WO2022129040A1

WO2022129040A1 - Device and method for examining deoxyribonucleic acid fragments

Info

Publication number: WO2022129040A1
Application number: PCT/EP2021/085680
Authority: WO
Inventors: Michael Kempe; Ralf Wolleschensky
Original assignee: Carl Zeiss Ag; Carl Zeiss Microscopy Gmbh
Priority date: 2020-12-18
Filing date: 2021-12-14
Publication date: 2022-06-23
Also published as: DE102020134261A1

Abstract

Devices and methods for examining DNA fragments are disclosed. In this case, DNA fragments (12) are arranged on a plasmonic chip (41) which is illuminated from a side facing away from the DNA fragments (12) and are scanned using slot-shaped apertures (42, 42A, 42B, 42D) that extend perpendicular to a preferred direction (44) of the DNA fragments (12), wherein the slot-shaped apertures (42, 42A, 42B, 42D) have a width of less than 100 nm and a length that is at least twice the width, and the slot-shaped apertures are aligned with the direction of their length substantially perpendicular to a preferred direction of the DNA fragments (12).

Description

description

Device and method for examining deoxyribonucleic acid fragments

The present application relates to devices and methods for examining deoxyribonucleic acid (DNA) fragments.

Determining genetic information encoded in DNA is required for various applications. There are numerous different technologies for this, for example for reading out an entire genetic code (ie the sequence of so-called base pairs in the DNA) of an organism or a part of it. For these technologies, the DNA must be extracted from cells of the respective organism and cut into smaller sections, so-called fragments. These fragments are then examined. One application of such studies is the identification of organisms, such as viruses or bacteria, based on their genetic code.

A method used to identify genes or organisms is what is known as optical mapping. The extracted DNA fragments are labeled at a specific sequence of bases, which is typically four to seven base pairs long, with one or more different fluorescent dyes (dye molecules 13) that bind selectively only to this sequence of bases. In one form of optical mapping, the DNA fragments are then applied to a substrate and fixed on the substrate. The orientation of the fragments on the substrate is essentially in one spatial direction, referred to as the preferred direction in the context of this application. This is illustrated in FIG. A substrate 10 with DNA fragments 12 fixed thereon is shown here. A section 11 from the substrate is shown enlarged. In this enlargement in particular, it can be seen that the DNA fragments 12 are essentially aligned in the preferred direction, in FIG. 1 in the vertical direction in the image. Substantially aligned can mean alignment in an angular range of less than ±20° or at least ±10° for at least 80%, for example at least 90% of the DNA fragments. As indicated by an arrow 14, the DNA fragments typically have a length in the range of 10 μm and are marked with the dye molecules 13 mentioned.

The sequence of the dye molecules 13 is then determined by optical imaging. The sequence of dye molecules 13, also known as the "barcode" of the DNA fragment is typical for a specific gene or gene segment. As shown in FIG. 1, the barcode of this DNA fragment can be determined, for example, from the distance between the dye molecules 13, which depends on the distances at which the respective specific binding sequence occurs in the DNA fragment. To identify an organism or the large number of organisms contained in a complex sample, such as a smear, or the traces of organisms contained therein, it is necessary to analyze a large number of DNA fragments, for example up to several 100,000 fragments depending on the length eg 10 correspond to ¹⁰ base pairs. For commercial applications in particular, it is therefore necessary to enable the detection of the dye molecules and thus the reading of this "barcode" with the highest possible throughput.

To detect the dye molecules, they are stimulated to fluoresce by illumination, and the position of the dye molecules is determined using optical imaging. The resolution required for this depends on the mean distance between the dye molecules and can be well below this mean distance, depending on the application. For example, if the dye molecules are sensitive to a specific sequence of four base pairs, the mean distance in a statistical distribution of the possible base pairs is 4 ⁴ = 256 base pairs, which corresponds to about 256 x 0.34 nm = 87 nm. A stretching of the DNA fragments can be associated with the fixation on the substrate 10, as a result of which this distance can be greater by a factor of about 1.5, for example. In any case, this distance in the visible wavelength range, in which at least some of the fluorescent dyes used emit, cannot be resolved with conventional microscopy due to the diffraction limit.

Known methods for microscopy beyond the diffraction limit are, for example, microscopy with structured illumination (SIM, Structured Illumination Microscopy), microscopy with stimulated emission depletion (STED, Stimulated Emission Depletion) or localization microscopy (PALM, Photoactivated Localization Microscopy). These methods all have the disadvantage that they do not allow rapid imaging in a large field of view, which is required for high throughput. Therefore, analysis of a substrate 10 having DNA fragments 12 arranged thereon as shown in Fig. 1 by such methods requires a very long time.

WO 2019/122161 A1 discloses a method and a device for super-resolution microscopy beyond the diffraction limit with a plasmonic chip, the chip being an individually addressable array of nano-apertures that illuminates an object located on the chip, allows in the near field. In this case, nano-apertures are surface sections on the array whose state can be controlled in a specifically addressable manner with regard to the excitation of surface plasmons. The nano-apertures on the lithographically produced plasmonic chip can reach a size of 25 nm or smaller. A corresponding device 20 is shown schematically in FIG.

In the device 20 of FIG. 2 , a sample 23 to be examined is provided on a substrate 24 . The thickness of the substrate 24 can be in the range of 25 nm. The substrate contains a plasmonic chip 29 with a multiplicity of individually controllable nano-apertures, closed nano-apertures (surface plasmons cannot be excited by incident light) being denoted by reference numeral 27 and open nano-apertures (surface plasmons can be excited by incident light) being identified by reference numeral 26 . The plasmonic chip 29 rests on a glass substrate 210.

For the measurement, illumination is carried out by means of a light source from the side of the glass substrate 210, as indicated by arrows 28. The nano-apertures 26, 27 can have a diameter of the order of 25 nm, for example. The light wavelength of the illumination is higher and can be in the visible range, for example.

The functionality of the nanoscreens 26, 27 is based on the fact that the excitation of surface plasmons by the incident light in the plasmonic chip 29 is sensitively dependent on the physical properties of the environment, e.g. the temperature, which can be set by appropriate control electronics. Thus, the nano shutters can be controlled from closed to open.

The open nano-apertures 26 allow the illumination light to reach the object 23 to be examined in accordance with the arrows 28 . However, due to the size of the nano-apertures being smaller than the wavelength of the light, a light field 25 that is created for illuminating the object 23 is merely an evanescent light field that propagates only a few 100 nm into the sample 23 and has a lateral extension that is essentially determined by the size of the nano-apertures 26 , 27 is determined. This achieves a localized excitation with a spatial resolution of the order of 25 nm. Fluorescence excited by the light field 25 is then detected by means of a detection device such as a detection microscope, of which a detection objective 21 is shown schematically, and can be determined with diffraction limitation. The high spatial resolution does not result here a high-resolution detection - this is diffraction-limited but by a corresponding spatially resolved excitation. In some applications, an immersion liquid 22 , for example water, which surrounds the sample 23 , is located between the detection objective 21 and the substrate 24 .

An entire image field, for example the substrate 10 of FIG. 1, can then be scanned by switching the nano-apertures 26, 27 sequentially. Because of the small size of these nano-apertures, however, a relatively long time is required here.

A parallelization of this scanning is possible in that for each chip area of the size of a diffraction disc of the detection optics used (detection lens 21) a nano diaphragm 26, 27 is opened and each object field of the size of the diffraction switching is scanned, but this for all such object fields in parallel happens. The diffraction-limited imaging of a pixel of the detector into the object plane by means of lens 21 is to be understood as the “diffraction disc of the detection optics used”. This is the area within which no separation of different objects can be detected due to diffraction limitation. This is visualized in FIG. A large number of diffraction discs 30 are shown here. The minimum diameter d of each diffraction disc is approximately given by d=λ/NA, where λ is the wavelength to be detected and NA is the numerical aperture of the objective (in the example of FIG. 2 objective 21). The number N of images required to scan a single diffraction disc with a nanoaperture diameter b is then given by

For a lens with NA=1.2, a wavelength of 550 nm and a nanoaperture diameter b of 25 nm, approximately 340 exposures per diffraction disc are accordingly required. Since the diffraction disks can be processed in parallel, a total of 340 exposures are necessary to scan an entire image field.

Even if this means a significant time saving compared to scanning an entire image field, it is nevertheless desirable to further reduce this time.

One possibility for further parallelization is the switching of the nano-apertures, which can take place at high frequencies in the kilohertz range, for parallel heterodyne detection to use. This means that different apertures that are opened close to each other (within a diffraction disc) are modulated with different frequencies, so that the corresponding fluorescent light, which is excited by the evanescent light field, is also coded with a corresponding frequency and can therefore be specifically detected . However, such a detection generally requires a special camera with direct demodulation, for example a so-called time-of-flight camera, which usually only has a small number of pixels, which in turn limits the resolution. In addition, the dynamic range and the signal-to-noise ratio (SNR) of the detection decrease sharply. With such an approach, within a diffraction disk, the following applies to N contributions with different frequencies at 100% modulation depth

where S is the measured signal and v _n are the frequencies used. This means that any signal component with an amplitude of 2 arbitrary units has to be filtered out of a background with amplitude 2N arbitrary units, which reduces the dynamic range of detection and the SNR substantially.

It is therefore an object of the present invention to provide options for keeping the measurement time for the optical measurement of fluorescence-labeled DNA fragments as short as possible with relatively little effort.

This object is achieved by a device according to claim 1 and a method according to claim 11. The subclaims define further embodiments.

According to a first aspect, a device for examining deoxyribonucleic acid, DNA fragments is provided, comprising: a plasmonic chip on which DNA fragments can be arranged, are an illumination device for illuminating the plasmonic chip from one of the side facing away from the DNA fragments, a Detection device for detecting fluorescence from the DNA fragments in response to illumination by the illumination device, and a controller which is set up to control the plasmonic chip for scanning the DNA fragments with slit-shaped apertures, the slit-shaped apertures having a width of less than 100 nm and a length that is at least that Is twice the width, and are aligned in the direction of their length substantially perpendicular to a preferred direction of the DNA fragments.

A plasmonic chip is a chip in which nano-apertures can be opened and closed as described in the introduction to the description, these nano-apertures forming slit-shaped aperture openings, in contrast to the prior art. The fact that DNA fragments can be arranged on this plasmonic chip means in particular that the DNA fragments can be brought into contact with the plasmonic chip or at least can be brought so close to the plasmonic chip that they are in an evanescent field that is at Illumination forms at the slit-shaped apertures. The DNA fragments can be arranged on a substrate that is different from the plasmonic chip.

By using such slit-shaped diaphragm openings, faster scanning and thus faster examination of a sample is possible. This exploits the fact that the fragments are present with a known orientation and only the resolution along the main direction of the fragment has to be high, in particular beyond the diffraction limit. Substantially perpendicular here can mean in a range from 70° to 110°, for example from 80° to 100°. The preferred direction of the DNA fragments is the direction in which the strands of the DNA fragments are mainly or on average arranged.

Slit-shaped apertures can be possible by interconnecting several nano-apertures as explained above, or the plasmonic chip can be set up to provide slit-shaped apertures. The use of individual nano-apertures, which are switched together to form the slit-shaped aperture, allows greater flexibility than fixed slit-shaped apertures, but is sometimes more complex in terms of control.

The controller can be set up to carry out the scanning for a number of areas in parallel. The areas can have the size of diffraction disks of the detection device. In this way, the throughput can be increased.

The length of the slit-shaped diaphragm openings can extend over several such areas. Alternatively, the length of the slit-shaped diaphragm openings can correspond to a diameter of the areas ±20% or ±10%, ie approximately the diameter of the areas. In the latter case, simultaneously opened slit-shaped diaphragm openings of adjacent areas can be offset from one another in the direction of the width of the slits, which can make it easier to separate signals from different areas.

The controller can be set up to carry out the scanning sequentially, at least within the ranges when they are used, i.e. in each range only one slit-shaped aperture is then open at a time.

Alternatively, the controller can be set up to open and close a plurality of slit-shaped diaphragm openings in parallel with different frequencies for scanning, ie to carry out a heterodyne detection method. Due to the use of the slit-shaped aperture openings, significantly fewer frequency components are required here than with conventional methods, which makes detection easier.

More specifically, only N'=^N frames (in the case of sequential scanning) or frequencies (in the case of parallel heterodyne detection) are required due to the slit-shaped aperture openings, instead of N with conventional approaches. This means a shorter recording time in the case of sequential scanning or lower noise and higher detection dynamics in the case of heterodyne detection.

The device can be set up to record an overview image before scanning and to determine at least one of the following on the basis of the overview image:

- the preferred direction,

- Areas where there is a fluorescent signal from DNA fragments, and

- a layer of individual DNA fragments.

This can help to optimize the subsequent scanning with the slit-shaped apertures. The term "territory" is used here to avoid confusion with the areas mentioned above.

The controller can be set up to carry out the scanning with the slit-shaped diaphragm openings only in the areas in which fluorescence is present. In this way, the evaluation can be facilitated.

The controller can be set up to adapt an alignment of the slit-shaped apertures to the position of the respective DNA fragments. So here the general Alignment are essentially adjusted perpendicular to the preferred direction of the actually detected position. The position can be determined by determining the center of gravity.

According to a second aspect there is provided a method for examining DNA fragments, comprising:

Arranging DNA fragments on a plasmonic chip,

illuminating the plasmonic chip from a side facing away from the DNA fragments, and driving the plasmonic chip to slit the DNA fragments

to scan apertures, with a width of the slit-shaped apertures being less than 100 nm and a length of the slit-shaped apertures being at least twice the width and extending essentially perpendicularly to a preferred direction of the DNA fragments, and

detecting fluorescence from the DNA fragments.

The explanations given for the device apply accordingly to the arrangement on the plasmonic chip.

Variations and additional features are possible for the method corresponding to the variations and additional features explained above for the device.

The invention is explained in more detail below with reference to the accompanying drawings using exemplary embodiments. Show it:

1 shows a sample preparation of DNA fragments according to the prior art,

2 shows a device for super-resolution microscopy according to the prior art,

3 shows a diagram to illustrate the parallelization of the device of FIG. 2 according to the prior art,

4A shows a device for analyzing DNA fragments according to an embodiment,

4B shows a device for examining DNA fragments according to a further exemplary embodiment, 5A and 5B diagrams to illustrate different embodiments,

6 shows a flow chart to illustrate a method according to an embodiment,

Fig. 7 is a diagram to illustrate some embodiments, and

8 shows a diagram to illustrate some exemplary embodiments.

Various exemplary embodiments are explained in detail below. These embodiments are provided by way of example only and are not to be construed as limiting.

In contrast to conventional methods, slit-shaped apertures are used in exemplary embodiments. Apart from the use of slit-shaped diaphragm openings and the possibilities and techniques associated therewith, the devices and methods used can be configured in accordance with conventional devices and methods, for example devices and methods as explained in WO 2019/122161 A1. Therefore, these conventional parts will not be described in detail.

Features (elements, components, method steps and the like) of different exemplary embodiments can be combined unless otherwise stated. Variations and modifications that are described for one of the exemplary embodiments are also applicable to other exemplary embodiments and are therefore not described repeatedly.

Identical or corresponding elements in different figures are denoted by the same reference symbols and are therefore not repeatedly explained in detail.

4A shows a device 40A for examining DNA fragments 12 according to an embodiment. The device 40A of FIG. 4A is based on the device for super-resolution microscopy described in the introduction to the description with reference to FIG. 2, and the same elements bear the same reference symbols and are not explained in detail again. Instead, mainly the differences to the device 20 of FIG. 2 are presented below. In the device 40A of FIG. 4A, in contrast to the general sample 23 of the device 20 of FIG. For the optical examination of the DNA fragments 12 , slit-shaped apertures 42 are opened in a plasmonic chip 41 controlled by a controller 45 , which extend perpendicularly to the preferred direction 44 . Other apertures 27 remain closed. The slit-shaped diaphragm openings 42 can be opened in that a plurality of nano-diaphragms of the device 20 lying in a row are controlled by the controller 45 to open them. In other exemplary embodiments, the plasmonic chip 41 is set up from the outset in such a way that the nano-apertures are slit-shaped. A slit shape is understood to mean a shape that is at least twice as long, for example at least three times as long as a width in the transverse direction in a longitudinal direction, the transverse direction being visible in the cross-sectional view of FIG. 4A. The width of the slit-shaped aperture can be well below the diffraction limit, for example less than 100 nm or less than 40 nm, for example about 25 nm A/NA, where A is the wavelength to be detected and NA is the numerical aperture. For example, for a wavelength of 550 nm and a numerical aperture NA of 1.2 as used as an example above, it can be of the order of 450 nm. In the order of magnitude of the diameter of the diffraction disk, the diameter of the diffraction disk can mean ±20% or ±10%, although a greater length is also possible. In other embodiments, the length in the longitudinal direction can also extend over a large number of diffraction discs or even over the entire field of view.

Examples of this are shown in Figures 5A and 5B. 5A and 5B each show a DNA fragment 12 with a multiplicity of fluorescent molecules 13. As in FIG. 3, diffraction disks are designated 30, in which a detection takes place in parallel.

In FIG. 5A, slit-shaped diaphragm openings 42A are used, which extend perpendicularly to the preferred direction 44 over many diffraction disks, for example over the entire image field. In the case of FIG. 5B, slit-shaped diaphragm openings 42B are used, the extent of which in the longitudinal direction is in the order of magnitude of the diameter of a diffraction disk and which, as shown, are offset for adjacent rows of diffraction disks in which separate detection is carried out are. The staggered arrangement makes it easier to separate signals from different diffraction disks.

In some exemplary embodiments, as will be explained in more detail later, the controller 45 can also obtain an overview image, as indicated by an arrow 43, which was created using the image recording device with the detection lens 21, and from this the preferred direction 44 can be determined. In the case of individual nano-apertures, in which rows are opened to form slit-shaped apertures, the longitudinal direction of the slit-shaped apertures can then be adjusted accordingly. In addition, the controller 45 can also receive the image recordings obtained during the actual measurements and evaluate them accordingly, for example in order to determine the exact position and assignment of individual detection events. In this case, the controller is also used for evaluation. However, a separate evaluation device, for example a separate computer, can also be provided, which receives the measurements (image recordings). Such a separate evaluation device can also be arranged at a distance from the devices shown and receive the image recordings, for example via a network connection.

Before additional details of the detection of fluorescence by means of slit-shaped diaphragm openings in the case of examining DNA fragments are discussed in more detail, a variant of the embodiment of FIG. 4A is explained with reference to FIG. 4B. For the most part, device 40B of Figure 4B corresponds to device 40A of Figure 4A. Only the illumination takes place instead of the illumination 28 of FIG. 4A as oblique illumination 28' in total reflection. This means that the illumination light is totally reflected at an interface between the plasmonic chip and the glass substrate 210, whereby surface plasmons can be effectively excited at open slit-shaped apertures, as a result of which an evanescent light field 25 extends into the DNA fragments 12. Otherwise, the activation takes place as slit-shaped diaphragm openings 42 as described for FIG. 4A or FIGS. 5A and 5B.

The effects of slit apertures such as slit apertures 42, 42A and 42B of Figures 4A, 4B, 5A and 5B will now be discussed.

First of all, it should be noted that the use of slit-shaped diaphragm openings with a corresponding extension in the longitudinal direction can also result in a propagating light field in addition to the evanescent light field. For an object that has a thickness of more than a few microns, resolution beyond the diffraction limit would be lost. However, in the present application to DNA fragments, this does not matter because DNA fragments are only a few nanometers thick, so the propagating field has no effect on imaging.

When using the slit-shaped apertures on DNA fragments, use is also made of the fact that during sample preparation, as explained in the introduction to the description with reference to FIG. 1, the distance between the individual DNA fragments perpendicular to the preferred direction is relatively large and with a high probability lies at the diffraction limit. This means that there is at least a very high probability that only one DNA fragment runs through a diffraction disk 30 , ie a detection area, and it is very unlikely that two DNA fragments will run through the same diffraction disk 30 . A high resolution is therefore only necessary in the preferred direction 44, since individual dye molecules can also be very close together here, depending on the arrangement of the bases on the DNA fragment, and this must be resolved in order to be able to analyze the fragments accordingly.

Due to the slit-shaped diaphragm openings, the diffraction disks only have to be scanned in one direction. As a result, the number of N image recordings used in the introduction to the description is reduced to N ->/N. The same also applies to the heterodyne methods mentioned, where instead of N frequencies, only N'=^N different frequencies are necessary. In order to implement a simultaneous, demodulated detection, N' frequencies are sufficient, which can be implemented in practice with typical image recording times. For example, for a typical fluorescent label on a four base pair sequence, an aperture size (width of the slit-shaped apertures in the transverse direction) of 80 nm can be used, giving with the other parameters N' = 550/(1.2 x 80) ~ 6 . With a frame rate of 50 images per second, a complete, high-resolution image of a sample is obtained in around 120 ms. It is preferred to scan the diffraction disks sequentially, in that the diaphragm with slit-shaped diaphragm openings scans the diffraction disks sequentially in each case.

Additional possible features and modifications will now be described with reference to Figures 6-8.

FIG. 6 shows a flowchart of a method according to an embodiment, and FIGS. 7 and 8 show diagrams for explaining the method of FIG. In step 60 of FIG. 6, an overview image of the sample is recorded, ie an overview image of the substrate 10 with the DNA fragments arranged thereon. This overview image can be recorded by means of the detection device represented by the detection objective 21, in that all nano-apertures of the plasmonic chip 41 are opened and thus complete illumination takes place. In other exemplary embodiments, incident light illumination can also take place, for example through the detection objective 21 . The overview image can be a fluorescence image in which the fluorescence molecules are excited to fluoresce, or it can be a bright field or phase contrast image, for example. In step 61, the preferred direction 44 is then determined from the overview image. Additionally or alternatively, the relevant areas can also be determined, ie those diffraction discs 30 within which fluorescence occurs or can occur (because fragments are located there). Even if, in the case of the overview image, this is not yet recorded with the resolution required for the final analysis, it can still be recognized on the basis of an overview image in which diffraction discs relevant areas are located at all.

In step 62, the relevant areas are then scanned with slit-shaped apertures. This is shown in FIG. 7, where slit-shaped apertures scan only those diffraction discs 30 in which it was determined by means of the overview image in step 61 that DNA fragments are present. As previously discussed, the scanning can be performed sequentially within each diffraction disk, or all slits in the diffraction disk can be driven in parallel to open and close at different frequencies to heterodyne signals from different slits to discriminate.

Restricting the scanning to those diffraction disks 30 in which there is actually a signal can be advantageous for the unambiguity and reliability of the evaluation, since signals from different DNA fragments can be separated even better in this way.

In FIG. 7, all of the slit-shaped diaphragm openings are aligned perpendicularly to the preferred direction 44. In other exemplary embodiments, the individual position of the individual fragments can also be estimated at 61 . For this purpose, the position of the individual DNA fragments can be determined from a fluorescence overview image by determining the center of gravity of the diffraction-limited signals over several diffraction disks. The diffraction-limited fluorescence signals present in the overview image are then fitted, for example, with a line 80 in FIG. 8 in order to approximate the position of the corresponding DNA fragment 12 . Even without determining the center of gravity, the orientation of the individual fragments can be approximated from the position of the diffraction-limited recorded signals using several diffraction disks. Determining the center of gravity allows the orientation of the fragment to be determined beyond the diffraction limit, since two fluorescent molecules perpendicular to the preferred direction cannot contribute to the signal at the same time. In other exemplary embodiments, this improved determination of position by means of determination of the center of gravity is not implemented, since as a rule the accuracy is sufficient for the method due to a "string of pearls" of diffraction discs per fragment in which fluorescence occurs.

If the position of the individual fragments is known, the alignment of the slit-shaped aperture openings can be adapted to the respective fragment 12 by correspondingly controlling adjacent nano-apertures in the plasmonic chip, so that it runs perpendicular to the direction of the fragment. This is shown in Figure 8 for slots 42D for two different DNA fragments 12. In this way, the accuracy can be further increased in some exemplary embodiments. However, it should be noted that an alignment perpendicular to the general preferred direction 44 as shown in FIG. 7 can already result in high accuracy with a corresponding throughput.

Even if the preferred direction of the DNA fragments is determined by an overview image in the method in FIG. 6, this is not necessary in other exemplary embodiments. For example, the preferred direction 44 can also be known from the sample preparation of the substrate 10 shown in FIG. 1, ie it can be defined by the type of sample preparation. In this case, for example, the substrate 10 can simply be inserted into the device 40A or 40B in a corresponding direction.

The lighting 28 or 28', which is used to carry out the method, has a lighting power. The required lighting power depends not only on the parameters of the dye but also on the size of the image field. The image field size should be as large as possible for a high throughput, but depends not only on the image field of the lens 21 but also on the size of the image sensor (camera chip) and the pixel size on the chip. For typical, high-quality cameras with 2000 x 2000 pixels and a diffraction disk 30 with a Diameter 550 nm/1.2-450 nm results in an image field of (0.45 μm×2000) ² =(920 μm) ² . Again, a wavelength of 550 nm and a numerical aperture of 1.2 are assumed. With a lens magnification of 20x, this requires an intermediate image size of 18mm and a total magnification of approx. 6pm/0.45pm = 13x with typical 6pm camera pixels. To have a signal to noise ratio of at least 10 in each

To reach diffraction disks, one needs about 100 emitted photons during a respective measuring time. With 50 images (frames) per second, a measurement time of approx. 10 ms can be assumed, which means 10 ⁴ emitted photons/s. Neglecting enhancement effects, the incident light intensity required for this for typical fluorescent molecules is approximately 10 ⁴ ×10′ ¹⁹ W/(10′ ¹⁶ cm ² ), ie approximately 10 W/cm ² . With a chip area of 1 mm ² this means a required light output of around 100 mW. This can easily be achieved with appropriate light sources such as cw (continuous wave) lasers. As can be seen from the explanations above, various variations are possible. Therefore, the illustrated embodiments are not to be construed as limiting.

Claims

patent claims

1. Device (40A, 40B) for examining deoxyribonucleic acid, DNA, fragments

(12), comprising: a plasmonic chip (41) on which DNA fragments (12) can be arranged, an illumination device (28, 28') for illuminating the plasmonic chip (41) from one of the DNA fragments (12) side facing away, a detection device (21) for detecting fluorescence from the DNA fragments (12) in response to illumination by the illumination device (28, 28'), and a controller (45) which is set up to control the plasmonic chip ( 41) to scan the DNA fragments (12) with slit-shaped apertures (42, 42A, 42B, 42D), wherein the slit-shaped apertures (42, 42A, 42B, 42D) have a width of less than 100 nm and a length that is at least Is twice the width, and are aligned in the direction of their length substantially perpendicular to a preferred direction of the DNA fragments (12).

2. Device (40A, 40B) according to claim 1, wherein the controller (45) is set up that

To carry out scanning for several areas (30) in parallel.

3. Device (40A, 40B) according to claim 1, wherein the areas (30) the size of

Have diffraction discs of the detection device (21).

4. Device (40A, 40B) according to claim 2 or 3, wherein the length of the slit-shaped

Aperture openings (42, 42A, 42B, 42D) extends over several areas (30).

5. Device (40A, 40B) according to claim 2 or 3, wherein the length of the slit-shaped

Aperture openings (42, 42A, 42B, 42D) correspond to a diameter of the areas (30) ± 20%.

6. Device (40A, 40B) according to one of claims 1 to 5, wherein the controller (45) is set up to carry out the scanning sequentially.

7. The device (40A, 40B) according to any one of claims 1 to 5, wherein the controller (45) is set up to open and close a plurality of slit-shaped apertures (42, 42A, 42B, 42D) in parallel with different frequencies for scanning.

8. The device (40A, 40B) according to any one of claims 1 to 7, wherein the device (40A, 40B) is set up to record an overview image before the scanning and to determine at least one of the following on the basis of the overview image:

- the preferred direction (44),

- Areas where there is a fluorescent signal from DNA fragments (12), and

- a layer of individual DNA fragments (12).

9. Device (40A, 40B) according to claim 8, wherein the controller (45) is set up that

Perform scanning with the slit-shaped apertures (42, 42A, 42B, 42D) only in the areas where fluorescence is present.

10. Device (40A, 40B) according to claim 8 or 9, wherein the controller (45) is set up to adjust an alignment of the slit-shaped apertures (42, 42A, 42B, 42D) to the position of the respective DNA fragments (12).

11. A method for examining DNA fragments (12), comprising:

Arranging DNA fragments (12) on a plasmonic chip (41),

illuminating the plasmonic chip (41) from a side facing away from the DNA fragments (12), and

Driving the plasmonic chip (41) to scan the DNA fragments (12) with slit-shaped apertures (42, 42A, 42B, 42D), a width of the slit-shaped apertures (42, 42A, 42B, 42D) being less than 100 nm and a length of the slit-shaped apertures (42, 42A, 42B, 42D) is at least twice the width and extends essentially perpendicular to a preferred direction (44) of the DNA fragments (12), and

detecting fluorescence from the DNA fragments (12).

12. The method (40A, 40B) according to claim 11, wherein the scanning for a plurality of regions (30) is performed in parallel.

13. The method (40A, 40B) according to claim 11, wherein the areas (30) the size of

Having diffraction disks of a detection device (21) used for detection.

14. The method (40A, 40B) according to claim 12 or 13, wherein a length of the slit-shaped apertures (42, 42A, 42B, 42D) extends over a plurality of regions (30).

15. The method (40A, 40B) according to claim 12 or 13, wherein the length of the slit-shaped

16. The method (40A, 40B) according to any one of claims 11 to 15, wherein the scanning is sequential.

17. The method (40A, 40B) according to any one of claims 11 to 15, wherein a plurality of slit-shaped apertures (42, 42A, 42B, 42D) are opened and closed in parallel with different frequencies for scanning.

A method (40A, 40B) according to any one of claims 11 to 17, further comprising:

Acquiring an overview image before scanning , and

Determine at least one of the following based on the overview image:

- the preferred direction (44),

- Areas where there is a fluorescent signal from DNA fragments (12), and

- a layer of individual DNA fragments (12).

19. The method (40A, 40B) according to claim 18, wherein the scanning with the slit-shaped

apertures (42, 42A, 42B, 42D) only in the areas where fluorescence is present.

The method (40A, 40B) of claim 18 or 19, further comprising:

Adapting an alignment of the slit-shaped apertures (42, 42A, 42B, 42D) to the position of the respective DNA fragments (12).

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