阅读说明：本技术 用于分析粒子的设备和方法 (Apparatus and method for analyzing particles ) 是由 S·魏德利希 M·施密特 于 2019-07-25 设计创作，主要内容包括：本申请涉及用于分析粒子的设备和方法。为了在此基础上提供分析设备,其中毛细管可以用作测量单元并且其同时允许以高信噪比进行可靠、可再现的测量,本发明建议所述中空通道的内径D<Sub>H</Sub>在10μm至60μm的范围内,所述光束有具备最小光束直径D<Sub>L</Sub>的径向光强度分布,其中以下关系适用于直径比D<Sub>L</Sub>/D<Sub>H</Sub>：0.05<D<Sub>L</Sub>/D<Sub>H</Sub><2.00,以及进入所述中空通道时,所述光束相对于所述中空通道的纵轴的入射角小于2度。(The present application relates to an apparatus and a method for analyzing particles. In order to provide an analytical device on this basis, in which a capillary can be used as a measuring cell and which at the same time allows reliable, reproducible measurements with a high signal-to-noise ratio, the invention proposes that the inner diameter D of the hollow channel be H In the range of 10 μm to 60 μm, the light beam has a minimum beam diameter D L Wherein the following relationship applies to the diameter ratio D L /D H ：0.05<D L /D H <2.00 and an angle of incidence of the light beam with respect to a longitudinal axis of the hollow channel is less than 2 degrees when entering the hollow channel.)

1. An apparatus for analyzing particles, comprising:

a glass capillary as a measuring cell having a hollow channel for receiving or passing a test sample containing the particles, the hollow channel having a hollow channel longitudinal axis and a hollow channel inner wall,

a light source for generating a light beam and an optical device at an input point for coupling the light beam into the hollow channel for illuminating the test sample, and

a detector for detecting scattered light exiting the hollow channel,

it is characterized in that

Inner diameter D of the hollow passage _HIn the range of 10 μm to 60 μm,

the beam having a minimum beam diameter D _LWherein the following relationship applies to the diameter ratio D _L/D _H：0.05<D _L/D _H<2.00, and

-an angle of incidence (Ψ) of the light beam with respect to a longitudinal axis of the hollow channel is less than 2 degrees upon entering the hollow channel.

2. The apparatus of claim 1, wherein the glass capillary is straight at least along the signal detection length.

3. The apparatus according to claim 1 or 2, characterized in that the glass capillary has hollow channel walls with a wall thickness of at least 100 μ ι η, at least 500 μ ι η and preferably at least 1000 μ ι η and a wall thickness of not more than 10mm, not more than 5mm and preferably not more than 2mm, and that the hollow channel walls, viewed in radial direction, have a uniform refractive index profile.

4. The apparatus according to claim 1 or 2, characterized in that the glass capillary consists of quartz glass.

5. The apparatus of claim 1 or 2, wherein the optical device is configured as an optical fiber in the form of a multimode optical fiber or a single-mode optical fiber, the optical fiber having an optical fiber core and a cladding surrounding the optical fiber core, and wherein the optical fiber has a numerical aperture, NA, that applies the relationship: NA < 0.05.

6. Device according to claim 1 or 2, characterized in that the hollow channel has an inner cross-section with at least one flattened portion and/or the capillary has an outer cross-section with at least one flattened portion.

7. The apparatus of claim 6, wherein the capillary is formed in a plate-like body having flat sides opposite one another, wherein the flat sides of the body form outer walls of the capillary.

8. The apparatus of claim 1 or 2, wherein the detector is configured such that scattered light energy is detected along a signal detection length of up to 20 cm.

9. The apparatus according to claim 1 or 2, characterized in that the glass capillary (1) has a hollow channel wall (3), the wall thickness of the hollow channel wall (3) being at least 100 μ ι η, at least 500 μ ι η and preferably at least 1000 μ ι η and the wall thickness not exceeding 10mm, not exceeding 5mm and preferably not exceeding 2mm, and the hollow channel wall (3) has a uniform refractive index profile as viewed in the radial direction.

10. The apparatus according to claim 1 or 2, wherein the surface roughness of the inner surface of the hollow channel wall is defined as the mean roughness depth R _aIs less than 1 nm.

11. A method for analyzing particles, comprising the following method steps:

providing a measuring unit in the form of a glass capillary having a hollow channel with a hollow channel longitudinal axis and a hollow channel wall,

introducing a test sample containing said particles into said hollow channel (4), wherein said test sample hasRefractive index Deltan _M，

The use of a light source to generate a light beam,

coupling the light beam into the hollow channel by an optical input device at an input point for illuminating the test sample, and

detecting scattered light exiting the hollow channel using a detector,

it is characterized in that

Using glass capillaries, wherein the internal diameter D of the hollow channel _HIn the range of 10 μm to 60 μm,

the beam having a minimum beam diameter D _LWherein the following relationship applies to the diameter ratio D _L/D _H：0.05<D _L/D _H<2.0，

-the angle of incidence (Ψ) of the light beam with respect to the longitudinal axis of the hollow channel is less than 2 degrees upon entering the hollow channel (4).

12. Method according to claim 11, characterized in that the glass capillary has hollow channel walls with a wall thickness of at least 100 μ ι η, at least 500 μ ι η and preferably at least 1000 μ ι η and a wall thickness of not more than 10mm, not more than 5mm and preferably not more than 2mm and that the hollow channel walls have a uniform refractive index profile as seen in radial direction.

13. Method according to claim 11 or 12, characterized in that a glass capillary consisting of quartz glass is used.

14. Method according to claim 11 or 12, characterized in that as the optical means an optical fiber in the form of a multimode fiber or a single mode fiber is used, said fiber having a fiber core and a cladding surrounding the fiber core, and wherein the fiber has a numerical aperture NA applying the following relation: NA < 0.05.

15. Method according to claim 11 or 12, characterized in thatSaid test sample having a refractive index Δ n _MAnd the glass of the capillary has a refractive index Deltan _KWherein the following relationship applies: Δ n _M<Δn _KPreferably Δ n _M<Δn _K-0.1。

Technical Field

The invention relates to an apparatus for analyzing particles, comprising:

a glass capillary as a measuring cell having a hollow channel for receiving or passing a test sample containing the particles, the hollow channel having a hollow channel longitudinal axis and a hollow channel inner wall,

a light source for generating a light beam and an optical device at an input point for coupling the light beam into the hollow channel for illuminating the test sample, and

a detector for detecting scattered light exiting the hollow channel.

Furthermore, the invention relates to a method for analyzing particles, comprising the following method steps:

providing a measurement unit in the form of a glass capillary having a hollow channel with a hollow channel longitudinal axis and a hollow channel inner wall,

introducing a test sample containing said particles into said hollow channel, wherein said test sample has a refractive index Δ n _M，

The use of a light source to generate a light beam,

coupling the light beam into the hollow channel by an optical input device at an input point for illuminating the test sample, and

detecting scattered light exiting the hollow channel using a detector.

Background

Methods of characterizing samples in fluid media are standard practice in basic medical and biological research and are routine diagnostic methods in many medical fields in hospitals.

One commonly used method is flow cytometry, in which light scattered from particles is analyzed. The required equipment complexity is low and in principle it can be used for cost effective analysis. A suitable scattered light measuring assembly is known from DE 102013210259 a 1. The flow measuring unit here is in the form of a hollow quartz glass cylinder which is equipped with a central longitudinal bore. A liquid stream with particles to be characterized is passed through the hole and irradiated with a laser beam, which is injected through the cladding of the hollow cylinder. At various angles around the cylindrical measuring cell, detectors are arranged which absorb the scattered light. The molecules or colloidal substances contained in the liquid are analyzed, for example with respect to their size, mass or structure.

However, the measurement sensitivity obtained here is often low due to the high background signal and poor signal-to-noise ratio. Thus, it is often desirable to observe fluorescence rather than scattered light to obtain a stronger signal that can be separated from background. However, very few sample particles will exhibit natural fluorescence, so in these cases it is necessary to prepare the sample by adding fluorophores.

Higher measurement sensitivity is obtained using an analytical device and analytical method according to the above-described type as described in WO 2016/038015 a 1. Here, a light-conducting hollow channel is used as a measuring cell, into the end face of which the illuminating light scattered by the excitation light is coupled. The hollow channel is located in the core of the optical waveguide composed of quartz glass, with a non-uniform refractive index profile in the radial direction, so that the coupled illumination light propagates by total internal reflection in the core of the optical fiber and to some extent also in the hollow channel itself along the longitudinal axis of the hollow channel.

Scattered light emerging through the walls of the hollow channel is detected by a detector. This is configured to measure, for example, coherent scattering intensity of the scattered light, incoherent scattering intensity of the scattered light, spectral distribution of the scattered light, spatial distribution of the scattered light and/or dynamic motion of the particle to be measured. Furthermore, the detector may also be configured to detect scattered fluorescence generated by illuminating the particles to be measured.

Restricted to pore cross-sections of less than 0.2 μm ²Is filled with a liquid containing the particles to be investigated. The nature of the interaction between the light and the material results in the irradiated light being scattered in the quartz glass only to a small extent, but significantly more strongly from the particles present in the liquid. The scattered measurement light is captured by a camera and processed for analysis. The small core cross-sectional area and the large refractive index difference between the core and the cladding promote spatial confinement of the illumination light in the core region of the optical waveguide and improve illumination of the hollow channel. It is mentioned that the hollow passage may be in the form of a capillary tube. Similar analytical devices and analytical methods are described in WO 2016/038108A 1.

JP 2006-125901A describes a method and apparatus for capillary electrophoresis. The apparatus includes a plurality of capillaries including irradiation sites, wherein the irradiation sites are arranged in a planar manner. In order to measure a large number of capillaries simultaneously, an array of capillaries is arranged in which laser irradiation positions are formed in a common line. The laser is divided so that both sides of each array can be irradiated with high efficiency laser light. In one example, 384 capillaries are bundled to form a capillary array. Each capillary was made of quartz and coated on the outer surface with a fluorocarbon material, and had a total length of 40cm, an outer diameter of 130 μm and an inner diameter of 50 μm.

US 2014/2960689 a1 describes a biological sample processing device comprising a cytometry station comprising an imaging device and a platform for receiving a microscope cuvette, and a detection station. In another embodiment, a capillary electrophoresis method is described, wherein a buffer filled capillary is suspended between two reservoirs filled with buffer. An electric field is applied across the capillary. A sample containing one or more components or species is typically introduced at a high potential end and under the influence of an electric field. The capillary array may be held in a guide and the inlet end of the capillary immersed in a vial containing the sample. After the sample is drawn in by the capillary, the end of the capillary is removed from the sample vial and submerged in a buffer, which may be in a common container or in a separate vial. The sample moves to the low potential end. During migration, the components of the sample are separated by electrophoresis. After separation, the components are detected by a detector. Detection may be performed while the sample is still in the capillary or after it exits the capillary. The inner diameter of the capillary may be in the range of about 5 to 300 μm, and preferably about 20 to 100 μm. The length of the capillary tube may typically be in the range of about 100 to 3000 mm. Which is typically constructed of a non-conductive material so that a high voltage can be applied to the capillary without generating excessive heat. Inorganic materials such as quartz, glass, fused silica, and the like may be advantageously used to fabricate the capillary tube. In the case of excitation and/or detection via the capillary wall, a particularly advantageous capillary is a capillary composed of a transparent material.

Technical problem

The measurement rate in such analytical methods is limited by the pore size cross section of the hollow channel. According to WO 2016/038015A 1, this is limited to less than 0.2 μm ². Compared to such narrow hollow channels, capillaries with a relatively large diameter in the range of a few micrometers may not only allow higher measurement rates, but also multiple parallel measurements and detection of larger particles.

The use of a capillary as a hollow channel in the analysis of particles, for example in flow cytometry, is therefore in principle ideal, but causes many other technical problems, for example a sufficiently high signal-to-noise ratio, which have not been addressed nor solved in the prior art.

It is therefore an object of the present invention to provide a device for analyzing particles, wherein a capillary can be used as a measurement cell and which allows a reliable, reproducible measurement with a high signal-to-noise ratio.

Furthermore, it is an object of the present invention to specify a method for analyzing particles which allows reliable, reproducible measurements with a high signal-to-noise ratio.

Disclosure of Invention

With respect to said device, according to the invention, this object is achieved starting from a device of the type described above by the fact that:

inner diameter D of the hollow passage _HIn the range of 10 μm to 60 μm,

the angle of incidence of the light beam with respect to the longitudinal axis of the hollow channel is less than 2 degrees when entering the hollow channel, and

the beam having a minimum beam diameter D _LWherein the following relationship applies to the diameter ratio D _L/D _H：0.05<D _L/D _H<2.00。

In the analysis device according to the invention, the measurement unit is configured as a glass capillary, the inner bore of which forms a hollow channel for receiving or passing through the test sample to be analyzed. The test sample is confined in the hollow channel or is passed through the hollow channel in a continuous flow.

In the simplest case, the glass capillary is configured as a hollow cylinder made of an optically homogeneous glass material, so that the capillary wall has a homogeneous refractive index profile in the radial direction. In contrast to known measuring units, the hollow channel therefore has no light guide which is based on total internal reflection and which facilitates illumination of the hollow channel in the form of an optical waveguide structure. Such a hollow cylinder walled waveguide structure may allow theoretically non-dissipative light guiding transverse to the hollow cylinder axis and thus allow uniform illumination over a long distance in the inner bore formed by the inner wall of the hollow cylinder. However, a capillary tube having a uniform refractive index profile (as here) will generally not direct any light in the interior volume of the hollow channel formed by the capillary wall. Nevertheless, in order to achieve measurements with high signal-to-noise ratio and high measurement sensitivity, it is desirable that the light beam is efficiently coupled into the hollow channel and that the transmission loss is low enough for the application.

Thus, in the device presented herein it is provided that by selecting and adjusting the design measures for beam guiding, light is introduced into the hollow channel such that a defined intensity distribution (so-called mode) is formed in a plane perpendicular to the longitudinal axis of the hollow channel, which is guided along the longitudinal axis of the hollow channel but which physically inherently experiences energy losses due to lateral dissipation of energy. In order to minimize this energy loss, it must be ensured that the light is coupled into the hollow channel of the capillary, so that the majority of the power, in particular the largest part of the power, is guided in the so-called fundamental mode. The intensity distribution of the fundamental mode on the one hand contributes to a particularly homogeneous irradiation of the hollow channel volume compared with all other possible modes, so that particularly trouble-free measurements of the particles to be analyzed can be carried out. On the other hand, this ensures that the light is guided along the longitudinal axis of the capillary, and in particular along the predetermined measuring distance, with as low losses as possible. The reason for this is that the fundamental mode has lower losses due to energy dissipation transverse to the longitudinal axis of the hollow channel than all other modes.

Furthermore, the optical power is preferably injected into the fundamental mode, reducing the adverse effects of so-called "mode mixing". In this case, the distribution of light energy from one mode to another occurs along the optical waveguide. This results in an intensity distribution that varies in its normal plane along the longitudinal axis of the optical waveguide, where a number of factors make it difficult to accurately determine the intensity distribution. However, it is helpful to know this intensity distribution to qualify the particles to be analyzed.

The above-mentioned design measures will be explained in more detail below:

(1) the relatively weak light guide in the hollow channel of the capillary leads to a high optical attenuation of the coupled-in light beam. It has been shown that the attenuation depends on the inner diameter of the capillary, and the smaller the inner diameter, the greater the attenuation.

Thus, the inner diameter D of the hollow passage _HIn the range of 10 μm to 60 μm. With hollow channels having an internal diameter of less than 10 μm, high optical attenuation occurs, which makes it difficult to perform reliable, repeatable measurements with high signal-to-noise ratios. Therefore, preferably, the inner diameter of the hollow channel is at least 20 μm. The hollow channel contains sample particles to be analyzed, which are able to move freely in the available hollow channel volume. The mobility in the transverse direction (perpendicular to the longitudinal axis of the hollow channel) is limited by the inner diameter of the hollow channel. In the case of hollow channels with an internal diameter of more than 60 μm, the volume and in particular the mobility of the particles in the lateral direction is so large that the detector is difficult to detect reliably due to the limited depth of field.

The light beam generated by the light source enters the hollow channel at an end-face input point (the end-face aperture of the hollow channel) and, as it further propagates through the hollow channel, it scatters from the sample particles contained therein, emitting scattered light, and is therefore attenuated. The scattered light is detected by a detector. Scattered light detection can start directly at the input point, although additional reflections and parasitic scattering effects can occur at the input point, making accurate detection and particle scattering assessment more difficult. The scattered light detection therefore preferably starts downstream of the input point viewed in the direction of illumination, for example over a length of at least 2 mm.

"scattered light" is understood here to mean the illuminating light which leaves the hollow channel through the capillary wall and is detected by the detector. The wall is transparent to the illuminating light.

(2) In order to transmit the light beam from the light source to the input point, light transmission means are provided. This includes, for example, arrangements of optical fibers or optical components for transmitting the free light beam. The coupling of the light beam into the hollow channel is substantially determined by the numerical aperture (hereinafter "NA") of the light delivery device and the ratio of the minimum beam diameter to the inner diameter of the hollow channel. In the device according to the invention, optical transmission means with a relatively small NA are used. This is achieved byThe following facts indicate: the light beam having a minimum beam diameter D _LRadial light intensity distribution, minimum beam diameter D _LApproximately corresponding to the inner diameter D of the hollow passage _HAre as large; more precisely, the following relationship applies to the diameter ratio D _L/D _H：0.05<D _L/D _H<2.00, preferably: 0.1<D _L/D _H<1.00, and particularly preferably: 0.2<D _L/D _H<0.5。

The minimum beam diameter D is determined from the beam waist width in the beam focus in the case of a free beam and from the core diameter at the light output end of the optical fiber in the case of fiber feeding _L。

Having a minimum beam diameter D _LIs as small as possible from the end face aperture of the hollow channel; it is preferably less than 10mm and ideally zero.

(3) In principle, the measures described in (2) above can be achieved by sufficiently strong focusing of the light beam. However, strong focusing is associated with a high divergence of the light beam. However, it has been shown that a smaller divergence angle is required and therefore a low focusing of the light beam is desired. If the light beam impinges at a flat angle on the end face aperture of the hollow channel, a portion of the light will be reflected at the inner wall of the hollow channel as a function of the angle of incidence, while another portion will penetrate into the capillary wall and leave the capillary with a high degree of energy loss as loss light. In order to facilitate a high proportion of reflected light and as low as possible loss light associated with the largest possible detection length, the angle of incidence of the light beam upon entering the hollow channel with respect to the longitudinal axis of the hollow channel is less than 2 degrees, and preferably less than 1 degree. The incident angle is defined herein as a hollow channel side acceptance angle (corresponding to half of the aperture angle).

The requirement for a relatively small beam diameter on the one hand and a small divergence angle on the other hand is somewhat contradictory. However, the combination of these measures in the device according to the invention leads to the beam guiding explained above, wherein a high proportion of the coupled optical power is transferred to the fundamental mode.

In this respect, an embodiment of the device is preferred, wherein the glass capillary consists of quartz glass.

Quartz glass is substantially transparent over a wide wavelength range between about 150nm and 3000 nm. Thus, glass capillaries allow for illuminating radiation in the wavelength range from UV to infrared, with less scattering contribution from the capillary walls. Furthermore, the quartz glass material makes it easy to achieve a particularly smooth inner wall even if the bore cross section of the hollow channel is small, since the temperature range over which the capillary tube can be drawn by thermoforming is relatively large. The quartz glass of the capillary may be undoped. It may also contain one or more dopants.

Glass refractive index Deltan of capillary _KPreferably with the refractive index deltan of the sample to be absorbed according to the description or other terms of use _MAnd (6) matching. Δ n _KPreference ratio Δ n _MLarge, and particularly preferred, ratio Δ n _MAt least 0.1 greater (measured in each case at a measurement wavelength of 532nm and a measurement temperature of 20 ℃). The test sample is typically absorbed in an aqueous medium. For the refractive index of water compared to air, a value of about 1.33 is given in the literature (measured parameter as described above). The refractive index of quartz glass is approximately 1.45 and therefore, in principle, a capillary made of quartz glass satisfies the above-mentioned preferred dimensional rule Δ n _M<Δn _K-0.1。

In terms of low transmission losses in the hollow channel, it has proven useful for the glass capillary to be as inflexible as possible and, on the contrary, straight along its entire length, in particular at least along the signal detection length. "straight" here is intended to mean a profile in which the positions of the longitudinal axes of the capillaries at the beginning and end of the signal detection length are at a distance of less than 1mm from each other in a direction perpendicular to the irradiation direction. A portion of the signal detection length corresponding to the length of the capillary tube along which scattered light detection is performed; this portion is at least 2mm and not more than 20cm in length, measured from the input point.

The adverse effects of "mode mixing" are also exacerbated by bending in the capillary and locally acting mechanical tensile or compressive stresses. By establishing a high bending stiffness and/or a sufficiently high area moment of inertia (in particular caused by a capillary wall of high thickness), it is possible to absorb mechanical stresses and to suppress bending.

On the other hand, the walls of the hollow channel may contain scattering centers and which contribute to the optical attenuation of scattered light to be detected by the detector.

It has therefore proved useful if the glass capillary has hollow channel walls with a wall thickness of at least 100 μm, at least 500 μm and preferably at least 1000 μm and a wall thickness of not more than 10mm, not more than 5mm and preferably not more than 2mm, wherein the hollow channel walls have a uniform refractive index profile, viewed in the radial direction.

In a particularly preferred embodiment, the optical device is configured as an optical fiber in the form of a multimode or single-mode optical fiber having an optical fiber core and a cladding surrounding the optical fiber core, and wherein the optical fiber has a numerical aperture NA applying the following relation: NA < 0.05.

The very low NA fiber enables the flattest possible angle of incidence to be achieved, thereby contributing to improved coupling efficiency and guiding of the light beam in the hollow channel of the capillary.

The optical modes guided in the multimode optical fiber have different refractive indices and attenuate differently in the test sample. For single mode of a single mode fiber, the coupling parameters can be optimized to obtain relatively high coupling efficiency, especially in the fundamental mode of the capillary.

Nevertheless, if the optical fiber has an inner diameter D with respect to the hollow passage _HMode field diameter D having the following relationship _MIt proves advantageous in particular in terms of high coupling efficiency: 0.05<D _M/D _H<2.00, preferably: 0.1<D _M/D _H<1.00, and particularly preferably: 0.2<D _M/D _H<0.5。

Mode field diameter is a parameter used to characterize the light distribution of the fundamental mode in a single mode fiber. For a radial light intensity distribution that can be approximated by a Gaussian curve (Gaussian curve), the mode field diameter is the diameter at which the magnitude of the light intensity drops to 1/e (about 37%).

The detector is preferably configured such that it detects scattered light along a signal detection length having a length of at least 2mm and not more than 20cm from the input point.

For detection lengths exceeding 20cm, uniform illumination of the sample volume becomes more difficult due to beam attenuation along the detection length.

For a higher measuring accuracy it has proved advantageous if the hollow channel has an inner cross section with at least one flat portion and/or the capillary has an outer cross section with at least one flat portion.

In the case of a circular inner cross section and a circular outer cross section, the measurement window of the detector for detecting scattered light is curved. The curvature leads to distortion of the image and undesired deflection of scattered light and/or optical errors in the image and must be taken into account in the analysis. The analysis is facilitated if at least the inner or at least the outer measurement window boundary surface is flat, and in particular if both measurement window boundary surfaces are flat. This is achieved by a flat portion on one or both sides of the capillary wall. The flat on one or both sides can also be realized by a polygonal inner and/or outer cross section.

In a particularly preferred embodiment of the device, the hollow channel is formed in a plate-like body having flat sides opposite to each other, wherein the flat sides of the body form the outer walls of the capillary.

The plate-like body has, for example, a rectangular shape, in particular the shape of a microscope slide, and is preferably distinguished by a high bending stiffness. The plate-like body forms or is integrated in the capillary. Which may for example extend in the longitudinal direction within a rectangle.

Capillaries are typically produced by elongating a hollow cylinder. During this operation, drawing marks and other surface structures can form on both the inner wall of the hollow channel and the outer wall of the capillary, which represent disturbances in the measuring path of the scattered light measurement and negatively influence the measurement result by reflection and scattering. In order to avoid as much as possible the falsifications caused by parasitic scattering effects and interfaces, it is desirable that the inner walls of the hollow channel are as smooth as possible and that the surface roughness is defined as the mean roughness depth R _aIs less than 1 nm.

Advantageously, the surface roughness of the outer wall of the capillary is also defined as the mean roughness depth R _aIs less than 1 nm.

The depth of the roughness was measured by Atomic Force Microscopy (AFM). The average roughness depth is determined from the measurement values according to DIN 4768 (2010).

With regard to the method for analyzing particles, according to the invention, the above technical problem is solved with a method comprising the following method steps:

using a measuring cell, wherein the internal diameter D of the hollow channel _HIn the range of 10 μm to 60 μm,

the beam having a minimum beam diameter D _LWherein the following relationship applies to the diameter ratio D _L/D _H：0.05<D _L/D _H<2.00, and

-the angle of incidence of the light beam with respect to the longitudinal axis of the hollow passage is less than 2 degrees when entering the hollow passage.

In the analysis method according to the invention, the measurement unit is configured as a glass capillary, the inner bore of which forms a hollow channel for receiving or passing through the sample to be analyzed. The test sample is confined in the hollow channel or is passed through the hollow channel in a continuous flow.

In the simplest case, the glass capillary is configured as a hollow cylinder made of an optically homogeneous glass material, so that the capillary wall has a homogeneous refractive index profile in the radial direction. In contrast to known measuring units, the hollow channel therefore has no light guide which is based on total internal reflection and which facilitates illumination of the hollow channel in the form of an optical waveguide structure. Such a hollow cylinder wall waveguide structure may allow theoretically non-dissipative light guiding transverse to the hollow cylinder axis and thus allow uniform illumination over a long distance in the inner bore formed by the hollow cylinder inner wall. However, a capillary tube having a uniform refractive index profile (as here) will generally not direct any light in the interior volume of the hollow channel formed by the capillary wall. Nevertheless, in order to achieve measurements with high signal-to-noise ratio and high measurement sensitivity, it is desirable that the light beam is efficiently coupled into the hollow channel and that the transmission loss is low enough for the application.

In the method according to the invention, it is therefore provided that, by selecting and adjusting the design measures for beam guidance, light is introduced into the hollow channel such that a defined intensity profile (so-called mode) is formed in a plane perpendicular to the longitudinal axis of the hollow channel, which is guided along the longitudinal axis of the hollow channel but which physically inherently experiences energy losses due to lateral dissipation of energy. In order to minimize this energy loss, it must be ensured that light is coupled into the hollow channel of the capillary, so that a large part of the power, in particular the largest part of the power, is guided in the so-called fundamental mode. The intensity distribution of the fundamental mode on the one hand contributes to a particularly homogeneous irradiation of the hollow channel volume compared with all other possible modes, so that particularly trouble-free measurements of the particles to be analyzed can be carried out. On the other hand, this ensures that the light is guided along the longitudinal axis of the capillary and in particular along the predetermined measuring distance with as low losses as possible. The reason for this is that the losses caused by energy dissipation transverse to the longitudinal axis of the hollow channel are lower for the fundamental mode than for all other modes.

Furthermore, injecting optical power into the fundamental mode preferably reduces the adverse effects of so-called "mode mixing". In this case, the distribution of light energy from one mode to another occurs along the optical waveguide. This results in an intensity profile that varies along the longitudinal axis of the optical waveguide in its normal plane, where a number of factors make it difficult to accurately determine the intensity profile. However, knowledge of this intensity profile helps to qualify the particles to be analyzed.

The above-mentioned design measures will be explained in more detail below:

(1) the relatively weak light guide in the hollow channel of the capillary leads to a high optical attenuation of the coupled-in light beam. It has been shown that the attenuation depends on the inner diameter of the capillary, and the smaller the inner diameter, the greater the attenuation.

Thus, the inner diameter D of the hollow passage _HIn the range of 10 μm to 60 μm. In case the inner diameter of the hollow channel is less than 10 μm, high optical attenuation occurs, which makes it difficult to perform with high signal-to-noise ratioReliable and repeatable measurement. Therefore, preferably, the inner diameter of the hollow channel is at least 20 μm.

The hollow channel contains sample particles to be analyzed, which are able to move freely in the available hollow channel volume. The mobility in the transverse direction (perpendicular to the longitudinal axis of the hollow channel) is limited by the inner diameter of the hollow channel. In the case of hollow channels with an internal diameter of more than 60 μm, the volume and in particular the mobility of the particles in the lateral direction is so large that the detector is difficult to detect reliably due to the limited depth of field.

The light beam generated by the light source enters the hollow channel at an end-face input point (the end-face aperture of the hollow channel) and, as it further propagates through the hollow channel, it scatters from the sample particles contained therein, emitting scattered light, and is therefore attenuated. The scattered light is detected by a detector. Scattered light detection can start directly at the input point, although additional reflections and parasitic scattering effects can occur at the input point, making accurate detection and particle scattering assessment more difficult. The scattered light detection therefore preferably starts downstream of the input point viewed in the direction of illumination, for example over a length of at least 2 mm.

"scattered light" is understood here to mean the illuminating light which leaves the hollow channel through the capillary wall and is detected by the detector. The wall is transparent to the illuminating light.

(2) In order to transmit the light beam from the light source to the input point, light transmission means are provided. This includes, for example, arrangements of optical fibers or optical components for transmitting the free light beam. The coupling of the light beam into the hollow channel is substantially determined by the numerical aperture (hereinafter "NA") of the light delivery device and the ratio of the minimum beam diameter to the inner diameter of the hollow channel. In the method according to the invention, an optical transmission device with a relatively small NA is used. This is indicated by the fact that: the light beam having a minimum beam diameter D _LRadial light intensity distribution, minimum beam diameter D _LApproximately corresponding to the inner diameter D of the hollow passage _HAre as large; more precisely, the following relationship applies to the diameter ratio D _L/D _H：0.05<D _L/D _H<2.00, preferably: 0.1<D _L/D _H<1.00, and particularly preferably: 0.2<D _L/D _H<0.5。

The minimum beam diameter D is determined from the beam waist width in the beam focus in the case of a free beam and from the core diameter at the light output end of the optical fiber in the case of fiber feeding _L。

Having a minimum beam diameter D _LIs as small as possible from the end face aperture of the hollow channel; it is preferably less than 10mm and ideally zero.

(3) In principle, the measures described in (2) above can be achieved by sufficiently strong focusing of the light beam. However, strong focusing is associated with a high divergence of the light beam. However, it has been shown that a smaller divergence angle is required and therefore a low focusing of the light beam is desired. If the light beam impinges at a flat angle on the end face aperture of the hollow channel, a portion of the light will be reflected at the inner wall of the hollow channel as a function of the angle of incidence, while another portion will penetrate into the capillary wall and leave the capillary with a high degree of energy loss as loss light. In order to facilitate a high proportion of reflected light and as low as possible loss light associated with the largest possible detection length, the angle of incidence of the light beam upon entering the hollow channel with respect to the longitudinal axis of the hollow channel is less than 2 degrees, and preferably less than 1 degree. The incident angle is defined herein as a hollow channel side acceptance angle (corresponding to half of the aperture angle).

The requirement for a relatively small beam diameter on the one hand and a small divergence angle on the other hand is somewhat contradictory. However, the combination of these measures in the method according to the invention leads to the beam guiding explained above, in which a high proportion of the coupled optical power is transferred to the fundamental mode.

Advantageous embodiments of the method according to the invention can be taken from the dependent claims. In case the embodiments of the method presented in the dependent claims are based on the embodiments mentioned in the claims relating to the device according to the invention, reference should be made to the statements made above with respect to the respective device claims for supplementary explanation. Further embodiments of the method according to the invention will be explained in more detail below.

For example, a proven process variantThe formation form is particularly advantageous, wherein the test sample has a refractive index Δ n _MAnd the capillary glass has a refractive index Deltan _KWherein the following relationship applies: Δ n _M<Δn _KPreferably Δ n _M<Δn _K-0.1。

Refractive index of capillary glass Deltan _KRefractive index delta n of sample to be received _MAnd (4) matching. In particular, Δ n _KPreferably the ratio Δ n _MLarge, and particularly preferably, the ratio Δ n _MAt least 0.1 greater (measurement wavelength: 532 nm; measurement temperature 20 ℃ C.). The test sample is typically absorbed in an aqueous medium. For the refractive index of water compared to air, a value of about 1.33 is given in the literature (measured parameter as described above). The refractive index of quartz glass is approximately 1.45 and therefore, in principle, a capillary made of quartz glass satisfies the above-mentioned preferred dimensional rule Δ n _M<Δn _K-0.1。

The analysis apparatus according to the invention and the analysis method according to the invention can be used for particle analysis in the medical and non-medical fields, in particular in flow cytometry. Here, the liquid is passed through a cuvette and the molecular or colloidal substances contained therein are analyzed, for example with regard to their size, mass or structure. In optical analysis, a light beam is focused on a liquid stream so that individual molecules can be analyzed. By this method, a large number of measurements per unit time (more than 1000 measurement events per second) can be achieved, and thus statistically reasonable conclusions about the sample can be quickly achieved.

Drawings

The invention is explained in more detail below with reference to exemplary embodiments and the accompanying drawings. The individual figures are shown below:

FIG. 1: the basic measurement set-up for flow cytometry using the device according to the invention,

FIG. 2: a glass capillary having a hollow channel in a schematic end-face top view,

FIG. 3: for explaining the sketch of the coupling of a light beam into the hollow channel of a capillary,

FIG. 4: a diagram explaining the coupling efficiency in relation to the inner radius of the hollow channel and the beam diameter,

FIG. 5: a diagram explaining the dependence of the effective refractive index on the radius of the hollow channel,

FIG. 6: a diagram explaining the dependence of the reflection angle of the capillary fundamental mode on the hollow channel radius,

FIG. 7: a diagram explaining the dependence of the Numerical Aperture (NA) on the radius of the hollow channel, and

FIG. 8: a diagram illustrating the optical attenuation of a light beam over the radius of a hollow channel is illustrated.

Detailed Description

Fig. 1 shows a basic measurement setup of flow cytometry using an apparatus according to the present invention.

The measurement principle is based on the optical detection of scattered or fluorescent light or light otherwise emitted as a result of illuminating the sample particles. The detection here may, but need not, be for a selected location, a selected frequency or a selected intensity. By means of suitable analysis optics and algorithms, properties of the sample particles under investigation, such as size, shape, diffusion rate, mobility or scattering cross section, can thus be recorded.

In the measuring setup of fig. 1, a flow measuring cell according to the invention is used, which is in the form of a quartz glass capillary tube 1 with a wall 3 and a hollow channel 4. A liquid flow passes through the hollow channel 4, said liquid flow containing the sample particles 5 to be characterized. A light source 2 in the form of a frequency-doubled Nd: YAG laser is used to illuminate the liquid stream. Laser light with a wavelength of 532nm is guided through an optical fiber 6 to a quartz glass capillary 1 and enters the hollow channel 4 as a light beam 63 at an end face input point 10.

The optical fiber 6 is configured as a single mode optical fiber. Having a core 61 and a cladding 62 surrounding the core, wherein the refractive index of the core 61 is higher than the refractive index of the cladding 62, such that the laser light is guided in the core 61 substantially by total internal reflection.

The core 61 has a diameter of 10 μm and a mode field diameter of 7 μm.

Instead of a monochromatic laser, polychromatic excitation radiation is used.

As an alternative to single mode optical fibres, multimode optical fibres are used. This also has a core and a cladding surrounding the core, wherein the refractive index of the core is higher than the refractive index of the cladding, such that the laser light is guided in the core 61 substantially by total internal reflection.

The diameter of the core here is 10 μm, with a higher refractive index step between the cladding and the core compared to a configuration as a single mode fiber.

The capillary 1 is coupled to a conventional microscope arrangement 7, the microscope arrangement 7 comprising a camera 8, the focal point or detection plane of the camera 8 being located in the region of the central axis 9 of the hollow channel, and by means of said camera 8 the test sample and the sample particles 5 contained therein are observed and transferred for data analysis. This involves detecting elastic light scattering (rayleigh scattering), which is emitted as scattered light by the sample particles at the same frequency as the excitation frequency. Due to the low attenuation of the capillary 1, hardly any background scattering occurs in the fiber material itself. The camera 8 may detect the scattered light for a detection length of between 2mm and 20 cm. Over this length, the capillary 1 extends completely straight. In other words, the positions of the longitudinal axis 9 of the capillary at the beginning and at the end of this length are at a distance of less than 1mm from each other in a projection perpendicular to the direction of illumination.

By means of the sCMOS camera 8 as detector, the coherent scattering intensity of the scattered light, the incoherent scattering intensity of the scattered light, the spectral distribution of the scattered light, the spatial distribution of the scattered light and/or the dynamic motion of the particles 5 to be measured can be detected. In addition, the camera is further configured to detect scattered fluorescence generated by illuminating the particles to be measured.

Fig. 2 shows a schematic top view of an end face of a measuring cell in the form of a quartz glass capillary 1 with a wall 3 and a hollow channel 4. The capillary wall 3 consists of synthetically produced, undoped quartz glass with a refractive index of 1.4607. This value is based on measurements at a wavelength of 532nm and a measurement temperature of 20 ℃. Unless explicitly stated otherwise, these measurement conditions are also used for the refractive index values given below.

The outer diameter of the capillary 1 was 400. mu.m. The diameter of the hollow channel 4 is 30 μm. The hollow channel 4 extends coaxially with the central axis 9 of the capillary (see fig. 1) and with the main propagation direction of the laser beam 63. The cladding 3 has no interfaces or other structural discontinuities or non-uniformities that would cause any significant scattering.

The capillary 1 is produced by drawing a hollow cylinder made of synthetically produced, undoped quartz glass. From the average roughness depth R _aThe characterized surface roughness is less than 1 nm.

The sketch of fig. 3 serves to illustrate that the laser beam 63 at the input point 10 is coupled into the hollow channel 4 filled with the test sample. Upon entering the hollow channel 4, the angle of incidence of the light beam 63 with respect to the longitudinal axis 9 of the hollow channel is less than 1 degree. The incident angle phi (phi) is obtained by solving the beam equation of the fundamental mode 31. Which corresponds to the hollow channel side acceptance angle (corresponding to half the aperture angle) of the basic mode 31 and, at the same time, to the angle at which the basic mode is guided in the hollow channel 4 using the beam modeling method.

In the diagram of fig. 4, the coupling efficiency W (in%) of the beam 63 at 532nm wavelength with respect to the minimum beam diameter D occurs as a gaussian beam focused on the end face aperture (input point 10) of the capillary for the case where the hollow channel 4 is water-filled and the illumination occurs as a gaussian beam _LDifferent radii D of the function (in μm) _HAnd/2 (in μm) is plotted. The refractive index of water is 1.33 and thus a refractive index step 0.1307 for the hollow channel wall is obtained.

Accordingly, the coupling efficiency of each hollow channel radius has a significant maximum at a particular beam diameter. As the radius of the hollow channel increases, the maximum expands and at the same time moves to a larger beam diameter.

Hereby, an optimal beam diameter of about one third of the inner diameter of the hollow channel is obtained for hollow channel radii of up to about 30 μm. For diameter ratio D _L/D _HThe optimum value is in the range of 0.3 and a limit value of at least 20% of the coupling efficiency is used.

This indicates that optimum coupling cannot be achieved by bringing the beam waist of the beam 63 to the diameter of the inner wall of the capillary, but that the relationship follows a complex path depending on the diameter of the capillary.

In the diagram of fig. 5, the effective refractive index Re (n) of the light beam at a wavelength of 532nm in the case of the water-filled hollow channel 4 _eff) Radius D relative to hollow channel _HAnd/2 (in μm) is plotted.

This value describes the parameters of the propagation shape and type of the capillary fundamental mode and is calculated from a complete solution of the fundamental dispersion equation. This allows the calculation of curves of illustrative variables of numerical aperture and divergence angle for the same mode, as shown in fig. 6 and 7 below. In the diagram of fig. 6, the reflection angle phi (in degrees) of the fundamental mode of the water-filled hollow channel 4 is relative to the hollow channel radius D _HAnd/2 (in μm) is plotted.

The angle is obtained here by solving the underlying equation, which gives n _effAnd can be translated to this angle. In the beam model, the angle can be explained here as the angle at which the guided beam of the fundamental mode is reflected by the walls within the channel and, at the same time, as the exit angle of this beam from the capillary after complete passage. In order to couple with maximum efficiency, the incident light should also be directed to the capillary at exactly this angle.

It can be seen here that the reflection angle decreases as the diameter of the channel increases. This means that a gradually flat angle of incidence is required for coupling into the capillary as its diameter increases in order to couple into the fundamental mode with constant efficiency.

In the diagram of fig. 7, for a water-filled hollow channel 4, the numerical aperture NA of the fundamental mode is relative to the hollow channel radius D _HAnd/2 (in μm) is plotted. Accordingly, the optimum NA decreases with increasing hollow channel radius and reaches a value of 0.007 at a hollow channel radius of 30 μm. As can be seen from the figure, as the diameter of the capillary increases, the NA of the light to be coupled must become smaller and smaller in order to couple into the fundamental mode with the same efficiency, as in the case of the curve of the reflection angle.

In the diagram of fig. 8, for the case of a water-filled hollow channel 4, the light attenuation L (in dB/cm) of the light intensity guided in the hollow channel 4 in the basic mode is relative to the hollow channel radius D _HAnd/2 (in μm) is plotted. Accordingly, attenuation increases rapidly as the hollow passage narrows. For a hollow channel radius of 30 μm, the theoretical attenuation of the beam is about 0.2dB/cm,whereas for a hollow channel radius of 10 μm the theoretical attenuation of the light beam is about 5 dB/cm.

For the measurement, the particles 5 to be investigated are absorbed in an aqueous medium and introduced as droplets into one end of a capillary. By the suction effect of the capillary force, the liquid is drawn into the interior of the capillary together with the contained particles and thus through the measurement area imaged with the microscope. The numerical aperture of the optical fiber 6 is configured such that the incident angle of the laser beam 2 is less than 2 degrees.

The test sample is guided through the hollow channel 4 in a continuous flow, wherein the particles 5 are free to diffuse in the aqueous medium within the hollow channel 4, except for a directed movement through the filling. At the same time, a light beam 2 from a Nd: YAG laser is coupled into the hole 10 of the hollow channel 4 through the optical fiber 6 and used to irradiate the particles 5 in the hollow channel 4. Depending on the polarizability and size of the particles, the illumination produces fluorescence and coherent and/or incoherent light scattering. Part of the scattered and fluorescent light leaves the hollow channel 4 through the capillary wall 3 and is detected by the sCMOS camera 8. The detected scattered light is then processed and evaluated using software.

Due to the small incident angle of the laser beam being less than 2 degrees and the diameter ratio D _L/D _H0.3, the laser light that is not scattered by the particles 5 is reflected at the inner wall of the hollow channel and guided along the hollow channel 4. Due to this light guiding and due to the low surface roughness of the inner wall of the hollow channel 4, the optical attenuation along a signal detection length of up to about 20cm is reduced, and this is related to the signal-to-back ratio and the signal-to-noise ratio that can be evaluated, such that each irradiated particle 5 can be detected within the signal detection length.

Instead of operating with a continuous flow of the test sample, the hollow channel 4 also offers the possibility of one-dimensional or two-dimensional accommodation of the sample volume, so that the sample particles 5 to be investigated can be kept within the measurement range for a longer measurement time.