DE102011008788B3 - Arrangement for performing a single-sample analysis and manipulation for Raman microspectroscopy - Google Patents

Abstract

The present invention relates to an arrangement for carrying out a single sample analysis and manipulation for Raman microspectroscopy as well as a method for operating such an arrangement. The object of the invention to provide an arrangement for carrying out a single sample analysis and manipulation for Raman microspectroscopy, which does not require a microscope and only a laser and enables lower power densities compared to the prior art, is achieved in that the arrangement has a planar substrate (1 ), in which a channel (11) for the transport of samples and channels (12) in which optical fibers (4; 6) are located are introduced, the channel (11) having an inlet (111) which is provided with means (81) is connected for sample supply, and an outlet (112) which is connected to means (9) for sample removal, and in the channel (11) between the inlet (111) and the outlet (112) a laser trap (5 ) is arranged, in which the fibers (4; 6) are optically coupled. The optical fibers (4) are at least two excitation and capture fibers (41) and the optical fibers (6) are a plurality of collection fibers (61) which run centrally towards the laser trap (5), with an excitation and capture fiber (41 ) is formed from a single fiber, via the excitation and capture fibers (41), excitation and capture laser light can be coupled into the laser trap (5) at the same time, via the collection fibers (61) scattered light can be coupled out of the laser trap (5) through the means (81) Individual samples can be generated by means of (9) the individual samples can be sorted, and the collecting fibers (61) in a Raman spectrometer (7) and the excitation and capture fibers (41) in a laser (2) with beam splitter (3) open out, whereby unfocused laser beams can be generated by the laser (2).

Description

The invention relates to an arrangement for carrying out a single-sample analysis and manipulation for Raman microspectroscopy according to the preamble of the claims, in particular microchip-based for individual particles, individual biological cells or individual liquid compartments, and a method for operating such an arrangement.

The Raman effect (inelastic light scattering) used in Raman spectroscopy is an intrinsically weak process that requires a measurement duration in the range of several seconds. During this time, a single particle must be kept in solution in the excitation area. For the implementation of Raman microspectroscopy various arrangements are already known which operate with optical traps.

Thus, for example, discloses. WO 2007/141539 A1 a microfluidic system for Raman microspectroscopy, which comprises a radiation source for exciting the Raman effect on a particle (sg excitation radiation) and means for optically trapping the particle in an optical trap, which means the particles in the optical Raman process Fix the trap. The means for forming the optical trap consist of a double-beam arrangement in which laser beams are used to fix the particle (sg capture radiation). The excitation radiation (with a wavelength of 785 nm), which is generated by a laser diode, is at an angle of 90 ° relative to the capture radiation (with a wavelength of 1070 nm), which is generated by at least two laser sources (orthogonal two-beam arrangement ). The excitation radiation is focused via a Raman microscope on the fixed in the optical trap sample, which is located in a channel (capillary), which runs in a planar glass substrate.

The scattered light generated by the excitation is focused by a microscope objective perpendicular to the fixation plane and fed to a multi-channel spectrometer (Raman spectrometer). The disadvantage of this technical solution according to WO 2007/141539 A1 is that at least two lasers and a Raman microscope are needed, resulting in a complex and expensive construction of the system. In addition, the Rarnan excitation radiation is focused on the individual particles, for example a biological cell, which can lead to radiation damage due to the high power density in the focus.

From the publication "Dual beam fiber trap for Raman microspectroscopy of single cells" (PRT Jess, V. Garcés-Chávez, D. Smith, M. Mazilu, L. Paterson, A. Riches, CS Herrington, W. Sibbett and K. Dholakia ; in Optic Express 2006; 14; 5779-5791) is one opposite WO 2007/141539 A1 similar arrangement for Raman microspectroscopy is known, in which the excitation radiation (with a wavelength of 785 nm) to the capture radiation (with a wavelength of 1070 nm), also arranged at an angle of 90 ° (orthogonal two-beam arrangement). In this technical solution as well, the excitation radiation is focused on the sample fixed to an optical trap via a Raman microscope and the scattered light generated by the excitation is focused through a microscope objective perpendicular to the fixation plane and fed to a Raman spectrometer, so that even in this arrangement WO 2007/141539 A1 named disadvantages exist.

It is known from the publication "Fiber sample based microfluidic raman spectroscopy" (PC Ashok, GP Singh, KM Tan and K. Dholakia, in Optic Express 2010; 18; 7642-7649) that biological cells located in a microfluidic channel of a chip are in Direction of the channel (ie, in the flow direction of the cells) are irradiated via an optical fiber with a Raman excitation laser, wherein perpendicular to the collected light via a further optical fiber, the light scattered by the cells and forwarded to a Raman spectrometer.

The disadvantage of this technical solution is that only a large number of cells of a sample can be measured and a single cell analysis and manipulation (eg downstream cell sorting) by means of this solution is not possible.

In addition, optical tweezers are known, for example, from the publication "An integrated optofluidic platform for Raman-activated cell sorting" (AY Lau, L P. Lee and JW Chan, in Lab Chip, 2008; 8; 1116-1120). The disadvantage of using these optical tweezers for the conventional Raman spectroscopy according to the prior art is that the disclosure of WO 2007/141539 A1 already discussed standing problem of high power densities in single cells, which can lead to radiation damage this.

From the book by Dochow, S; Henkel, Th .; Kraft, C. and Popp, J: Manipulation and identification of individual cells by Mkrofluidik in combination with optical traps and Raman spectroscopy, in: 2009 Annual Report of the IPhT, Jena, 2009, pp. 34-35, an arrangement is known a planar substrate in which a channel for the transport of samples and channels, in which optical fibers lie, are introduced, wherein the channel an inlet, which is connected to means for sample introduction, as well as an outlet, which is connected to means for sample removal, by which individual samples can be generated and sorted. In this case, a laser trap is arranged around the channel between the inlet and the outlet, into which the fibers are optically coupled, wherein the optical fibers are at least two excitation and trapping fibers. These excitation and catching fibers lead into a laser with a beam splitter, so that unfocussed laser beams can be generated by the laser. The light coupled out of this arrangement is picked up by a lens of a microscope, the numerical aperture of the lens exerting an influence on the yield of the light supplied to the Raman spectrometer.

The publication by Neugebauer, U .; Dochow, S .; Kraft, O. and Popp, J: Chip-based Spectroscopic Diagnosis for the Identification of Circulating Tumor Cells, In: Photonik, 2011, Vol. 1; P. 17 discloses novel microfluidic systems for the rapid diagnosis and efficient therapy of cancer cancers by means of Raman spectroscopy, in which micro-manipulation and sorting of individual cells takes place on the chip and the light coupled out of the chip is picked up by a microscope objective and transmitted to the Raman. Spectrometer is forwarded. The micromanipulation is carried out with a Raman excitation laser, which serves as optical tweezers to keep the cell to be examined in focus during the Raman spectroscopic measurement. The cell sorting takes place in a square capillary in the chip, wherein the capture of the cells takes place separately from the Raman measurements. For this purpose, the capture lasers exert a greater force on the cells than the excitation laser.

The disadvantage of the above arrangement and the previous method is that a microscope is required, at least two lasers are needed and the yield of the light to be examined by Raman spectroscopy depends on the numerical aperture of the microscope objective.

The invention is therefore based on the object of specifying an arrangement for carrying out a single-sample analysis and manipulation for Raman microspectroscopy, in particular microchip-based for individual particles, individual biological cells or individual liquid compartments, which avoids the disadvantages of the prior art, in particular no microscope and requires only one laser and allows lower power densities compared to the prior art.

Furthermore, the object of the invention is that the arrangement allows manipulation (for example addressable sorting of the single-pobe) according to the Raman spectrometric measurement.

In addition, the object of the invention is to provide a method for operating such an arrangement for performing a single-sample analysis and manipulation for Raman microspectroscopy.

This object is achieved by an arrangement according to the first claim and a method for operating this arrangement according to claim 7. Advantageous embodiments of the invention are specified in the dependent claims.

According to the invention, the arrangement for carrying out single-sample analyzes and manipulations for Raman microspectroscopy comprises a planar substrate in which a channel for the transport of samples and channels in which optical fibers are located, wherein a laser trap is arranged around the channel for the individual samples is.

The fluidic channel for the discrete samples has an inlet connected to sample delivery means and an outlet connected to sample removal means.

The laser trap, in which the optical fibers are optically coupled, is thereby positioned between the inlet and the outlet.

The optical fibers are at least two excitation and trapping fibers, whereby both Raman excitation and trapping laser light can be conducted simultaneously through each of these fibers. At the same time both Raman excitation and capture laser light can be coupled into the laser trap via these excitation and capture fibers.

In addition, the optical fibers also include a plurality of collection fibers which converge towards the laser trap. Scattering light can be coupled out of the laser trap via these collecting fibers.

It is essential to the invention that an optical fiber is designed as an excitation and trapping fiber which simultaneously transports the excitation laser light and the trapping laser light, wherein unfocused laser light of a wavelength of 785 nm is fed into the optical fiber. However, it is also within the scope of the invention that other wavelengths, such as 633 nm or 1064 nm, may be used.

Alternatively, however, there is also the possibility that laser light having a wavelength of 785 nm and 1070 nm is simultaneously fed into an optical fiber. However, it is also within the scope of the invention that other wavelengths, such as, for example, 633 nm or 1064 nm, can be used.

The collecting fibers open into a Raman spectrometer and the excitation and trapping fibers open into a single laser with a beam splitter, it being possible to generate unfocussed laser beams by the laser.

According to the invention, means are provided for producing individual samples (means for separating particles, biological cells or drops of liquid) connected to the inlet of the fluidic channel, wherein a generator (for example a syringe pump) generates a fluid flow through the channel the individual particles, biological cells or drops of liquid carries in itself.

According to the invention, at the outlet of this channel are means for sorting the individual samples, which is carried out according to the Raman spectroscopic measurement (for example in the form of a passage of the channel into a multiplicity of branched channels in capillary form, which can open into individual vessels).

Particularly advantageously, the substrate is designed as a chip made of quartz glass and the channel as a capillary, wherein the non-fluidic channels are designed as guides for the optical fibers.

In this case, two excitation and catching fibers are arranged to the fluidic channel in each case at an angle of 90 °.

When the inventive arrangement is operated as intended, laser light from a laser is coupled into the excitation and capture fibers (i.e., excitation and capture radiation through a fiber) via a beam splitter (having a wavelength of 785 nm or wavelengths of 785 nm and 1070 nm).

The individual components of the arrangement, such as. Lasers, beam splitters, pump, Raman spectrometers are signal-conducting connected to a computer, so that control and regulation of the components and data acquisition (in particular the Raman spectra) can be done.

The advantage of the inventive arrangement for carrying out a single sample analysis and manipulation for Raman microspectroscopy and the inventive method is that no Raman microscope is required, only one laser is needed and that individual particles, individual biological cells or individual liquid drops (eg aqueous system in an oily system for the serial flow of single cells or droplets) can be measured by Raman spectroscopy and then sorted, whereby lower power densities for the Raman effect are required compared to the prior art. The reduced power density according to the invention, which substantially reduces the risk of radiation damage, especially in the case of biological cells, is achieved by working not with focused but with unfocussed laser light in the laser trap compared with the prior art. As a result, in contrast to the prior art, the entire single particle, single cell or single liquid drop volume is illuminated by the excitation radiation, which allows a higher specificity of the Raman classification, since not only the excitation volume of the focused laser beam is used. (According to WO 2007/141539 A1 Only one signal can come out of focus, which is small in relation to the particle, the cell or the droplet.) Through the use of divergent laser beams according to the invention, the entire particle, the entire cell or the entire droplet is illuminated, resulting in a scattered signal of the entire Particles, the entire cell or the entire drop according to the invention with a plurality of collection fibers can detect. (This makes it possible, for example, for biological cells to disregard lateral fluctuations in the concentration of the cytoplasm and its cell-specific distribution, which makes cell identification much more accurate.) The plurality of collection fibers positioned around the excitation volume is for the arrangement of the invention over the prior art no microscope / lens required, which greatly reduces the cost of the arrangement.

As a result of the use according to the invention of a large number of collection fibers, the solid angle for the scattered light to be recorded is also increased compared with the prior art, so that an increase of the collected scattering signal takes place, so that the signal-to-noise ratio is decisively improved.

A further advantage is that the laser trap and the Raman spectroscopic measurements can be generated by a laser suitable for Raman spectroscopy (for example with a wavelength of 758 nm or 633 nm), whereby the absorption by the individual sample compared to the state of the art Technology (capture laser with 1070 nm) is reduced.

The invention will be explained in more detail below with reference to the embodiment and the figure. Showing:

1 : A schematic representation of an inventive arrangement for performing a single sample analysis and manipulation for Raman microspectroscopy and

2 : a schematic detail of a substrate according to 1 ,

In the 1 and partly in clipping in 2 The inventive arrangement for carrying out single-sample analysis and manipulation for Raman microspectroscopy comprises a planar substrate ( 1 ), for example a chip in which a channel ( 11 ) for the transport of samples and channels ( 12 ), in which optical fibers ( 4 ; 6 ) are introduced, wherein the channel ( 11 ) an inlet ( 111 ), which with funds ( 81 ) is connected to the sample supply, and an outlet ( 112 ), which with funds ( 9 ) is connected to the sample discharge, and in the channel ( 11 ) between the inlet ( 111 ) and the outlet ( 112 ) a laser trap ( 5 ) into which the fibers ( 4 ; 6 ) are optically coupled.

The optical fibers ( 4 ) are at least two excitation and capture fibers ( 41 ) and the optical fibers ( 6 ) are a plurality of collection fibers ( 61 ), which are centered on the laser trap ( 5 ), with an excitation and trapping fiber ( 41 ) is formed of a single fiber. About the excitation and trapping fibers ( 41 ) is simultaneously excitation and capture laser light in the laser trap ( 5 ) einkoppelbar.

About the collection fibers ( 61 ) is from the laser trap ( 5 ) Scattered light can be coupled out.

By the means ( 81 ) in the form of a device for separating particles, biological cells or liquid drops, for example a microfluidic focusing, individual samples can be produced and by means of a generator ( 8th ) as individual samples through the channel ( 11 ) transportable.

By the means ( 9 ), for example, a branch of the channel ( 11 ) into a plurality of capillaries, which can open into individual vessels, not shown, the individual samples are sortable.

The collection fibers ( 61 ) flow into a Raman spectrometer ( 7 ) and the excitation and trapping fibers ( 41 ) enter a laser ( 2 ) with beam splitter ( 3 ), whereby by the laser ( 2 ) unfocused laser beams can be generated.

The substrate ( 1 ) is in this example as a fused silica chip and the channel ( 11 ) performed as a capillary.

The channels ( 12 ) are used as guides for the optical fibers ( 4 ; 6 ), wherein two excitation and trapping fibers ( 41 ) to the channel ( 11 ) are each arranged at an angle of 90 °, wherein the excitation and trapping fibers ( 41 ) Laser light with a wavelength of 785 nm or with wavelengths of 785 nm and 1070 nm is coupled.

All features described in the description, the exemplary embodiments and the following claims may be essential to the invention both individually and in any combination with one another.

LIST OF REFERENCE NUMBERS

1

planar substrate

11

Sampling channel

111

inlet

111

outlet

12

Channels for optical fibers

2

laser

3

beamsplitter

4

optical fiber

41

Fang fibers

5

laser trap

6

optical fiber

61

collecting fibers

7

Raman spectrometer

8th

Generator for sample movement

81

Means for sample delivery

9

Means for sample removal

Claims (8)

Arrangement for carrying out single-sample analyzes and manipulations for Raman microspectroscopy comprising a planar substrate ( 1 ), in which a channel ( 11 ) for the transport of samples and channels ( 12 ), in which optical fibers ( 4 ; 6 ) are introduced, wherein the channel ( 11 ) an inlet ( 111 ), which by first means ( 81 ) is connected to the sample supply, and an outlet ( 112 ), which with second means ( 9 ) is connected to the sample discharge and by the first means ( 81 ) Individual samples can be generated and by the second means ( 9 ) the individual samples are sortable, and around the channel ( 11 ) between the inlet ( 111 ) and the outlet ( 112 ) a laser trap ( 5 ) into which the fibers ( 4 ; 6 ) are optically coupled, wherein the optical fibers ( 4 ) at least two excitation and trapping fibers ( 41 ), the excitation and trapping fibers ( 41 ) into a laser ( 2 ) with beam splitter ( 3 ), so that through the laser ( 2 ) are generated unfocussed laser beams, characterized in that the optical fibers ( 6 ) a plurality of collection fibers ( 61 ) which are centered on the laser trap ( 5 ) and the excitation and trapping fiber ( 41 ) is formed of a single fiber, so that via the excitation and trapping fibers ( 41 ) simultaneously excitation and capture laser light in the laser trap ( 5 ) can be coupled, whereby over the collecting fibers ( 61 ) from the laser trap ( 5 ) Scattered light can be coupled out, and the collecting fibers ( 61 ) into a Raman spectrometer ( 7 ). Arrangement for carrying out single-sample analyzes and manipulations for Raman microspectroscopy according to claim 1, characterized in that substrate ( 1 ) as a fused silica chip and the channel ( 11 ) is designed as a capillary. Arrangement for carrying out single-sample analyzes and manipulations for Raman microspectroscopy according to claim 1, characterized in that the channels ( 12 ) as guides for the optical fibers ( 4 ; 6 ) are executed. Arrangement for carrying out single-sample analyzes and manipulations for Raman microspectroscopy according to claim 1, characterized in that two excitation and trapping fibers ( 41 ) to the channel ( 11 ) are each arranged at an angle of 90 °. Arrangement for carrying out single sample analyzes and manipulations for Raman microspectroscopy according to claim 1, characterized in that the first means ( 81 ) Devices for separating particles, biological cells or drops of liquid, by means of a generator ( 8th ) the samples are transportable. Arrangement for carrying out single sample analyzes and manipulations for Raman microspectroscopy according to claim 1, characterized in that the first means ( 9 ) are formed by branched capillaries, which open into individual vessels. Method for operating an arrangement according to one or more of the preceding claims, in which the excitation and trapping fibers ( 41 ) Laser light with a wavelength of 785 nm or with wavelengths of 785 nm and 1070 nm is coupled. Method for operating an arrangement according to one or more of the preceding claims, in which the movement of the samples by a generator ( 8th ) is produced.

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