Method and device for determining a critical angle of an excitation light beam

A method for determining a critical angle of total reflection based upon images captured at different angles of incidence of a light beam includes illuminating a sample with an excitation light beam, capturing images of at least part of the sample at a plurality of different angles of incidence of the excitation light beam, and determining a critical angle of total reflection at an interface of the sample based upon analysis of the images. An apparatus for determining a critical angle of total reflection at an interface of a sample includes a light source arrangement to illuminate a sample with an angle of incidence, an image capturing arrangement to capture an image of the sample, and a processing arrangement to determine the critical angle of total reflection at an interface of the sample on the basis of an analysis of images captured at a plurality of different angles of incidence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority, under 35 U.S.C. § § 119 and 371, of German patent application No. DE 10 2012 102 983.0, filed Apr. 5, 2012; the prior application is herewith incorporated by reference in its entirety.

Not Applicable

FIELD OF THE INVENTION

The present application relates to methods and apparatuses for determining a critical angle of total reflection of an excitation light beam in a microscopy arrangement, in particular in TIRF-microscopy arrangements (“Total Internal Reflection Fluorescence”).

BACKGROUND OF THE INVENTION

Total internal reflection fluorescence microscopy is a particular measurement method in light microscopy in which an excitation light beam is typically totally reflected at an interface between a cover glass or slide and a sample. This generates a so-called evanescent (decaying) field in the sample which may excite the sample to fluorescence. The penetration depth of the evanescent fields depends on the exact angle of incidence of the excitation light beam.

As the critical angle for total reflection, i.e. the threshold angle at which a transmitted light beam vanishes, depends on the indices of refraction of the materials involved, this angle depending on the type of samples used, may not be known from the start. Therefore, it is desirable to be able to determine the critical angle.

United States Patent Publication No. 2011/0057093 A1 discloses a method evaluating the intensity of an optical response of a sample depending on an angle of incidence of irradiation light. To increase robustness of the method, an analysis is performed in a pupil plane which sometimes requires additional optical elements.

Therefore, a need exists for methods and devices for determining such a critical angle which should be easy to implement, preferably without using additional optical elements, and which are robust.

Thus, a need exists to overcome the problems with the prior art systems, designs, and processes as discussed above.

SUMMARY OF THE INVENTION

According to an embodiment, a method is provided, comprising:illuminating a sample with an excitation light beam,capturing images of the sample for a plurality of different angles of incidence of the excitation light beam, anddetermining a critical angle of total reflection of the excitation light beams depending on the captured images, i.e. based on the captured images.

Due to such a determination of the critical angle on the basis of the images it is not necessary to view a pupil plane for increasing robustness. Furthermore, the capturing of images in some embodiments may be performed with an image sensor which is provided already for other purposes such that no additional elements are needed.

In an embodiment, the determination of the critical angle may be performed on the basis of a contrast analysis of the images. For this determination it may be used that, when passing the angle of total reflection, contrast ratios, in particular intensity ratios, may change.

In an embodiment, images of different image planes within the sample are captured, for example of two different image planes, and the determining of the critical angle may be performed on the basis of the images of the different image planes, for example by comparing of images at equal angles of incidence and different image planes. In an embodiment, in this way a result may be verified which was obtained on the basis of images of a single image plane.

The method in some embodiments may also comprise a comparing of image parameters, for example a mean captured fluorescence intensity in an area, between successive angles of incidence of the plurality of different angles of incidence. Additionally or alternatively, in embodiments at least in some of the images different areas, for example an area within an evanescent field and an area outside an evanescent field, may be analysed.

A ratio of image intensities may, for example be determined along one or more line profiles, using overall images or predetermined areas of an image.

The method may be performed automatically or semi-automatically. For example a change of the angle of incidence and/or a change of image plane of detection, i.e. image capturing, may be performed automatically, but may also be performed by a user in an interactive manner.

DETAILED DESCRIPTION OF THE INVENTION

In the following, embodiments will be described in detail. These embodiments serve merely illustration purposes and are not to be construed as limiting the scope of the present application. Features of different embodiment may be combined with each other to form further embodiments unless noted otherwise. On the other hand, a description of an embodiment with a plurality of features is not to be construed as indicating that all these features are necessary for practising the invention. In particular, in other embodiments some features of the described embodiments may be omitted and/or replaced by alternative features.

As common in the field of optics an angle of incidence of a light beam in the context of this application indicates the angle between this light beam and a direction perpendicular to a surface of an object through the point of incidence of the light beam on the object. In other words, an angle of incidence of 0° corresponds to a light incidence perpendicular on the illuminated object, while an angle of incidence of 90° corresponds to a glancing incidence parallel to a surface of the object.

InFIG. 1a TIRF microscopy apparatus according to an embodiment is schematically shown. It is to be noted thatFIG. 1is to be regarded as being schematic only and serves for illustrating some features of some embodiments. However, additional elements, in particular lenses, mirrors or other optical elements which are used in conventional TIRF microscopy arrangements may also be used in the embodiment ofFIG. 1without being explicitly shown and described.

In the embodiment ofFIG. 1a light source10, for example a laser light source, generates an excitation light beam11. Excitation light beam11is guided to a sample arrangement13-15via a mirror12. Sample arrangement13-15in particular comprises a slide14made of glass or other material which is transparent to excitation light beam11and to light to be detected from the sample. On a first side of slide14a sample15, for example a suspension with particles to be examined, is provided. On a second side opposing to the first side, the second side facing light source10, an oil immersion layer13is provided. Excitation light beam11is refracted at an interface between the surrounding medium (for example air) and oil immersion layer13and then impinges on an interface between slide14and sample15with an angle of incidence α117. In the embodiment shown, the indices of refraction of oil immersion layer13and slide14are chosen such that at the interface between oil immersion layer13and slide14no or only a negligible refraction of excitation light beam11takes place.

Excitation light beam11is reflected at the interface between slide14and sample15and, as reflected light beam17, reaches a light trap18, which absorbs reflected light beam17. In other embodiments, reflected light beam17may be analysed by corresponding devices if this is helpful for a respective application.

When angle of incidence117of excitation light beam11at the interface between slide14and sample15exceeds the critical angle of total reflection for this interface, excitation light beam11is totally reflected, and in sample15a so-called evanescent field which is exponentially decaying is formed, which is schematically shown in dashed lines inFIG. 1and provided with reference numeral16. The critical angle of total reflection depends in particular on the index of refraction of slide14and on index of refraction of sample15. Evanescent field16typically has an extension in the order of 100 nm in the direction perpendicular to the above mentioned interface (in the following, this direction is also referred to as z-direction), such that in this manner a high spatial resolution in the z-direction may be obtained. The z-direction therefore corresponds to the direction of perpendicular118inFIG. 1.

Excitation light beam11may excite fluorescence in sample15or parts of sample15. Corresponding fluorescence light19originates from the sample and is detected in the embodiment ofFIG. 1via an image sensor110, for example a CCD-sensor (Charged Coupled Device) or a CMOS-sensor (Complementary Metal Oxide Semiconductor), this image sensor110capturing a fluorescence image of the sample or part thereof, which corresponds to a spatially resolved detection. To this end, an imaging optic (not shown inFIG. 1) which images the sample onto image sensor110, and/or a corresponding filter arrangement which cancels light in wavelength ranges which do not correspond to the fluorescence to be detected may be provided.

The information captured by image sensor110is processed in a processing arrangement111, for example analysed to determine the critical angle of total reflection as will be described in the following in more detail. Processing arrangement111may for example be implemented by programming a processor device with one or more processors, for example a computer, correspondingly. Furthermore, an input/output arrangement112is provided via which information may be output to a user, for example via a monitor or other kind of display, and/or data may be input by the user.

In the embodiment shown, mirror12is both pivotable as well as movable in a direction parallel to the above-mentioned interface as indicated by arrows113, to be able to vary angle of incidence117. Mirror12constitutes merely one possible example for a device to change the angle of incidence, and other implementations are also possible. For example, a mirror used may only be movable in a linear direction, and, additionally, a curved mirror, for example a hyperbolic mirror, may be provided. In this case, for example via a linear translation movement of the mirror different points of the curved mirror are illuminated and, thus, the excitation light beam is reflected to the interface between slide14and sample15by the reflected mirror under different angles, therefore varying in angle of incidence117.

It is to be noted that in the example ofFIG. 1via the translation movement of mirror12parallel to the interface, a change of the location at which excitation light beam11falls on the interface caused by a pivoting of mirror12may be compensated.

As indicated by an arrow114, the movement of mirror12or another device for changing the angle of incidence may be controlled by processing arrangement111. In other embodiments the control of mirror12or another device may also be performed manually, and processing arrangement11is such cases may, for example, instruct a user via input/output arrangement112to change the angle of incidence accordingly.

Additionally, in the embodiment ofFIG. 1slide14and therefore sample arrangement13,14,15may be displaced as indicated by an arrow115in the z-direction. Through this, an image plane in which the fluorescence is detected by image sensor110, i.e. an area from which a focused image of the sample is present on the image sensor (depth of field of the imaging) may be moved in z-direction. As an alternative, in some embodiments also image sensor110may be movable in z-direction, or an imaging optic (not shown inFIG. 1) between image sensor110and sample15may be changed accordingly to change the image plane, i.e., the plane where the image is captured, of image sensor110.

Also this movement or displacement may be controlled by processing arrangement111as indicated by an arrow116. In other embodiments, also in this case the adjustment may be performed manually by a user, the processing arrangement111, for example, giving instructions to change the position of slide14in z-direction to the user via input/output arrangement112.

To determine the critical angle of total reflection, the apparatus ofFIG. 1is configured to vary angle of incidence117automatically or interactively using a user and to determine the critical angle of total reflection through analysis of images captured by image sensor110at the different angles of incidence117. Optionally, additionally the image plane may be changed, for example by moving slide14in z-direction as explained above, and images from different image planes additionally may be used for determining the critical angle of total reflection. In particular, a contrast of the images may be evaluated, information at different areas of the images may be evaluated, and/or information for different image planes may be compared. Details of implementation examples will be explained later in more detail.

InFIG. 2, a corresponding method is shown which may, for example, be implemented in the apparatus ofFIG. 1, but which also may be implemented independently therefrom. At20, an angle of incidence of an excitation light beam, i.e., an illumination angle, on an interface to a sample is varied. When changing the angle of incidence in an embodiment, the directional vector and/or a location where the light beam illuminates the sample is kept as constant as possible. Optionally, at21additionally, an imaging plane of an image detection is varied. At22, the actions at22being performed for each angle of incidence and for each image plane of20and21in some embodiments, corresponding images are captured. At23, a critical angle of total reflection is determined by analyzing the captured images, for example, by analyzing an image contrast.

Various possibilities for determining the critical angle will now be explained with reference toFIGS. 3 to 7.

InFIG. 3schematic sample images which are captured by an image sensor like image sensor110ofFIG. 1are shown. InFIG. 4for illustration purposes a corresponding sample with an image plane z44and an image plane z+143is shown. InFIG. 4, 40indicates a slide on which a sample41with an object42which is present only in image plane43is provided.

The distance between image planes43,44is chosen such that when total reflection is present the evanescent field does not reach to image plane43, i.e., a depth of field when capturing image plane43does not include the evanescent field. This causes a different behaviour for the two image planes as will now be explained.

The distance between image planes43and44is therefore in embodiments, for example, chosen such that it is greater than the penetration depth of the evanescent field, such that image plane43is outside the evanescent field.

In the upper row ofFIG. 3images from image plane z+1, i.e., image plane43ofFIG. 4, are schematically shown, and in the lower row images of image plane z, i.e., image plane44, are shown. Reference numeral30inFIG. 3denotes where, when the critical angle of total reflection is exceeded, the evanescent field is present within sample41ofFIG. 3.

In the columns ofFIG. 3, images for different angles of incidence α are shown, namely α=0, α=45°, α=62°, α=68° and α=73°. The critical angle of total reflection αKin this example is slightly below 62°.

To determine the critical angle of total reflection in the example shown, two areas are evaluated which are designated A and B inFIG. 3, area B being within area30and area A being outside area30.

In the images shown inFIG. 3, different light intensities, for example fluorescence intensity detected by the respective image sensor, in areas A and B are represented by different types of lines surrounding the letters “A” and “B”.

For angles α smaller than the critical angle of total reflection, inFIG. 3the complete image except element42appears light. This corresponds to the first two columns inFIG. 3. When the critical angle is exceeded in image plane z+1, the complete image is dark. This is caused by the fact that in this case the excitation light beam is total reflected and in image plane z+1 no evanescent field is present, such that here no excitation takes place and therefore no light is emitted by the sample, in particular no fluorescence light.

In image plane44, i.e., in image plane z, when exceeding the critical angle (last column inFIG. 3) the signal outside of area30becomes weaker, whereas the signal in area30increases slightly and then decays slowly. This is caused by the fact that, when exceeding the critical angle basically only in area30an evanescent field is present which may cause an excitation. With increasing angle of incidence this evanescent field becomes weaker, and therefore the intensity in area30decreases.

These results are graphically shown inFIG. 5. A curve50shows a development of a densiometric mean value of the intensity in area B for image plane z, a curve51shows the development for area B in image plane z+1, a curve52shows the development for area A in image plane z, and curve53shows the development for area A in image plane z+1. A line54indicates dark noise of the image sensor used.

As can be seen, all curves show a strong change at the critical angle of total reflection, curve50increasing to then decrease again, whereas all other curves decrease strongly at the critical angle.

For further illustration, inFIGS. 6 and 7further measurements are shown, the measurements ofFIG. 6having been measured in image plane z, whereas the measurement ofFIG. 7have been measured in image plane z+1, i.e. outside the evanescent field. The lines shown an intensity distribution in y-direction, i.e., in the image plane of the image sensors used, the dots between the lines corresponding to background signals. The signal, represented by the lines, in the example shown is generated by fluorescence beads of 1.2 μm diameter, whereas the background signal may, for example, be caused by fluorescein or fluorescent markers in the liquid in which the beads are provided. As can be seen inFIG. 6, when exceeding the critical angle (marked with αK) the background signal strongly decreases, whereas the signal from the beads remains. In contrast thereto, inFIG. 7, when exceeding the critical angle, both background and the signal itself decrease strongly.

From the above explained properties of the recorded images with varying image plane and/or varying angles of incidence, various possibilities for determining the critical angle result. In a simple case, simply the bending or the step in the various graphs ofFIG. 5may be detected. In another embodiments, image intensities Iz/Iz+1along one or more line profiles or based on complete images are evaluated, I indicating the intensity, Izthe intensity of a line profile or the complete image for image plane z, and Iz+1the corresponding quantity for image plane z+1. Through the strong decrease when exceeding the critical angle in the image plane z+1 together with the constant or increasing behavior in the image plane z, an exceeding of the critical angle causes a strong increase of this ratio, which may be detected. Additionally or alternatively, in some embodiments, in order to reduce a sample dependence (for example, to reduce an influence of sample bleaching) in a first step a ratio Iato Ia+1may be determined, I being the fluorescence intensity of an area of interest or of the complete image and a being an index of a measurement for a specific angle of incidence, i.e., a+1 denotes the measurement with the next greater angle of incidence compared to measurement a. This may be performed, for example, in image plane z. Also, here the ratio changes when the critical angle is exceeded. Finally, it is possible, for example for fine-tuning, to evaluate two areas in an image, for example an area where a sample to be measured is located and an area the signal of which may suppressed by illumination with an angle of incidence above the critical angle, for examples areas B and A inFIG. 3, for example the complete areas or line profiles in the respective areas. Also, an out-of-focus area may be selected for area A, i.e., the area which is not of interest for a later measurement. When here the intensity ratios are formed for image plane z when the critical angle is exceeded, a change by a specific factor occurs. For example, a threshold value may be selected, and when this factor exceeds the threshold value from one angle of incidence to the next, this corresponds to detection of the critical angle. The threshold value may, for example, be determined by a user or may be set to a typical value, for example a value of 10.

Thus, various possibilities have been provided how the critical angle may be determined on the basis of contrast information of images to be analyzed, for example on the basis of intensity ratios.

The above methods may also be combined with each other, for example to verify a result obtained by one of the methods with another one of the methods or techniques.

The above apparatuses and methods are merely to be seen as examples. For example, in the apparatus ofFIG. 1, fluorescence light originating from sample15is detected on the same side from which also excitation light beam11is provided. In other embodiments, fluorescence light may additionally or alternatively be detected on the opposite side. Furthermore, the present invention is not limited to the detection of fluorescence light, but, generally, light emitted (originating) from the sample in response to the excitation light beam, for example Raman-scattered light, may be detected.

Therefore, the application is not limited to the embodiments shown.