector may be a CMOS sensor which is capable of detecting fluorescence light independently of wavelength in different layers (channels). Alternatively, the detector may include a plurality of CCD detectors to which detection light of different wavelength ranges is fed with dichroic beam splitters. In particular for a detection of fluorescent light of different wavelengths, the excitation light beam may include a plurality of excitation light wavelengths, for example a multi-color laser or a plurality of individual lasers or a white light source.

As already mentioned, the illuminating light beam may be circular in cross section. In such an embodiment, the impact surface on the interface between the optically transparent medium and the sample is naturally elongate and elliptical. However, as already mentioned, it is also possible that the illuminating light beam has the shape of a light stripe. Such a beam shape can be achieved, for example, with a cylinder optic arranged in the beam path of the illumination light bundle. Alternatively, it is also possible to produce a quasi-light strip in that a cross-sectionally substantially round illumination light bundle, for example with the aid of a beam deflection device in a spatial direction, is moved back and forth in such a rapid manner that it can no longer be generated by a light strip generated by means of a cylinder optics is different.

The use of an illumination light beam in the form of a light strip makes it possible to illuminate the interface between the optically transparent medium and the sample over a large area.

A particularly flexible and precisely working arrangement for carrying out the method according to the invention results when the optical axis of the illumination lens and the optical axis of the detection lens are aligned parallel or coaxial with each other, preferably the detection lens and the illumination lens with their respective front lenses facing each other. In such an embodiment, the sample to be examined can be arranged in the intermediate space between the illumination objective and the detection objective, with advantageously a lot of space for guiding and directing the illumination light bundle, in particular with regard to illuminating the interface from different directions or with different angles of incidence, remains. This has the particular advantage that there are a large number of possibilities for adapting the examination conditions to the respective requirements.

In a very particularly advantageous embodiment of the method according to the invention, the same sample is examined with the same device and another examination method. Such a procedure has the very special advantage that information about the sample can be obtained independently of one another with different examination methods, for example in order to compare this information with one another or to gather as much information about the sample as possible. In this case, the sample preferably remains in the space between the illumination objective and the detection objective.

In addition to a study in which an evanescent illumination of the specimen as described above takes place, a single plane illumination microscopy (SPIM) examination can advantageously be carried out, in which the illuminating light bundle, in particular in the form of a light stripe or a quasi light stripe, directed directly and without total reflection on and through the sample. By means of such a light stripe or quasi-light stripe, a layer of the sample is completely transilluminated, with detection of the detection light emanating vertically from this layer and collimated by the detection objective. In order to be able to obtain location information regarding the individual sample locations of the layer, an area detector, for example a CCD detector, is preferably used. By successive shifting of the light strip relative to the sample, it is possible to screen-through layer by layer and to produce a layer stack of spatially resolved detection signals and thus a three-dimensional reconstruction of the sample.

In particular for a SPIM examination, it may be advantageously provided that the illumination light bundle is deflected directly to the sample by means of a further deflection means arranged on the illumination objective after passing through the illumination objective. This is preferably such that the light strip propagates at an angle other than zero to the optical axis of the detection objective, in particular at an angle of 90 degrees to the optical axis of the detection objective. The plane in which the deflected light strip or quasi-light stripe propagates is preferably aligned perpendicular to the optical axis of the detection objective. Such an alignment allows a particularly precise image reconstruction, in which complex geometric correction calculations are largely avoided.

In a particular embodiment, at least two deflecting means are arranged on the detection objective, one of which serves for deflecting the illuminating light bundle for evanescent sample illumination, while the further deflecting means serves to redirect the illuminating light bundle, in particular to a light stripe or quasi-stripe, for a SPIM examination ,

In order to be able to switch between an evanescent sample illumination and a direct sample illumination, the deflection means can be movably arranged on the detection objective such that either the one or the further deflection means can be introduced into the beam path of the illumination light beam. Alternatively or additionally, it is also possible to direct the illuminating light bundle to the respectively desired deflection means with the aid of a beam deflecting device which can be adjusted with respect to the deflection angle.

In most cases, it is advantageous if the illumination light beam is coupled into the illumination objective in such a way that it passes eccentrically through the illumination objective because such a beam path makes it possible to position the sample on or at least close to the optical axis of the illumination objective and / or the detection objective, while being able to illuminate the sample from very different directions.

In a particularly advantageous embodiment of a device suitable for carrying out the method according to the invention, the transparent optical medium itself is the deflection means or at least part of the deflection means. In particular, such an embodiment makes it possible to initially mechanically completely unloaded the sample, for example, to position in a water-filled Petri dish between the illumination objective and the detection objective, wherein Preferably, the illumination objective is arranged in an inverted microscope arrangement spatially below the sample while the detection objective is located above the sample. Only in such an arrangement can the transparent optical medium, together with the detection objective, be moved in the direction of the sample until immediately before the actual examination, until it rests against the sample, thus creating the prerequisite for evanescent sample illumination. In this way, it is effectively avoided that the sample is pressure-loaded long before the actual examination, for example by pinching between coverslips, whereby just the edge areas of the sample to be examined can be sustainably damaged, which is an enormous disadvantage especially for living samples.

The deflection means may in particular comprise a block of transparent material, in particular a prism. In this case, it can be provided, for example, that the illuminating light bundle emerging from the illumination objective is coupled into the prism through a hypotenuse surface of the prism and reflected on a mirrored catheter surface of the prism in the direction of the specimen attached to the hypotenuse surface in such a way that the illuminating light bundle at the hypotenuse surface serves as an interface is totally reflected to the sample. The fluorescent light emanating from the sample passes through the prism to the detection objective, which collimates the detection light.

Between the optically transparent medium, which may be formed, as mentioned, for example, from a block of transparent material, and the detection objective is preferably a means for adjusting the refractive indices, such as an immersion oil.

Such an arrangement makes it possible, in particular, to be able to move the detection objective relative to the sample and relative to the adjacent block of transparent material, for which purpose a corresponding adjustment device for adjusting the distance of the block of transparent material relative to the detection objective can be present. Such an adjustment, for example, with a screw, has the advantage that occurring in the evanescent lighting effects, such as the Goos chicken effect, can be compensated.

In another advantageous embodiment, the optically transparent medium, which also acts as deflection means, is coupled in particular directly to a front lens of the detection objective. The coupling can be realized for example by the use of an optical kit between the front lens of the detection lens and the corresponding counter-molded block of transparent material. For example, the front lens may be formed as a hemispherical lens, to which a correspondingly counter-molded with a hemispherical, concave coupling surface block of transparent material, for example with an optical kit, is coupled. Alternatively, it can also be provided that the optically transparent medium, which simultaneously acts as a deflection means, includes a front lens of the detection objective and / or that the deflection means functions as a front lens of the detection objective. These solutions have the very particular advantage that losses of detection light due to unwanted reflections on the light path of the detection light via the optically transparent medium and the front lens of the detection objective are avoided or at least minimized.

As already mentioned, it may be advantageously provided that the deflection means comprise a block of transparent material, wherein at least one outer surface of the block, in particular the outer surface of the block, which is designed and arranged to abut a sample, acts as a coupling window for the illumination light bundle ,

As also already mentioned, it can be provided that one, preferably another, outer surface of the block is designed as a mirror or has a mirror in order to deflect the illumination light bundle. As already mentioned, in particular for an examination of the same sample on the basis of another examination method, a further deflecting means for deflecting the illuminating light bundle, in particular an illuminating light bundle in the form of a light stripe or quasi-light stripe, may be present, in particular arranged on the detection objective. In particular, the deflecting means and / or the further deflecting means can be movably fastened to the detection objective, for example in order to be able to change the point of incidence and / or the angle of incidence and / or the direction of incidence of the illuminating light bundle and / or geometric corrections, for example for correcting the Goos chicken Effect to be able to make. In particular, for these purposes, as already mentioned, the device for carrying out the method according to the invention may have a beam deflection device which can be adjusted with respect to the deflection angle.

In particular, the beam deflection device may also be the beam deflection device of a scanning microscope, in particular a confocal scanning microscope.

An apparatus which is suitable for carrying out the method according to the invention can advantageously be based on a scanning microscope, in particular a confocal scanning microscope, be constructed. In particular, the use of an inverted microscope stand is suitable. Of particular advantage in this respect is the use of a (possibly already present in a laboratory anyway) scanning microscope for carrying out the method according to the invention.

In the drawing, the subject invention is shown schematically and will be described with reference to the figures below, wherein the same or equivalent elements are usually provided with the same reference numerals. Showing:

1 a detailed view of a first embodiment of an apparatus for carrying out the method according to the invention with a deflecting means having a mirror,

2 a detailed view of a second embodiment of an apparatus for carrying out a method according to the invention, in which the deflection means is formed as a block of a transparent material,

3 a detailed view of a third embodiment of a device according to the invention for carrying out the method according to the invention, in which the deflection means additionally assumes the function of a front lens of the detection lens,

4 a detailed view of a fourth embodiment in which both an evanescent sample illumination, as well as a direct sample illumination is made possible. Shown is the position for an evanescent sample illumination,

5 the detail view of the fourth embodiment, in the setting for direct sample illumination for SPIM examination,

6a and 6b a schematic representation of the incident surface of the illumination light beam,

7a and 7b a schematic representation of the change of the impingement of the illumination light beam,

8th a detailed view of an embodiment with a deflection means having a facet mirror,

9 a detailed view of an embodiment with a deflection means having a cone-shaped mirror, and

10 a detailed view of an embodiment with a beam deflecting device for changing the place of impact and / or the angle of incidence and / or the direction of incidence.

1 shows a detailed view of a first embodiment of an apparatus, based on a possible embodiment of the method according to the invention will be explained below.

The device has an illumination objective 1 and a detection lens 2 , which is designed as Ölobjektiv on. The illumination lens 1 and the detection lens 2 are arranged coaxially with each other and opposite to each other with respect to their optical axes. At the detection lens 2 is a diverter 3 attached, which is a cone-shaped mirror surface 4 having.

The sample to be examined 5 is between a first coverslip 6 and a second coverslip 7 arranged in an aqueous nutrient medium. The coverslips 6 . 7 are by means of a circumferential seal 9 sealed against each other, so that the aqueous nutrient medium 8th can not escape.

Between the cover glass 6 that the detection lens 2 facing, and the detection lens 2 is immersion oil 11 , in which also the deflection 3 is immersed.

The cover glass 6 that the detection lens 2 facing, serves as an optically transparent medium 12 having a higher refractive index than the sample 5 , An illumination light bundle 13 is from the illumination lens 1 focused and passes through after exiting the front lens of the illumination lens 1 the coverslips 6 . 7 without the sample 5 to interact and then passes to the deflection 3 , From this becomes the illumination light beam 13 in the direction of the sample to be examined 5 deflected so that the illumination light beam 13 on the interface 10 between the optically transparent medium 12 and the sample 5 meets and there for evanescent illumination of the sample 5 is totally reflected.

That from the sample 5 outgoing fluorescent light 14 goes through the detection lens 2 and then passes to a non-drawn area detector for detecting a two-dimensional image, such as a CCD detector. With the help of a (not shown) beam deflecting the illuminating light beam 13 to different positions of the frusto-conical mirror surface 4 be steered to the illumination light beam 13 from different directions of incidence on the interface 10 between the optically transparent medium 12 and the sample 5 to steer. This is shown only schematically in the figure by a dashed line, changed beam path of the illumination light beam 13 indicated.

It is especially important for a good resolution that the focus of the Illuminating light beam 13 , in particular taking into account geometric corrections, such as to compensate for the goos-chicken effect or to compensate for a deviation of a thickness of the optically transparent medium 12 , especially the cover glass 6 , of a nominal thickness, exactly on the interface 10 between the optically transparent medium 12 and the sample 5 lies. To set this exactly, the coverslips 6 . 7 together with the sample 5 relative to the illumination lens 1 be adjusted in Z direction. For this purpose, for example, an adjustable in the Z direction displacement table may be present.

In addition, it is also possible the coverslips 6 . 7 along with the interposed sample 5 in the X-direction and / or Y-direction to the place of incidence of the illumination light beam 13 on the interface 10 to be able to adjust. Alternatively or additionally, a change in the point of incidence and / or the angle of incidence and / or direction of incidence can also be effected with the aid of the beam deflecting device which can be adjusted with respect to the deflection angle.

In particular, to ensure that the numerical aperture of the detection lens 2 for collecting the detection light 14 is also exploited, the detection lens 2 also be adjusted in the Z direction and / or in the XY direction relative to the sample.

2 shows a detailed view of a second embodiment, in which the optically transparent medium 12 is designed as a prism, which has both a mirrored surface 15 for redirecting the of the illumination lens 1 coming illumination light beam 13 has, as well as an outer surface 16 , which is designed and arranged with a sample 5 to get in contact, so that between the outer surface 16 and the sample 5 an interface 10 to the total internal reflection of the illumination beam 13 forms.

The sample 5 is in one with an aqueous nutrient medium 8th filled, to the detection lens 2 open container 17 arranged. Such a design has the very special advantage that the sample 5 only immediately before the actual examination with the optically transparent medium 12 comes into contact and only then is pressure loaded. Damage to the sample 5 By continuous pressure load before the actual examination is avoided or at least greatly reduced in this way.

As with the in 1 illustrated embodiment, the detection light runs 14 through the detection lens 2 to a non-illustrated area detector for detecting a two-dimensional image, such as a CCD detector.

Also at the in 2 illustrated embodiment, it is possible, for example by means of an adjustable with respect to the deflection angle beam deflecting device, the impact location and / or the angle of incidence and / or the direction of incidence of the illumination light beam 13 on the interface 10 between the optically transparent medium 12 and the sample 5 precise and flexibly adjusted for the respective examination.

In this embodiment, the optically transparent medium 12 For example, with an optical kit or with an immersion oil, directly to the front lens of the detection lens 2 coupled. To examine the sample 5 becomes the optically transparent medium 12 into the water 8th dipped and sent to the sample 5 created.

3 shows a detail view of a third embodiment, which differs from the in 2 illustrated embodiment characterized in that the optically transparent medium 12 is formed such that it additionally the function of the front lens of the detection lens 2 can take over. In particular, the optically transparent medium 12 a front lens and one acting as a deflection and an outer surface for contact with the sample 5 comprising block, both made in one piece and together. Such a design is particularly robust and less prone to failure.

The 4 and 5 each show a detailed view of a fourth embodiment of an apparatus for carrying out a method according to the invention. The device has a first deflection means 18 and another deflecting means 19 on. Both the first deflection 18 , as well as the further deflection 19 are at the detection lens 2 attached. The first deflection 18 includes an optically transparent medium 20 that has a mirrored surface 21 for redirecting an illumination light beam 13 having. In addition, the optically transparent medium 20 an outer surface 22 on which is designed and arranged with a sample 5 for the formation of an interface 10 for a total internal reflection in contact.

4 shows one of the two possible settings of the device, namely the setting for performing an examination with evanescent sample illumination. Also, with this setting, as described in detail above, a change in the place of impact and / or the angle of incidence and / or the direction of incidence of the illumination light beam 13 on the interface 10 respectively.

The device is additionally designed to perform an examination on the same sample 5 by means of another examination procedure, in particular SPIM. For this purpose, the illumination light beam 13 , in particular in the form of a light strip or a quasi-light strip, on the further deflection means 19 directed. The further deflection 19 has a second mirror surface 23 on.

The further deflection 19 deflects the illumination light beam 13 in such a way that, after the deflection, it is in the direction of the optical axis of the detection objective 2 vertical plane spreads. In this way, a layer of the sample 5 transilluminated and the detection light emanating from the sample 14 that through the detection lens 2 runs, preferably spatially resolved, detected. For spatially resolved detection of the sample 5 outgoing detection light 14 For example, an area detector can be used to acquire a two-dimensional image, in particular a CCD detector or SCMOS detector. The detector is not shown in the figures for the sake of clarity.

To set a correct focus position and / or to compensate for special geometrical effects, as they can occur in particular in an evanescent sample illumination, are the first deflection 18 and the further deflecting means 19 movable and in terms of their relative position to the detection lens 2 adjustable on the detection lens 2 attached. For this purpose, an adjustable fastening device is used 24 ,

Also in this embodiment, it is advantageous if the individual components can be adjusted relative to each other in terms of their spatial position, for example, to adjust the focus position correctly and / or to be able to adjust the point of incidence and / or the angle of incidence and / or the direction of incidence.

The 6a and 6b show a schematic representation of the impact surface 25 of the illumination light beam 13 on the interface 10 between the optically transparent medium 12 and the sample 5 for an evanescent sample illumination.

To get the largest possible illumination spot 25 to get the focus of the illumination beam 13 in the illumination light propagation direction as long as possible and has a large focus diameter. In the case of an illumination light bundle which is circular in cross-section perpendicular to the propagation direction, the incident surface is 25 on the interface 10 between the optically transparent medium 12 and the sample 5 naturally elliptical. In that regard, the focal length should, in particular by using a lighting objective 1 low numerical aperture, preferably greater than twice the major half axis 26 the ellipse of the impact surface 25 be. The focus diameter corresponds approximately to twice the small semi-axis 27 the elliptical impact surface 25 , In this direction can increase the impact area 25 for example, by using an illumination light beam 13 in the form of a light stripe or a quasi-light stripe instead of a cross-sectionally circular illumination light beam 13 be achieved.

The 7a and 7b show a schematic representation of the change of the place of impact 28 of the illumination light beam 13 on the interface 10 between the optically transparent medium 12 and the sample 5 ,

By moving the sample 5 relative to the illumination light beam 13 , for example with an adjustable sample table, and / or by moving the illumination light bundle 13 relative to the sample 5 , For example, with an adjustable with respect to the deflection angle beam deflecting device, and / or by moving the deflecting means 3 can be the place of impact 28 be successively changed, so the impact surface 25 be moved relative to the sample. This, for example, in order to obtain several two-dimensional images of different sample areas, which can then be combined to form an overall image. For example, the sample 5 based on 7a perpendicular to the plane of the drawing relative to the illumination light beam 13 postponed, can the impact locations 28 as in 7b shown schematically lined up and created a two-dimensional image for each place of impact.

8th shows a detailed view of an embodiment with a deflection 3 that has a facet mirror 29 having. The facet mirror 29 has several different facets with different spatial position and orientation.

The sample to be examined 5 is between a first coverslip 6 and a second coverslip 7 in an aqueous nutrient medium 8th arranged. The coverslips 6 . 7 are by means of a circumferential seal 9 sealed against each other, so that the aqueous nutrient medium 8th can not escape.

In this embodiment, for example, with a (not shown in this figure) beam deflecting the illumination light beam 13 on different facets 30 be steered to the illumination light beam 13 at different locations 28 and or at different angles of incidence and / or with different directions of incidence of the illumination light beam 13 on the interface 10 between the sample 5 and the optically transparent medium 12 namely the cover glass 6 to meet. Alternatively, instead of changing the spatial position and / or orientation of the illumination light beam 13 also the facet mirror 29 For example, by means of a (not shown in this figure) positioning unit relative to the illumination light beam 13 to be moved.

9 shows a detailed view of an embodiment with a deflection 3 holding a frustoconical mirror 31 of which, for the sake of clarity, only a section is shown.

The sample to be examined 5 is between a first coverslip 6 and a second coverslip 7 in an aqueous nutrient medium 8th arranged. The coverslips 6 . 7 are by means of a circumferential seal 9 sealed against each other, so that the aqueous nutrient medium 8th can not escape.

The cone-shaped mirror 31 allows, for example, the illumination light beam 13 , in particular continuous and descriptive of a cone surface, through the point of impact 28 To be able to rotate extending axis.

10 shows a detailed view of an embodiment with a deflection angle adjustable adjustable beam deflector 32 for example, which may include one or more galvanometer mirrors for altering the location of impact 28 and / or the angle of incidence and / or the direction of incidence of the illumination light beam 13 on the interface between the sample 5 and the optically transparent medium 12 namely the cover glass 6 ,

In this embodiment, the deflection means 3 as a level mirror 33 educated. By changing the place of impingement of the illuminating light beam 13 on the level mirror 32 can be the place of impact 28 and / or the angle of incidence and / or the direction of incidence of the illumination light beam 13 on the interface between the sample 5 and the optically transparent medium 12 to be changed.

The illumination lens 1 and the course of the illumination light beam 13 is shown in this figure only schematically and not completely in accordance with reality to illustrate the principle of changing the lighting conditions.

LIST OF REFERENCE NUMBERS

1

lighting lens

2

detection objective

3

deflecting

4

Truncated cone-shaped mirror surface

5

sample

6

First cover glass

7

Second cover glass

8th

aqueous nutrient medium

9

poetry

10

interface

11

Immersion oil

12

Transparent optical medium

13

Illumination light beam

14

detection light

15

Mirrored surface

16

outer surface

17

container

18

First deflection

19

Further deflection

20

Optically transparent medium

21

Mirrored surface

22

outer surface

23

Second mirror surface

24

fastening device

25

incident

26

big half-axle

27

small half-axle

28

impingement

29

facet mirror

30

facets

31

cone-shaped mirror

32

adjustable beam deflection device

33

level mirror

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 10344410 A1 [0005]

Claims (19)

Method for microscopically examining a sample ( 5 ), characterized by the following steps: a. Contacting the sample ( 5 ) with an optically transparent medium ( 12 ), which has a higher refractive index than the sample ( 5 b. Generating an illumination light bundle ( 13 c. Steering the illumination light bundle ( 13 ) through an illumination objective ( 1 ), which illuminates the illumination light beam ( 13 ), d. Redirecting the illuminating light bundle ( 13 ), which illuminates the illumination objective ( 1 ), in the direction of the sample to be examined ( 5 ) with one on a detection objective ( 2 ) arranged deflecting means such that the illumination light beam ( 13 ) to an interface ( 10 ) between the optically transparent medium ( 12 ) and the sample ( 5 ) and there for evanescent illumination of the sample ( 5 ) is totally reflected, e. Detecting the from the sample ( 5 ) and through the detection lens ( 2 ) running fluorescent light. A method according to claim 1, characterized in that a. the interface ( 10 ) between the sample ( 5 ) and the optically transparent medium ( 12 ) at an angle different from zero degrees to the optical axis of the illumination objective ( 1 ) and / or the detection objective ( 2 ) or that b. the interface ( 10 ) between the sample ( 5 ) and the optically transparent medium ( 12 ) perpendicular to the optical axis of the illumination objective ( 1 ) and / or the detection objective ( 2 ) is aligned. A method according to claim 1 or 2, characterized in that a. the illumination light beam ( 13 ) after deflection in a plane which is at an angle different from zero degrees to the optical axis of the illumination objective ( 1 ), and / or that b. the illumination light beam ( 13 ) is deflected so that it is at an angle of incidence in the range of 55 to 70 degrees on the interface ( 10 ) between the sample ( 5 ) and the optically transparent medium ( 12 ), and / or that c. the illumination light beam ( 13 ) is deflected so that it is at an angle of incidence in the range of 60 to 64 degrees to the interface ( 10 ) between the sample ( 5 ) and the optically transparent medium ( 12 ) meets. Method according to one of claims 1 to 3, characterized in that a. the place of impact ( 28 ) and / or the angle of incidence and / or the direction of incidence of the illumination light beam ( 13 ) on the interface ( 10 ) between the sample ( 5 ) and the optically transparent medium ( 12 ), and / or that b. the place of impact ( 28 ) of the illumination light beam ( 13 ) on the interface ( 10 ) between the sample ( 5 ) and the optically transparent medium ( 12 ) is continuously changed along a scan path, and / or that c. the place of impact ( 28 ) and / or the angle of incidence and / or the direction of incidence of the illumination light beam ( 13 ) on the interface ( 10 ) between the sample ( 5 ) and the optically transparent medium ( 12 ) by means of a light beam ( 13 ), with respect to the deflection angle adjustable beam deflecting device is changed, and / or that d. the place of impact ( 28 ) and / or the angle of incidence and / or the direction of incidence of the illumination light beam ( 13 ) on the interface ( 10 ) between the sample ( 5 ) and the optically transparent medium ( 12 ) by moving the sample ( 5 ) relative to the illumination objective ( 1 ), and / or that e. the place of impact ( 28 ) and / or the angle of incidence and / or the direction of incidence of the illumination light beam ( 13 ) on the interface ( 10 ) between the sample ( 5 ) and the optically transparent medium ( 12 ) is changed by moving the deflecting means and / or that f. the deflection means ( 3 ) is designed as a multi-faceted mirror and the place of impact ( 28 ) and / or the angle of incidence and / or the direction of incidence of the illumination light beam ( 13 ) on the interface ( 10 ) between the sample ( 5 ) and the optically transparent medium ( 12 ) are changed by successively illuminating different facets. A method according to claim 4, characterized in that a. every place of impact ( 28 ) a sample area is assigned, and / or that b. for each place of impact ( 28 ) taking into account the angle of incidence and / or the refractive index of the sample ( 5 ) and / or the refractive index of the optically transparent medium ( 12 ) and / or the wavelength of the illumination light beam ( 13 ) and / or the diameter of the place of impact ( 28 ) a sample area is determined. Method according to claim 4 or 5, characterized in that each place of impact ( 28 ) and / or each assigned sample area is in each case associated with an image which is detected by detection of the light emitted during the lighting of the respective place of impact ( 28 ) from the sample ( 5 ) outgoing detection light was obtained. Method according to one of claims 1 to 6, characterized in that in each case the same place of impact ( 28 ) is illuminated in succession from different directions. Method according to one of claims 1 to 7, characterized in that the illumination light beam ( 13 ) is circular in cross-section or that the illuminating light beam ( 13 ) has the form of a light stripe or quasi light stripe. Method according to one of claims 1 to 8, characterized in that the optical axis of the illumination objective ( 1 ) and the optical axis of the detection objective ( 2 ) are aligned parallel to one another or coaxially and / or that the detection objective ( 2 ) and the illumination objective ( 1 ) are opposed to each other and aligned opposite each other. Method according to one of claims 1 to 9, characterized in that a. the same sample ( 5 ) is subjected to a further examination, in which its illumination with the illuminating light beam ( 13 ) directly and without total internal reflection at the interface ( 10 ), and / or that b. the same sample ( 5 ) is subjected to a further examination, in which its illumination for a single plane illumination microscopy (SPIM) examination with the illuminating light beam ( 13 ) directly and without total internal reflection at the interface ( 10 ), and / or that c. the same sample ( 5 ) is subjected to a further examination in which its illumination is combined with the illuminating light beam formed into a light stripe or quasi-stripe of light ( 13 ) directly and without total internal reflection at the interface ( 10 ) he follows. Method according to claim 10, characterized in that the illuminating light beam ( 13 ), after the illumination lens ( 1 ), for further examination by means of another, on the detection objective ( 2 ) arranged deflecting means to the sample ( 5 ) is deflected. Method according to one of claims 1 to 11, characterized in that the illumination light beam ( 13 ) into the illumination objective ( 1 ) that it is eccentrically by the illumination lens ( 1 ) runs. Device for carrying out a method according to one of Claims 1 to 12, having an illumination objective ( 1 ) and a detection objective ( 2 ), on which a deflection means ( 3 ) for redirecting an illumination light bundle ( 13 ) to an interface ( 10 ) between an optically transparent medium ( 12 ) and a sample ( 5 ) is arranged. Apparatus according to claim 13, characterized in that the transparent optical medium ( 12 ) the deflection means ( 3 . 18 ) or part of the deflection means ( 3 . 18 ). Device according to one of claims 13 or 14, characterized in that a. the deflection means ( 3 . 18 ) has a block of transparent material, in particular a prism, and / or that b. the deflection means ( 3 . 18 ), in particular directly, to a front lens of the detection objective ( 2 ), or that the deflection means a front lens of the detection lens ( 2 ), and / or that c. the deflection means ( 3 . 18 ) has a block of transparent material, wherein at least one outer surface of the block is formed as a mirror, and / or that d. the deflection means ( 3 . 18 ) has a block of transparent material, wherein an outer surface as a coupling window for the illumination light beam ( 13 ) is formed and arranged. Device according to one of claims 13 to 15, characterized in that a. another deflection means ( 19 ) for a further study in which the illumination of the sample ( 5 ) with the illumination light beam ( 13 ) directly and without total internal reflection at the interface ( 10 ), exists, and / or that b. another deflection means ( 19 ) for a further study in which the illumination of the sample ( 5 ) with the illumination light beam ( 13 ) directly and without total internal reflection at the interface ( 10 ), on the detection objective ( 2 ) is arranged. Device according to one of claims 13 to 16, characterized in that a. the deflection means ( 3 ) or the further deflection means as a mirror or as a facet mirror ( 29 ) or that b. the deflection means ( 3 ) or the further deflection means at least one mirror or a facet mirror ( 29 ), or that c. the deflection means or the further deflection means comprises a mirror with a frusto-conical mirror surface. Device according to one of claims 13 to 17, characterized in that the deflection means ( 3 . 18 ) and / or the further deflection means ( 19 ) movable on the detection objective ( 2 ) are attached. Device according to one of claims 13 to 18, characterized by a deflection device with adjustable beam deflection device ( 32 ) for changing the place of impact ( 28 ) and / or the angle of incidence and / or the direction of incidence of the illumination light beam ( 13 ) on the interface ( 10 ).

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