EP2165182A2

EP2165182A2 - Device and method for measuring static and dynamic scattered light in small volumes

Info

Publication number: EP2165182A2
Application number: EP08784613A
Authority: EP
Inventors: Christoph Janzen; Reinhard Noll; Walter Uhl; Kurt Hoffmann
Original assignee: Hoffmann Kurt Dr
Current assignee: Hoffmann Kurt Dr
Priority date: 2007-07-05
Filing date: 2008-07-04
Publication date: 2010-03-24
Also published as: JP2010532468A; US20100315635A1; WO2009003714A3; WO2009003714A2; DE102007031244B3

Abstract

The invention relates to a device for measuring scattered light, comprising at least one focusing element provided with electromagnetic radiation that can be focused on a sample, a detector and a detector optical system with which electromagnetic radiation scattered by the sample can be conducted to the detector. The device is characterised in that it comprises means for forming an annular beam such that said annular beam can be focused on a focus point inside the sample by the at least one focusing element and that electromagnetic radiation scattered by the sample can be detected by the detection optical system, said electromagnetic radiation dispersing inside the area surrounded by the annular beam.

Description

Apparatus and method for performing static and dynamic scattered light measurements in small volumes

Applicant: Fraunhofer-Gesellschaft for the promotion of applied research e.V.

Technical application

The present invention relates to a device, a measuring system and a method for carrying out scattered light measurements, in particular static and dynamic scattered light measurements. Preferred fields of application are those in which it is necessary to carry out a large number of measurements in automated processes, for example in the investigation of crystallization processes in the context of the growth of protein crystals.

State of the art

Scattered light measurements, in particular laser scattered light measurements, are already used in a number of fields of application, for example for the characterization of colloids. In the food industry, for cosmetic products or even for polymers or adhesives, the size distribution and stability of colloidal particles play an important role.

Another field of application is the elucidation of the structures of complex proteins and other biomolecules, which are required for the targeted development of new ones

Medications as well as the study of the biochemical function of these

Molecules are of great importance. Structure elucidation based on X-ray diffraction experiments relies on high-quality single crystals with diffractive properties, which are usually grown from solutions. Essentially, the crucial step in the growth of protein crystals is to be suitable for crystallization

To find solution conditions. By varying solution parameters such as pH, protein concentration, the composition and concentration of salts and precipitating reagents, temperature, etc., the interactions between the dissolved protein molecules are varied and attractive or repulsive interactions can be set. The investigation of the crystallization of proteins is usually carried out empirically and represents a bottleneck in the structural elucidation of the proteins.

Laser scattered light measurements allow a direct measurement of the interaction between dissolved proteins prevailing in a crystallization solution and can therefore be used for the targeted finding of optimum solution parameters for the crystallization. The so-called osmotic virial coefficient is a thermodynamic quantity that describes the real interaction behavior of dissolved particles as a deviation from the behavior of an ideal solution. Osmotic virial coefficients can be determined by means of static scattered light measurements. For the measurement method, the detection of the absolute scattered light intensity in different solutions with different particle concentrations is necessary. Light reflected by interfaces can very easily disturb the measurement. It is already known that the osmotic virial coefficient is of particular importance for the crystallization of proteins. The probability of a successful crystallization is particularly high in a value range of the osmotic virial coefficient called "crystallization window" and particularly low outside this crystallization window.

With the help of dynamic scattered light methods information about the diffusion behavior of dissolved particles and thus the particle size can be obtained. These quantities change sensitively through an aggregation process. The process of nucleation preceding crystallization can thus be recognized.

Especially for static scattered light measurements, it is of central importance to achieve optimal suppression of unwanted radiation components.

These are mainly caused by reflections at interfaces. The nearer the interfaces are at the measurement volume, the more difficult is the effective suppression.

It is already known to use laser scattering light methods for the characterization of macromolecules. Various devices are available for determining osmotic virial coefficients or molecular weights by means of static light scattering and also for determining hydrodynamic radii distributions by means of dynamic light scattering. The devices used today for carrying out scattered light measurements usually work with glass cuvettes in which sample solutions have to be filled. In most cases, a laser beam is focused into the cuvette, and the light scattered by the particles being examined is detected at a defined angle relative to the direction of irradiation (for example 90 °). There are also systems which make it possible to change the detection angle or to collect scattered light simultaneously with several detectors at different angles, as described for example in EP 0867711 A2. Typically, for such systems, round cuvettes are used which are positioned to further avoid reflectance in stray light in an index-matching bath (a vessel filled with a refractive index-matched liquid). Due to the small difference in the refractive index between the bath liquid and the cuvette wall, only a comparatively small proportion of reflected radiation is formed when the laser beam enters the cuvette.

However, automated high-throughput scattered light measurements can not be carried out with the known devices based on cuvettes, since a manual filling of the cuvettes is usually necessary. The cuvettes used are made of polished glass in the case of precision measurements and are expensive. For economic use, they often have to be reused and therefore be cleaned consuming. The high requirements of the measuring method - problematic is, for example, that surface contaminants on the glass and particles in the solution distort the scattered light intensity - make this step consuming and difficult to automate. The problem of cleaning can not be solved by the use of disposable cuvettes, such as plastic, as this is due to the poorer optical properties especially for the static scattered light measurements, where absolute light intensities with high precision must be determined by the scattered light generated by the plastic leads to distortions. Overall, the handling of the cuvettes (positioning in the index-matching bath, cleaning, refilling) is therefore complicated and time consuming.

The volumes of typical cuvettes for scattered light measurements are on the order of at least 5 -10 microliters, often even much higher. Although the production of cuvettes with even smaller volumes would be technically possible in principle, scattered-light cuvettes with nanoliter volumes have not hitherto been used and are currently not offered. The handling of the solutions necessary for the manual execution of individual measurements (manual pipetting, preparation of the solutions, filling of the solution in the cuvette, etc.) requires a minimum volume, which is typically in the microliter scale. A large reduction of the cuvette volumes does not seem to be necessary for single measurements, moreover, the excitation and detection optics of the commercial scattered light devices are not optimized for extremely small cuvette volumes.

In the case of screening hundreds or thousands of measurements for the automated crystallization of proteins, however, a further minimization of the sample volume and thus the protein consumption is necessary because the proteins often have to be laboriously recovered and are only available in small quantities. The large sample consumption thus prevents in the known scattered light measuring systems systematic investigations to optimize the crystallization conditions on the basis of static scattered light measurements.

In the context of crystallization experiments, protein solutions are often applied in the form of small droplets which are either suspended under a glass plate (so-called "hanging drop") or placed on the floor of a sample carrier ("sitting drop"). The curved interfaces caused by the droplet shape, when irradiated with a laser beam, cause reflections that are difficult to control, which is why static scattered light measurements on drops have hitherto not been carried out or have appeared impossible. Wyatt Technology Corporation offers a scattered light meter for use in microtiter plates, but it only allows dynamic measurements and no static measurements. For the recording of dynamic scattered light measurements signal fluctuations, which are due to particle movements, evaluated. A constant signal background due to reflections does not disturb the dynamic scattered light measurement, or does so little. Static measurements, on the other hand, are based on the recording of absolute scattered light intensities, here the influence of reflections on the scattering signals prevents the measurement. However, the Wyatt Technology Corporation device is unable to suppress disturbing reflections as much as would be necessary to perform static scattered light measurements.

The invention has for its object to provide an apparatus and a method for carrying out scattered light measurements, in particular absolute scattered light measurements to provide a particularly effective suppression of unwanted radiation components is achieved, which also has a systematic and particularly largely automated and thus fast examination of a variety of Samples is possible, and in particular also samples with a very small volume can be examined or used.

Presentation of the invention

The solution to this technical problem is provided by a device, a measuring system, as well as a method according to the independent claims. Advantageous embodiments and further developments are indicated by the dependent claims or can be taken from the following description and the exemplary embodiments.

The present device for carrying out scattered light measurements has at least one focusing element, with which electromagnetic radiation can be focused on a sample, a detector, and a Detection optics, with the scattered by the sample electromagnetic radiation can be passed to the detector. According to the invention, it has been recognized that the technical problem can be solved by the device additionally having a means for forming a ring beam, that the ring beam can be focused on a focal point within the sample by the at least one focusing element, and by the detection optics detected by the sample scattered electromagnetic radiation which propagates within the space surrounded by the ring beam.

By these features, a confocal optical system is realized, with the static or dynamic scattered light measurements are made possible in a backscatter geometry. That is, the detection of the scattered radiation is thus approximately in the same direction from which the excitation laser (or the light source used for the excitation) is irradiated (backscattering direction). This facilitates in particular a particularly compact construction of the device and makes the device particularly flexible with respect to the sample carrier used. In particular, this offers the possibility of carrying out scattered light measurements in measuring geometries with only one optical access. For example, it is possible to examine samples or sample drops in microtiter plates with glass plates. It can thus be achieved to perform a variety of measurements with a single sample carrier (e.g., microtiter plate). The use of microtiter plates simplifies the use of pipetting robots for automatic filling of individual sample positions. As a result, a comprehensive automation of sample preparation and scattered light measurement can be achieved. This offers considerable economic advantages over the devices known from the prior art, in particular because the use of the sample carrier as a disposable article is economical due to the large number of measurements that can be carried out with a sample carrier and thus a complicated cleaning, as is the case in the prior art For cuvettes is known, can be omitted.

The fact that with the device, the formation of a ring beam and a focusing of this ring beam is possible, in addition, on the one hand, a comparatively strong focus, and thus achieve a minimization of the measurement volume. In this way it is possible to measure too Samples with particularly small volumes (less than 1 μl, or on the order of (a few) 100 nl) perform. In addition, the intensity of the irradiation is particularly high due to the comparatively small size of the focus volume of the exciting beam for a given total power. Thus, a particularly high scattered radiation can be generated per volume element.

On the other hand, by scattered radiation, in particular exclusively scattered radiation, can be detected, which propagates within the space surrounded by the annular beam, at the same time ensures that a relatively strong suppression of unwanted, reflected by interfaces of the sample or the sample carrier radiation can be done. The exciting radiation may according to the invention be blasted into the sample at a very shallow angle. Direct reflections at the interfaces of air sample carrier and sample carrier sample liquid are thus reflected back at a very shallow angle. The scattered radiation propagating within the space surrounded by the ring beam, on the other hand, leaves the detection volume at a more acute angle. The difference in angle between stimulating light and scattered radiation thus leads to a strong suppression of reflections.

The ring beam is generated from a collimated beam with Gaussförmigen beam profile, as the beam source is preferably a laser, in particular a continuously operated semiconductor, solid state or gas laser used. The shaping of the ring beam can be achieved by a beam expansion in conjunction with a ring diaphragm. However, a particularly preferred embodiment provides for shaping the ring beam, a beam shaping optics, which comprises two positioned on the optical axis and with their tips facing each other Axicone (glass cone). These are illuminated on the input side by the laser beam in the middle. Such an arrangement has particularly high transmission values, so that the losses due to the formation of a ring beam are comparatively small.

The focusing element is preferably reflective and annular. This allows an advantageous arrangement of the detection optics. It has advantages if the focusing element is designed in such a way that an approximately radially symmetrical (and as small as possible) focus can be generated in the sample, since in this way a particularly high intensity per volume element can be achieved. Since only signals from the overlapping region of excitation and detection focus are recorded by the confocal detection, both foci should have the same order of magnitude. Of course, a fully filled aperture microscope objective may produce a smaller focus than a ring beam, so the excitation focus will typically be greater than the detection focus. For this reason, one gains signal intensity by generating the smallest possible excitation focus and providing the highest possible area-related excitation intensity, since only the inner part of the focus (from the overlap area) contributes to the signal. As a focusing element, for example, a (annular) parabolic mirror can be used. With such, it is possible in principle to produce an approximately radially symmetrical focus. However, it is important to note the boundary layers to be crossed by the radiation between the focusing element and the sample so that an optimum focusing element is slightly modified in shape compared to a parabolic mirror and thereby corrects the effect of refraction on the glass bottom or on entering the sample.

The focusing element should be designed so that a focus diameter smaller than 30 microns, preferably less than 20 microns, can be generated (the focus diameter should be defined so that at its limits, the laser intensity to the 1 / e-part of the maximum intensity in Focus center has dropped). Due to the small size of the focus diameter, it is possible to examine even extremely small samples of less than 1 μl and, moreover, this results in a particularly high scattered radiation per volume element and thus overall a high signal strength. The high degree of focusing allows measurements with a very small distance to interfaces.

Preferably, the detection optics is arranged within the space surrounded by the ring beam, in particular the detection optics is arranged centrally within this space, or in the center of the optical axis predetermined by ring beam or focusing element. The focus of the detection optics falls doing so together with the focus of the excitation optics. By such an arrangement, the detection optics is able to capture the largest possible proportion of the generated scattered radiation and to supply the detector without large losses. For the collection of scattered radiation, a microscope objective with a numerical aperture of at least 0.6, preferably at least 0.7 can be used.

For the majority of the proteins of interest for crystallization, the molecules are much smaller (typically a few nm) than the wavelength of the laser light used (several 100 nm). Therefore, the scattering intensity is, in a first approximation, independent of the considered angle and a collection of the scattered radiation over many angles is possible and, in principle, provides no other results than experiments in which scattered light is collected only at an angle. A detection optics with large numerical aperture thus has advantages, since a greater signal strength can be achieved.

In a particularly advantageous embodiment, the detection optics is designed such that it collects the scattered light emanating from the sample and in turn forms it into a collimated beam. This collimated beam is then focused with a further optical element, for example a lens, onto a small entrance aperture of the detection system, for example onto the core of an optical fiber, preferably a single-mode fiber. The system is designed such that only such radiation forms a collimated beam and is focused in the plane of the small entrance aperture of the detection system, eg the fiber front surface, which originates from a limited area within a predetermined detection plane. The detection plane corresponds to the focal plane of the detection optics in the reverse direction of the beam path. The limited area has an extension of less than 10 μm, preferably approximately 5 μm, in particular in the direction perpendicular to the detection axis. It lies completely within the focus of the stimulating radiation. Scattering radiation from planes other than the predetermined detection plane is focused by the focusing optics in front of or behind the plane of the fiber front surface and can therefore enter the fiber only to a very small extent. For the procedure to work the foci of excitation optics (parabolic mirrors) and Detection optics (microscope objective) as well as possible in all three spatial directions are superimposed.

By this measuring principle one obtains an extensive suppression of unwanted reflection components. In this way, both directed reflections and diffusely scattered radiation, which arises, for example, from scratches and defects in the glass bottom of the sample carrier, are effectively suppressed.

The detection optics can be formed, for example, by a multi-lens microscope objective with an adapted numerical aperture or comprise such. Here, the collection angle of the lens must be adapted to the angle under which is illuminated. By selecting the aperture of the microscope objective as large as possible, but somewhat smaller than the inner limit aperture of the illumination, it is ensured that, on the one hand, the highest possible signal component of the scattered light is detected and, on the other hand, no direct reflections from the glass surface can occur in the detection beam path.

A further aspect of the invention resides in a measuring system for carrying out scattered light measurements with a device which has at least one focusing element, with which electromagnetic radiation can be focused on a sample, a detector, and detection optics, with the electromagnetic radiation scattered by the sample can be passed to the detector. In addition, the measuring system comprises a flat sample carrier, which is designed such that a sample consisting of a single drop can form an interface with it. In addition, the at least one focusing element and the detection optics are arranged such that the beam path of the electromagnetic radiation impinging on the sample and the scattered radiation detected by the detection optical system thwart the interface.

The measuring system thus comprises, in addition to a device with which electromagnetic radiation can be emitted and scattered radiation detected, a flat sample carrier. This can be a flat plate in the simplest case. He must be in the areas designated for samples at least in be substantially transparent to the electromagnetic radiation used. Preferably, it is made of glass.

Excitation and detection takes place through the flat sample carrier and the interface that forms the sample with the flat sample carrier. The beam thus crosses only planar interfaces and is therefore relatively easy to control with respect to unwanted reflections.

According to an advantageous embodiment, the detection optics defines an optical axis (z). This is (at least substantially) perpendicular to the sample carrier (xy plane) and forms the central axis for the ring beam and the focusing element. The detection optics is capable of detecting the scattered radiation emitted by the sample starting from the detection volume in the direction of the z-axis and at an angle to the z-axis (half the opening angle) of up to 48 °, preferably up to 44 ° , A half opening angle of 44 ° leads to a numerical aperture of 0.7. The exciting radiation guided by the focusing element in the direction of the focus forms an angle between 51 ° and 59 ° with the z-axis. The exciting radiation thus crosses the sample carrier and the interfaces at a shallower angle than the scattered radiation from the detection volume detected by the detection optics.

The focusing element focuses the exciting ring beam below an upper numerical limit aperture of at least 0.84 (corresponding to 57.1 °), preferably 0.86 (corresponding to 59.3 °), or lower numerical limit aperture of at most 0.82 (corresponding to 55.1 °), preferably 0.78 (corresponding to 51.2 °), to the test. This ensures a sufficient angular distance to the detection optics.

The sample carrier is preferably a microtiter plate, i. a standard component with a multitude of isolated measuring fields in rows and columns.

With the measuring system according to the invention, it is possible to apply a large number of samples each as a single drop on a single flat sample carrier and thus perform a variety of measurements in a short time and automatically. The measuring system has particular advantages if it additionally has an automated, image-data-based positioning unit with which the diffraction-limited laser focus can be positioned approximately in the center of the small, preferably less than 1 μl, volume of the protein solutions to be examined. This allows a rapid change from sample to sample under reproducible measurement conditions and thus the use of the laser scattered light method in the form of a high-throughput method for the systematic crystallization of protein.

As a positioning, for example, a system of multiple precision sliding tables with spindle drive and microprocessor-controlled stepper motors in question. Alternatively, piezo-powered tables are also suitable. The positioning accuracy should be significantly smaller than the dimension of the sample droplets, a positioning accuracy of a few micrometers has proven successful.

A further aspect of the invention consists in a method for carrying out scattered light measurements, in which electromagnetic radiation is focused on a sample and radiation scattered by the sample is detected, wherein the sample is present as a drop, which has an interface with a flat sample carrier, and wherein the Exposure to the electromagnetic radiation and the detection of scattered radiation through the flat sample carrier and the interface is carried out. For the method, the use of the above-described device or the measuring system offers.

The sample is present as a single drop placed in connection with the sample carrier. This means that the sample forms a self-contained phase boundary surface, wherein a part of its surface forms an interface with the sample carrier, the remaining part forms a liquid / gaseous phase interface with the gaseous environment (air), whereby a stable state is present. The shape of the kiln is determined in a known manner by the cohesive forces within the droplet and the adhesion forces against the surface of the sample carrier.

Preferably, the sample, ie, the small aqueous liquid droplet in which the proteins to be examined are dissolved, is not covered under a layer of one pipetted with the sample miscible liquid, preferably an oil or paraffin layer directly on the glass bottom of the sheet element. The droplet displaces the paraffin or the oil from the glass bottom and sits in a hemispherical shape directly on the floor. This is feasible even for very small sample volumes, such as volumes below one μl or even for 100 nl, and can be carried out automatically with needle-based pipetting robots. The oil or paraffin layer protects the small quantities of liquid from drying out. Another advantage is that the sample carrier can also be made easily even for a large number of drops. For example, it may also have only a few larger separate cells, since not every single drop has to be placed in its own compartment of the sample carrier. The oil or paraffin surrounding the drop effectively replaces the walls of a separate sample compartment and prevents the individual drops from mixing.

The pobe, especially in the case of protein solutions in the context of crystallization experiments, often applied in the form of small droplets; These can either sit on the floor of a sample holder (so-called "sitting drop") or hang under the sample holder or glass plate (so-called "hanging drop"). The invention also offers the possibility of carrying out scattered light measurements for such an unconventional geometry of the sample. Because of the effective suppression of unwanted disturbances, the curved and near-focus interfaces of the droplets can also be tolerated.

In order to achieve a further reduction of unwanted radiation (reflections at interfaces, inter alia) in the detected signal, according to an advantageous embodiment of the invention, the electromagnetic radiation focused on the sample has a plurality of radiation components of different wavelengths, in particular two different light sources or laser sources can be selected for this purpose , In addition, different spectral components of the scattered radiation are detected separately. This allows the measurement of a difference intensity. For this measure causes a sufficient increase in accuracy, the electromagnetic radiation preferably contain at least two different radiation components whose wavelengths differ by at least 50 nm, preferably even by at least 120 nm.

The reflection of radiation at an interface can be described using Fresnel's equations. Thus, the reflectance of an interface between two materials having refractive indices n and n 'is calculated as follows:

R _ = -I ± «An - n) I B (» '+ »)

with: R = reflectivity, / _R = reflected intensity (incidence perpendicular to the surface), / E = incident intensity, n and n 'refractive indices of the adjacent materials.

For the transition of a laser beam of air (n = 1) in a typical glass (n '= 1, 5) results in a reflectance of about 4%. The wavelength has only a very small influence on the reflectance in the case of small displacements. At the same time, however, the wavelength enters the scattering intensity of small particles with the fourth power, so that small wavelength differences are already noticeable in different scattering intensities. If now not the absolute scattering intensity at one wavelength but the difference of the scattering intensities at two different wavelengths is used as the basis for a scattered light experiment, we obtain a measurand which is relatively insensitive to distortions of the measurement signal by reflection components. If, in successive measurements on the same sample, different amounts of reflected light enter the detection optics, the measurement results are greatly falsified in an experiment with only one wavelength. In an experiment with two wavelengths, the intensities of both wavelengths would be changed in the same way, but the difference of the two intensities would be only slightly disturbed. Therefore, this approach offers the possibility to perform scattered light measurements with high accuracy, even in the presence of unwanted reflections. For the embodiment with two beam sources, the device according to the invention is to be extended with an optical means which makes it possible to superimpose the beams of the two beam sources, for instance with a dichroic mirror, which reflects the wavelength of one beam and allows that of the other beam to pass , Other equivalent optical means are conceivable. This superposition of the two radiation components can be carried out particularly easily upstream of the beam-shaping optical system in the beam path. Furthermore, at least one further optical means is necessary in order to separate the radiation components in the detection beam path from one another or to guide them to different detectors. For this purpose, for example, a dichroic mirror can also be used.

At the beginning of a measurement, the power of the two laser beam sources is adjusted so that when measuring a standard sample without scattering particles, for example, a blank solution that consists only of solvent, buffer, etc. but contains no proteins, the signal strength registered at both detectors is identical. If the standard sample or dummy solution is replaced by a "true sample", i. e.g. Replacing a solution with proteins as scattering particles, the signal strength for the detection channel with a shorter wavelength increases more than for the detection channel with a longer wavelength. The difference represents a scattering intensity proportional measurement signal, which is only very slightly distorted by reflection effects.

An embodiment of the invention with more than two lasers of different wavelengths is also possible. For this purpose, only input and output optics and detectors for the other laser must be added. Through different dichroic mirrors, the individual wavelengths are superimposed and separated again. In order to compensate for different laser intensities and spectral sensitivities of the detectors, again an intensity measurement with a sample without scattering particles (eg without proteins) must first be carried out. All further measurements with the substances to be examined are related to this reference measurement, by division a relative scattering intensity is obtained. If a linear detector behavior is assumed, this results for the different detectors a sequence of relative scattering intensities which increase in proportion to v ⁴ (with v = frequency of the exciting laser radiation). The proportionality constant is a measure of the scattering cross section of the examined sample and is only slightly distorted by reflectance components.

An embodiment of the invention in which the exciting light has a continuous spectral distribution is also conceivable. In this case, a spectrometer is preferably used as the detector. Assuming that the sample shows no absorption in the observed wavelength range, a relative intensity profile, which increases in proportion to v ⁴ , can be expected after referencing a measurement on a sample without scattering particles. The scattering spectrum of a sample with scattering particles is divided by the scattering spectrum of a sample without scattering particles (eg without proteins). By means of a mathematical fit analysis (eg according to the principle of least squares), the course of the relative scattering intensity can then be approximated by an analytical expression. This results in a proportionality factor k, which is a measure of the scattering cross section of the solution.

Here are / _re ι (v) the relative wavelength-dependent scattering intensity, / ₀ (v) and / p _r (v) the intensities of the reference sample (without scattering particles) and the sample to be examined, v is the frequency and k is a proportionality factor, which corresponds to the scattering cross section of the sample.

With the described method, protein-protein interactions in solution can be determined without markings by means of laser scattered light measurements. In this case, a very narrow range of weakly attractive interactions sets limits within which protein crystals can form. These interactions are influenced by the properties of the solution. Laser scattered light measurements are used to measure the interactions of proteins under different solution conditions. In this case, those solution compositions are determined that a protein crystallization favor. Using mathematical optimization algorithms, new solution conditions can be calculated from the selected solution approaches, from which a further approximation to the sought-after crystallization window can be expected. A set of other different protein solutions is automatically set up in the computed compositions and analyzed for protein-protein interactions by laser scattered light measurements. The iteration of the procedure described is intended to bring the crystallization parameters closer to the crystallization window with each pass until a protein single crystal is formed.

Brief description of the drawings

The present invention will be explained in more detail below with reference to exemplary embodiments in conjunction with the drawings without limiting the scope of protection specified by the claims. Hereby show:

Fig. 1: an embodiment of the invention with a light source

2 shows an embodiment of the invention with two light sources

3 shows an embodiment of the invention with three light sources

4 shows an embodiment of the invention with a polychromatic light source

Ways to carry out the invention

In Fig. 1 a, the individual components of excitation and detection optics are shown schematically, Fig. 1b shows an enlarged section of Fig. 1 a and shows a sketch of sitting on the bottom of the microtiter plate 10 sample drop 11 and the beam paths of acting radiation 3 and detected radiation 15.

A collimated laser beam 1 is converted by a beam shaping optics 2 into a collimated ring beam 3. This can be done by a sequence of two identical axicon (glass cones), which are each arranged with their tips to each other, can be achieved. The laser beam 1 is generated by a temperature-stabilized diode laser, which is coupled into a single-mode fiber. After coupling the beam out of the fiber, the intensity distribution has a pure Gaussian profile (TEM-OO mode), the beam diameter being 6 mm, for example. After the beam shaping by the axicon 2 results in an annular beam 3 with an inner diameter of 20 mm and an outer diameter of 26 mm.

The collimated ring beam 3 impinges on the parabolic mirror 4 and is focussed into the area covered by a not liquid with the sample 1 1 miscible liquid layer 14 sample 11 on the focal point 12. He passes through the glass bottom of a

Microtiter plate 10 and traverses the from the bottom of the sample 11 and the

Microtiter plate 10 formed interface 13. The exciting ring beam 3 is irradiated at an angle range between 51 ° and 59 ° (half the opening angle), this corresponds to numerical Grenzaperturen of 0.78 to 0.86. The generated stray radiation 15 is placed from the center in the parabolic mirror 4

Microscope objective 5 at an angle between 0 ° and 44 ° (half

Opening angle), which corresponds to a numerical aperture of 0.7, collected and shaped again into a collimated beam 6. This is focused by a further optics 7 on a single mode fiber 8. The fiber 8 directs the

Scattered light signal to a sensitive photodetector 9, which is a

Photomultiplier or an avalanche photodiode can act.

FIG. 2 shows a device according to the invention with two beam sources 18, 18 '. The two laser beam sources 18 and 18 '(eg a diode laser at 658 nm and a frequency-doubled Nd: YAG laser at 532 nm) each emit a collimated laser beam 1, 1'. Via a dichroic mirror 16, which reflects one wavelength and allows the other wavelength to pass, the two beams 1, 1 'are superimposed. In a beam-shaping optical system 2, both beams are respectively transformed into a collimated ring beam 3. The two superimposed ring beams 3 are focused by the parabolic mirror 4 without the occurrence of a chromatic error in the sample volume 11, scattered radiation 15 is collected by the chromatically corrected microscope objective 5 and as a collimated beam 6 via a Auskoppelspiegel 17 to another Directed dichroic mirror 20. Here the separation of the wavelengths takes place, which are directed via two separate optics 7, 7 'onto two single-mode fibers 8, 8' and forwarded to two detectors 9, 9 '. In turn, photomultipliers or avalanche photodiodes are used as detectors 9, 9 '.

3 shows a device according to the invention with three (or more) beam sources 18, 18 ', 18 ". Incidentally, in comparison with FIG. 2, the number of dichroic mirrors 16, 16 \ 16" for superimposing the beams of the different lasers, and the number of dichroic mirrors 20, 20 ', 20 "for separating the radiation components of the scattered radiation 15, the number of optics 7, TJ", the number of fibers 8, 8', 8 "and the number of detectors 9, 9 '. , 9 ".

FIG. 4 shows a possible arrangement with an excitation with continuous spectral distribution. This has a polychromatic light source 18 and a detector 9 as a spectrometer. The radiation of the light source 18 is ideally guided via a fiber (21) and a coupling-out optical system (19) into the beam-shaping optical system 2. The light source 18 can be a classical light source (halogen lamp, discharge lamp) or a laser source (eg self-phase modulation of a femtosecond laser in an optical fiber).

LIST OF REFERENCE NUMBERS

1 laser beam

2 beam shaping optics

3 ring beam

4 parabolic mirrors

5 microscope objective

6 collimated scattered light beam

7 Focusing optics for the scattered light beam

8 optical fiber

9 photodetector

10 microtiter plate

11 sample

12 focus

13 interface

14 liquid layer

15 scattered radiation

16 dichroic mirror

17 output mirror

18 beam source

19 output optics

20 dichroic mirror

21 fiber optic fiber

Claims

claims

1. An apparatus for carrying out scattered light measurements, comprising at least one focusing element (4), with which the electromagnetic radiation (3) can be focused on a sample (11), a detector (9), as well as a detection optics (5, 7, 8), with the electromagnetic radiation (15) scattered by the sample (11) can be conducted to the detector (9), characterized in that there is provided a means (2) for forming a ring beam (3) through the at least one focusing element (3). 4) a focusing of the ring beam (3) on a focal point (12) within the sample (11) is effected and that by the detection optics (5,7,8) of the sample (11) scattered electromagnetic radiation (15) can be detected, which propagates within the space surrounded by the ring beam (3).

2. Apparatus according to claim 1, characterized in that the detection optics (5,7,8) is disposed within the space surrounded by the ring beam (3).

3. Device according to one of claims 1 to 2, characterized in that the detection optics has a numerical aperture of at least 0.6, preferably at least 0.7.

4. Device according to one of claims 1 to 3, characterized in that the detection optics comprises a microscope objective (5).

5. Device according to one of claims 1 to 4, characterized in that the detection optics (5,7,8) is formed so that substantially only from a predetermined detection volume in the detection optics (5) radiated electromagnetic radiation (15), for Detector (9) can get.

6. Apparatus according to claim 5, characterized in that the detection volume within the focal point (12) of the annular beam (3).

7. Device according to one of claims 1 to 6, characterized in that the focusing element (4) is reflective.

8. Device according to one of claims 1 to 7, characterized in that the focusing element (4) is annular.

9. Device according to one of claims 1 to 8, characterized in that the focusing element (4) is a parabolic mirror.

10. Device according to one of claims 1 to 9, characterized in that the focusing element (4) is designed such that in the sample (11) is an approximately radially symmetrical trained focus (12) can be generated.

11. Device according to one of claims 1 to 10, characterized in that a focus diameter less than 30 microns, preferably less than 20 microns, can be generated.

12. Device according to one of claims 1 to 11, characterized in that a beam-shaping optical system (2) for forming the ring beam (3) is present.

13. The apparatus according to claim 12, characterized in that the beam-shaping optical system (2) comprises two axles arranged with their tips to each other.

14. Device according to one of claims 1 to 13, characterized in that the sample (11) with electromagnetic radiation (1, 3) consisting of radiation fractions of at least two different wavelengths can be acted upon.

15. The apparatus according to claim 14, characterized in that a plurality of detectors (9, 9 ¹ ) and at least one means (20) are provided to separate mutually different spectral radiation components of the scattered radiation (6) on mutually different detectors (9, 9 ', 9 ").

16. Device according to one of claims 1 to 13, characterized in that the sample (11) with electromagnetic radiation (1, 3) of a continuous spectral distribution can be acted upon.

17. The apparatus according to claim 16, characterized in that the detector (9) is a spectrometer.

18 measuring system for carrying out scattered light measurements with a device, in particular according to one of the preceding claims, which at least one focusing element (4), with the electromagnetic radiation (1, 3) can be focused on a sample (11), a detector (9) , and a detection optics (5, 7, 8), with which electromagnetic radiation (6) scattered by the sample (11) can be conducted to the detector (9), characterized in that the measuring system has a planar sample carrier (10). which is configured such that a sample (11) consisting of a single droplet can form an interface (13) with it and that the at least one focusing element (4) and the detection optics (5, 7, 8) are arranged in this way in that the beam path of the electromagnetic radiation (3) impinging on the sample (11) and the scattered radiation (15) detected by the detection optics (5, 7, 8) cross the boundary surface (13).

19. Measuring system according to claim 18, characterized in that the flat sample carrier (10) is suitable for providing a plurality of individual separated from each other sample drops (11).

20. Measuring system according to one of claims 18 to 19, characterized in that the flat sample carrier (10) is a microtiter plate with one for the sample (11) acting on electromagnetic radiation (3) transparent bottom.

21. Measuring system according to one of claims 18 to 20, characterized in that a positioning unit is provided, with which the position of the flat sample carrier (10) relative to the focal point (12) is adjustable.

22. A method for carrying out scattered light measurements, wherein the electromagnetic radiation (1, 3) focused on a sample (11) and from the sample (11) scattered radiation (15) is detected, characterized in that the sample (11) as a drop is present, which has an interface (13) to a flat sample carrier (10) and the exposure to the electromagnetic radiation (3) and the detection of the scattered radiation (15) through the flat sample carrier (10) and the interface (13) therethrough.

23. The method according to claim 22, characterized in that the drop is covered with a liquid (14), preferably a paraffin or an oil, which counteracts the evaporation of the sample (11).

24. The method according to any one of claims 22 to 23, characterized in that the volume of the sample (11) is less than 1 ul.

25. The method according to any one of claims 22 to 24, characterized in that on the sample (11) focused electromagnetic radiation (3) has a plurality of Strahlungsaηteile different wavelength and that different spectral components of the scattered radiation (15) are detected separately.

26. The method according to any one of claims 22 to 25, characterized in that the sample (11) sits as drops on the flat sample carrier (10).

27. The method according to any one of claims 22 to 25, characterized in that the sample (11) hangs as a drop under the flat sample carrier (10).