Biological tissue analysis apparatus and method

Abstract

A biological tissue analysis device for analyzing biological tissue by laser-induced breakdown spectroscopy comprises a laser generator (1, 2), a spectrometer module (7, 8, 12) and a control unit (5, 6). The laser generator (1, 2) is configured to provide a laser beam pulse to a biological target tissue. The control units (5, 6) are connected to the laser generators (1, 2) and the spectrometer modules (7, 8, 12). The control unit (5, 6) is configured to operate the laser generator (1, 2) to provide at least one laser beam pulse as an analysis pulse that generates an analysis plasma at a laser pulse time, the analysis plasma comprising a reference element in at least a first excited state and a second excited state. The spectrometer module (7, 8, 12) is configured to collect emitted light of the analysis plasma at an acquisition time and to analyze emitted lines in the collected emitted light. The control unit (5, 6) is configured to maintain an emission line relationship between a first emission line of the reference element of the analysis plasma at a first wavelength associated with the first excited state and a second emission line of the reference element of the analysis plasma at a second wavelength associated with the second excited state within a predefined reference emission line range.

Description

Technical Field

[0001] The present invention relates to a biological tissue analysis device according to the preamble of independent claim 1 and, more particularly, to a method for detecting a specific type of cells or analyzing the content of a specific type of cells in a biological target tissue.

[0002] This biological tissue analysis device can be used to automatically analyze biological tissue by laser induced breakdown spectroscopy. This device may include a laser generator, which is configured to provide a laser beam pulse to a target tissue; a spectrometer module; and a control unit connected to the laser generator and the spectrometer module. The control unit can be configured to operate the laser generator to provide at least one laser beam pulse as an analysis pulse, which generates an analysis plasma within the laser pulse time, and the analysis plasma includes a reference element in at least a first excited state and a second excited state. The spectrometer module can be configured to collect emission light of the analysis plasma at a collection time and analyze emission lines in the collected emission light. Background Art

[0003] Laser induced breakdown spectroscopy (LIBS) is a well-established technique that is often used in metal processing or geology. In LIBS, short laser pulses (femtoseconds to nanoseconds) are delivered to the surface of a probe, which generates a hot plasma for a fairly short time. During this time, molecules break down into their atoms and become excited by electrons. Subsequently, the excited atoms drop to their ground state and emit emission light characteristic of their atoms. The emission light of a particular sample is collected and analyzed by a spectrometer and is usually concentrated into emission lines. From these atomically characteristic emission lines, the original atom can be identified and the amount of the corresponding atom relative to other atoms can also be quantified.

[0004] In recent years, attempts have also been made to apply LIBS to the characterization of biological samples, such as plants, leaves, bones, and other biological tissues. However, it has proven to be much more difficult to apply LIBS on biological tissues than on metals and minerals. While metals, alloys, and stones have a high density and are dry and solid, biological materials are much less dense, less uniform, and contain a lot of water. The low density leads to reduced signal intensity because there are fewer molecules and atoms. The water content leads to additional signal loss, as water leads to lower plasma temperatures and ultimately other quenching effects. Therefore, soft and / or wet materials often lead to quality-limited results. In addition, since biological tissues are often also quite inhomogeneous at fairly small scales, the plasma plume generated in LIBS may also be quite inhomogeneous. For example, different water contents may result in different temperatures when the LIBS laser is applied, thus generating plumes with different compositions and conditions over time when analyzing biological tissues.

[0005] Another challenge is that the differences in biological samples are often so small that only advanced statistical methods can observe these differences. For the reasons mentioned above, known LIBS systems are not often used to analyze biological samples. Instead, typical histological preparation of biological tissue obtained by biopsy remains the most common way of analyzing biological tissues in widespread use. However, this procedure is rather tedious and slow. Typically, these systems require up to hundreds of accumulated spectra to overcome the high variability of the signal intensity from biological tissues.

[0006] Since LIBS is advantageous for sample analysis for a variety of reasons, such as speed, accuracy, and variability, there is a need for a LIBS system that allows for efficient analysis of biological tissues. Summary of the invention

[0007] According to the invention, this need is solved by a biological tissue analysis device as defined by the features of independent claim 1, a method for detecting a specific type of cells in a biological target tissue as defined by the features of independent claim 33, and a computer program as defined by the features of independent claim 62. Preferred embodiments are subject matter of the dependent claims.

[0008] In particular, the present invention is a biological tissue analysis device, which is intended to analyze tissue by laser induced breakdown spectroscopy. The device comprises a laser generator, a spectrometer module and a control unit. The laser generator is configured to provide laser beam pulses to the biological target tissue. The control unit is connected to the laser generator and the spectrometer module. This connection particularly relates to a communication connection, through which the connected components can communicate. This connection can be a wired connection or a wireless connection.

[0009] The control unit is configured to operate the laser generator to provide at least one laser beam pulse as an analysis pulse, the analysis pulse generating an analysis plasma at the laser pulse time, the analysis plasma comprising at least a reference element in a first excited state and a second excited state. The spectrometer module is configured to collect emission light of the analysis plasma at the acquisition time and analyze emission lines in the collected emission light.

[0010] The control unit is configured to maintain an emission line relationship between a first emission line of a reference element of the analysis plasma at a first wavelength associated with a first excited state and a second emission line of the reference element of the analysis plasma at a second wavelength associated with a second excited state within a predefined reference emission line range.

[0011] The term "reference element" in connection with the present invention refers to a specific or selected element, which is an atomic species having a given number of protons in its nucleus, including pure substances consisting only of that species. Unlike compounds, elements cannot be decomposed into simpler substances by any chemical reaction. However, elements can have different degrees of ionization, i.e., include different numbers of electrons.

[0012] The reference emission line range may be a predefined trust range within which at least two emission lines of the reference element are targeted. Since the plasma generated by the analysis plume cannot be directly controlled, according to the present invention, the plasma conditions may be analyzed and taken into account. In particular, data may only be collected from plasma plumes that satisfy a given temperature profile, which plasma plumes can be effectively identified by emission lines (e.g., emission lines of Ca).

[0013] As used herein, the term "excited state" is associated with the ionization of a reference element. As mentioned, the same element can have different numbers of electrons, thereby producing different excited states. Such different excited states of the same element (i.e., the reference element) are associated with emission lines of different wavelengths or different sets of emission lines.

[0014] The device can be designed for immediate or real-time analysis. For example, the device can be configured to apply relatively low laser power to prevent undue high temperatures from impeding accurate analysis of biological tissue.

[0015] The device according to the present invention is capable of automatically analyzing tumors and other changes in fresh biopsy tissue or surgical extracts in a short time or in real time. The device mainly analyzes all intact cells within the volume of the laser spot, thereby obtaining the electrolyte composition of the cells themselves. Therefore, in a given tissue (biopsy tissue, tissue obtained by surgery or from a living body), the device can automatically analyze or identify the tissue mainly by its cell content or composition. The results can be visualized by optical overlay, CT images, etc., to make tumor structures visible, for example.

[0016] In particular, by automatically keeping the emission lines of the reference elements within a predefined reference emission line range, it can be achieved that the plasma plume and its characteristics or features are kept within a uniform range so that the results of the spectroscopic analysis can be reliably compared and evaluated.

[0017] Thus, due to the considerable speed of the spectral analysis, an instant characterization of the tissue can be achieved, i.e. allowing the next laser shot or pulse to be readjusted. This allows information about the tissue to be taken into account in real time in therapeutic applications. For example, when removing cancerous tissue, the limits of the cancerous tissue can be identified so that the cancerous tissue can be precisely removed, for example by a surgeon.

[0018] The control unit can be configured to map a given tissue onto a defined area, where the number of laser shots and the position to be analyzed are determined, for example by controlling the xyz sample stage. This ensures that there are enough spectra and data points of sufficient quality at the end. The criteria for such quality can involve plasma temperature being within a given range, the spectrum not being saturated, the spectrum or signal not being below a threshold, and no air shot data.

[0019] Furthermore, biological tissues or samples often show great variability in density and water content. This results in very different LIBS spectra even for the same tissue type. Even a slightly less amount of water can cause a dramatic change in the spectrum. It can also be observed that the plasma temperature may decrease when more water is present. All of this makes LIBS spectra difficult to compare, and therefore the identification of tumor states or even different tissues becomes a challenge. In order to perform a quantitative analysis of a given set of elements, spectral normalization or the ratio of two selected elements becomes an important part of the analysis. In biological samples, for example, the concentration changes of biomarkers in different disease states are usually very small. Therefore, the differences in the LIBS spectra are also small, and the measurements must be performed as accurately as possible without any technical changes.

[0020] However, the device according to the present invention can allow the effective identification of cancerous tissue. More specifically, when healthy cells are transformed into tumor cells, the electrolyte and element system (Mg, Fe, K, Na, Ca, CN, etc.) of the healthy cells will change. This change is measured by indirect methods such as Raman and fluorescence microscopy systems. Many tumor cells show a significant increase in potassium. For example, in mandibular tumor cells, the calcium content decreases, while potassium increases. In addition, other LIBS signals will also change during the transformation from a healthy state to a tumor state, such as CN. This molecule may be produced from organic molecules containing nitrogen-carbon double bonds, such as certain lipids, amino acids or proteins, by incomplete molecular decomposition. It should be noted that the biological tissue analysis device of the present invention can be constructed in a way that inhibits or minimally reduces the formation of CN by reaction with ambient air.

[0021] By means of the present invention, a direct, rapid and independent investigation of tissue probes or biopsies can be achieved to quickly distinguish healthy and diseased tissue areas of a given tissue. Intracellular electrolyte content and some minor intracellular electrolytes can be analyzed.

[0022] For example, detecting cancer cells in bone tissue samples remains a challenge in pathology today. First, the hard bone material must be slowly broken down without destroying the cells, and then the cells themselves can be seen after appropriate staining. This process may take several days to weeks until the patient will know the status of the tumor and perhaps a new operation will be required to remove all remaining tumor cells. Therefore, the surgeon should be able to identify tumor cells quickly so that the operation can be carried out without any interruption. LIBS is able to analyze the electrolyte and elemental composition of tissue samples. LIBS imaging (mapping of selected elements) guides the surgeon to further understand the growth of the tumor. The invention can also distinguish different tissue variations such as necrotic tissue, fibrotic tissue, cancerous tissue and other tissues more or less in real time, which will make a difference in the patient's health.

[0023] In other advantageous applications, the invention allows identification of the treated tissue. For example, in applications where laser ablation of tissue is performed, immediate or real-time identification of which type of tissue is ablated can be achieved. For example, when ablating bone tissue or cartilage tissue, the type of tissue actually ablated can be monitored, and if tissue that was not intended to be ablated is involved, appropriate actions can be triggered. In this way, other tissues can be prevented from being ablated, for example, and particularly precise ablation can be provided.

[0024] In order to achieve appropriate sensitivity in the entire spectral range of interest, the biological tissue analysis device advantageously comprises at least one additional spectrometer module.Thereby, each spectrometer module can be optimized for a specific part of the spectral range of interest.

[0025] The biological tissue analysis device according to the present invention allows analysis on a cell basis. Therefore, a fairly accurate analysis can be provided. In addition, by maintaining the emission light range, the emission light or spectrum quality can be ensured to ensure that the same conditions are maintained on all laser spots studied.

[0026] Furthermore, it is well known that in biological tissues, laser-induced plasma plumes lead to high signal variability. Therefore, the statistical comparisons provided can only produce poor results. However, according to the present invention and its preferred embodiments, high-quality spectra can be generated in a single analytical laser shot. This can be derived from i) the generation of a uniform plasma over a larger surface area; ii) a relatively long delay time for establishing the equilibrium of the emitting atoms; iii) a light collection system with a spectrometer that has a relatively high sensitivity to allow this relatively long delay time; iv) a novel automatic spectral quality recognition system to control the device; and v) a cleaning or drying laser pulse can be applied before the analytical laser pulse.

[0027] Preferably, the control unit is configured to adjust the laser generator and / or the spectrometer module so that the plasma temperature of the analysis pulse is within a predefined temperature range, and the plasma density of the analysis pulse is within a predefined density range, and the emission line relationship is maintained within a predefined reference emission line range. In this way, a relatively uniform plume can be effectively generated.

[0028] Preferably, the reference element is calcium (Ca). Alternatively, sodium (Na) can also be used as a reference element. Ca is an element that is usually present in bones, and bone ablation usually releases these elements. In this way, the biological tissue analysis device is particularly suitable for bone applications, such as removing cancerous bone tissue. In addition, these elements have suitable emission line pattern changes in appropriate temperature curves. Although Ca is preferred, especially when the target tissue is bone, other elements such as Na may also be suitable for other tissues or other temperature curves in particular. For example, pure Ca emission lines can represent bones (hydroxyapatite), while CaO and / or CaOH emission lines can represent cartilage tissue. Bones as hydroxyapatite have only a small amount of hydrate water in their crystals, while soluble Ca ions have huge hydrate balls. In the device according to the present invention, this hydrate ball of cartilage can mainly produce CaO and CaOH light emission bands or lines, while the hydroxyapatite of the bone provides a purer narrow Ca atomic emission line. In this way, the sources of the two Ca can be distinguished.

[0029] Preferably, when the reference element is Ca, the first wavelength is about 393 nanometers (nm), such as, for example, 393.39 nm or about 396 nm, such as, for example, 396.86 nm, and the second wavelength is about 422 nm, such as, for example, 422.71 nm. The first excited state represented by the first wavelength at about 393 nm or about 396 nm can correspond to doubly charged Ca. The second excited state represented by the second wavelength at about 422 nm can correspond to singly charged Ca. When the reference element is Na, the first wavelength can also be about 589 nm, such as, for example, 589.18 nm, and the second wavelength can be about 819 nm, such as, for example, 819.22 nm. Such wavelengths allow for efficient implementation of fairly uniform plumes and accurate analysis.

[0030] Preferably, the shot line relation is a ratio between the first shot line and the second shot line.Such a ratio allows providing a fairly accurate and reliable predefined reference shot line range.

[0031] Thus, when the reference element is Ca, the ratio between the first emission line and the second emission line is preferably the ratio between the sum of the emission line of the first wavelength of about 393 nm and the emission line of the second wavelength of about 422 nm, wherein the ratio is preferably greater than 0.6 or greater than 0.2 and less than 1. The sums involved can be calculated or determined in various ways. For example, such sums can be calculated by integrating the emission lines. Utilizing these sums allows efficient and accurate analysis and evaluation.

[0032] Advantageously, the control unit is configured to set the delay time, which is the difference between the acquisition time and the laser pulse time, such that the maximum background scatter is reduced below a certain value. In this way, interferences caused, for example, by the tissue environment can be reduced or eliminated, so that continuous emission is possible. In particular, non-specific radiation can be achieved.

[0033] Advantageously, the control unit is configured to keep the baseline of the spectrum as flat as possible. In particular, background scattering can be suppressed by increasing the delay time.

[0034] Preferably, the control unit is configured to maintain the emission line relationship within a predefined reference emission line range and / or eliminate backscattering to allow quantitative analysis of the reference element by adapting the delay time (i.e. the difference between the acquisition time and the laser pulse time). By this adaptation, effective maintenance of the emission line relationship within the predefined range can be established.

[0035] Thereby, the laser generator is preferably configured to provide a plurality of analysis pulses, wherein the control unit is configured to evaluate the emission line relationship of each analysis pulse of the plurality of analysis pulses and to adapt the delay time of subsequent analysis pulses to keep the emission line relationship within a predefined reference emission line range and / or to eliminate backscatter. Such on-the-fly adaptation allows to provide continuous and uninterrupted processing.

[0036] The delay time is preferably in the range of about 0.5 microseconds to about 25 microseconds, or in the range of about 2 microseconds to 10 microseconds. Such delay times have been proven to be suitable for most application areas of biological tissue analysis equipment, such as bone or other surgical procedures or analysis of tissue sections embedded in plastic or paraffin.

[0037] The control unit is preferably configured to adapt the delay time according to the reference element and / or the backscatter. In this way, the device can be adapted to a specific application and the involvement of a favorable reference element. Furthermore, the control unit can be configured to adapt the delay time to reduce or result in a lack of background scatter. In this way, the device can be adapted to a specific application and the involvement of a favorable tissue or probe.

[0038] Preferably, the control unit is configured to maintain the emission line relationship within a predefined reference emission line range by adapting the power of the analysis pulses. By such power adaptation, it can be ensured that the current laser power always provides enough photons to generate a similar plasma electron temperature. It can be used as an alternative or in addition to adapting the delay time. In this way, a particularly suitable relationship can be maintained throughout the process.

[0039] Preferably, the control unit is configured to maintain the emission line relationship within a predefined reference emission line range by adapting the integration time. Likewise, such adaptation may be provided as an alternative or supplement to adapting the delay time and / or the laser power. In this way, maintaining a suitable relationship may be further improved.

[0040] Preferably, the laser generator has beam guiding optics configured to focus the multiple laser beam pulses.By suitable optics, the laser beam pulses can be precisely configured so that efficient ablation and / or plume generation is possible.

[0041] Thus, the beam directing optics of the laser generator preferably have an emission lens having a focal length of at least about 50 millimeters (mm), at least about 400 mm, or in the range of about 150 mm to about 250 mm. The emission lens can be a lens in the more literal sense, i.e., a piece of transparent material with curved sides for concentrating light, or other structures suitable for focusing a laser beam, such as a mirror or the like.

[0042] Typical LIBS systems for analyzing biological tissue have rather small laser powers (typically 1 to 10 millijoules (mJ)) and rather short focusing lenses (typically less than 100 mm) to provide a small spot size and a high photon density. However, a small spot also means that fewer atoms can be excited, resulting in lower sensitivity and a more uniform plasma. The small size of the plasma plume produces a signal with large intensity variations, resulting in the need to average multiple laser shots. Typically, since the photon density is very high on a small spot size, the high photon flux results in a breakdown of the air or ambient gas, which is no longer sample-dependent (number and / or intensity of N and O emission lines). Therefore, it is sometimes necessary to use a shielding gas, such as argon or helium, to avoid these dense peaks, but this is not necessary according to the present invention.

[0043] The angle between the laser and the collection optics is also important for sensitivity (e.g. between 0° and 15°). The crater formed by the laser beam sends the plasma emission cloud more vertically out of the tissue. This effect becomes stronger as the crater becomes deeper. However, this effect makes it possible to collect LIBS signals from deeper areas beyond the surface (e.g. about 0.01 mm to 3 mm, or even deeper) without signal loss. In addition, it is important to track the expanding plasma for as long as possible to collect the emission light also after a long delay.

[0044] In terms of optics, it is important to note that for LIBS systems it is important to always have the sample surface in the focus of the laser lens. However, tissue samples and biopsies may not always have a flat surface, which can lead to variations in photon density and also different temperature and signal intensity variations. Therefore, it is important that the sample surface always sees the same number of photons to produce a good spectrum for comparison.

[0045] In this case, by having a transmitting lens as defined in the biological tissue analysis device, a relatively high depth of focus or depth of field can be achieved. The lens used allows to generate a relatively large area and a relatively uniform plasma with substantially the same photon density. In addition, the use of such a lens allows to keep the sample or biological tissue surface in focus, because it provides substantially the same photon density over a relatively long range or distance of the sample or biological tissue. In addition, the long focal length lens also allows to keep the light emission and the fast-moving shock wave in the focus or focal space for a relatively long time.

[0046] Preferably, the beam guiding optics is configured to generate a laser beam or laser beam pulse having a spot diameter in the range of about 0.1 mm to about 0.4 mm. Such a spot diameter allows to effectively provide a resolution or spot size suitable for analyzing tissue. Thus, averaging by spot size or area can be performed in such analysis, which allows to effectively identify tissue or its structure.

[0047] Preferably, the beam guiding optics are configured to produce a laser beam with a depth of focus (DoF) greater than 10 mm. DoF may be defined or referred to as twice the Rayleigh length. With such beam guiding optics, a fairly uniform beam profile may be achieved.

[0048] In a specific example, for a spot diameter in the range of 0.1mm to 0.4mm and a focal length in the range of 150mm to 250mm, the following configuration can be embodied: when the spot diameter is 0.1mm and the focal length is 150mm, a DoF of 24.4mm can be generated; when the spot diameter is 0.4mm and the focal length is 150mm, a DoF of 30.5mm can be generated; when the spot diameter is 0.1mm and the focal length is 250mm, a DoF of 12.7mm can be generated; when the spot diameter is 0.4mm and the focal length is 250mm, a DoF of 49.7mm can be generated.

[0049] Such biological tissue analysis equipment can produce uniform beam profiles and low beam divergence. In this way, it is possible to provide a larger target area with the same photon density. For quantitative problems, it may be necessary to treat all cells with approximately the same number of photons. If this can be achieved, then a fairly large plasma plume may disappear and be uniform after a given delay and background scattering, and provide sufficient emission light for single-shot sensitivity.

[0050] Preferably, the spectrometer module has a converging lens and the analysis pulse has a focus, wherein the converging lens of the spectrometer module is arranged in the projection of a cone, whose vertex is located at the focus of the analysis pulse, and the cone angle is less than about 60°, less than about 35°, preferably about 11°. The term "projection of a cone" here refers to a cone that can exist in reality in the form of an optical or physical structure. More specifically, the projection of the cone is a virtual cone that defines the space in which the lens is arranged. By this arrangement of the lenses, a highly sensitive collection of emitted light can be achieved.

[0051] Preferably, the biological tissue analysis device comprises an acoustic wave sensor configured to record the acoustic shock waves of the analysis pulse provided to the target tissue, wherein the acoustic wave sensor is connected to the control unit. Such an acoustic wave sensor allows to provide additional evaluation means, thereby improving the analysis of the target tissue. Furthermore, the acoustic wave sensor also allows to determine the exact focal distance, since the laser beam pulse generates the loudest acoustic signal at the focal point. Alternative distance measuring devices can also be used to determine the focal distance.

[0052] Preferably, the control unit is configured to receive the recorded acoustic shock wave from the acoustic wave sensor or data from an alternative distance measuring device and to keep the recorded acoustic shock wave within the shock wave range by adapting the distance between the beam steering optics and the target tissue, adapting the focus of the beam steering optics, and / or adjusting the power of the laser generator. The involvement of such shock wave evaluation allows providing a further improved plasma plume.

[0053] Preferably, the control unit is configured to position the beam guiding optics and the target tissue at a plurality of distances relative to each other, activate the laser generator to provide at least one analysis pulse at each of the plurality of distances, receive the recorded acoustic shock waves from the acoustic wave sensor for each of the plurality of distances, and position the beam guiding optics and the target tissue at one of the plurality of distances that meets a selection criterion.

[0054] Therefore, the selection criterion is preferably the height of the intensity of the recorded acoustic shock wave. Preferably, the control unit is configured to determine the quality by the acoustic shock wave from the acoustic wave sensor. This allows an efficient assessment of the quality and / or a signal normalization.

[0055] Preferably, the biological tissue analysis device includes a light collection optical device configured to direct light to the spectrometer module. The light collection optical device may include one or more converging lenses and / or reflective collimators, such as a 210 mm lens. Through such an optical device, light can be effectively guided to the detector of the spectrometer module or into the optical fiber (glass fiber) of the spectrometer module. In order to improve light conversion (transition) within a sufficiently large mass range (such as, typically 180 nm to 900 nm), more than one collimator can be used to collect more light or different coatings can be used. In addition, as described above, two or more spectrometers can be used, for example, one for ultraviolet to visible light (UV-VIS) and another for visible light to infrared light (VIS-IR).

[0056] Preferably, the spectrometer module is configured to switch on in one nanosecond or less and switch off in one nanosecond or less. Such a spectrometer module allows for a fairly accurate detector gating. Furthermore, it also allows for a precise laser pulse triggering, which can be very important when fast growing signals have to be measured, since large jitter in the triggering means high variations in signal intensity. Thus, precise laser pulse triggering can be very important for the quantification of the emitted light.

[0057] Advantageously, the spectrometer module is a low-resolution spectrometer, e.g. with a resolution of more than 0.2 nm per pixel, and the associated detector is configured in a sufficiently sensitive manner that a single plasma (laser emission) provides sufficient emission light (also of low-abundance elements) to obtain a complete and dense LIBS spectrum.

[0058] Preferably, the laser generator comprises a high power Q-switched Nd:YAG laser. Such a laser allows efficient implementation of both analytical beams and ablative laser pulses (such as bone ablation laser pulses). In particular, it can be embodied as a Q-switched crystal that switches rapidly or switches slowly between the preparation laser pulses and the analytical pulses.

[0059] Advantageously, Nd:YAG lasers can provide a laser beam of 1064 nm or 532 nm, or other laser beams with wavelengths between 200 and 1200 nm, with similar power and beam profile. Generally, it is important that the tissue is able to absorb the laser light to produce hot plasma. Therefore, the beam profile is very important to produce a given photon density so that a long focal length lens may be used.

[0060] Furthermore, it may be necessary that the laser beam has a low divergence and a uniform beam profile to provide an equal distribution of photons over the target area. For quantification purposes, it may be very important to produce a uniform plasma plume and that tissue cells within the laser spot (target) receive the same number of photons.

[0061] A problem that needs to be considered when measuring or detecting emission lines by a spectrometer module is that the measured spectral data includes background scattering or background noise. This background noise may be caused by elements present in the environment, such as elements in the air. Therefore, the later the plasma is measured or evaluated, the lower the background noise. However, at the same time, the later the plasma is measured or evaluated, the lower the signal.

[0062] Therefore, the spectrometer module comprises a relatively high sensitivity spectrometer, such as a gated spectrometer. With such a spectrometer, relatively low signals can be measured, which allows relatively late measurements to be made with no or only limited background noise present. Furthermore, such a spectrometer also allows providing appropriate performances required in many applications of biological tissue analysis equipment, such as speed and accuracy.

[0063] Preferably, the control unit is configured to operate the laser generator to provide at least one laser beam pulse as a preparation pulse sequence. The preparation pulse sequence may be a relatively long laser pulse, or a series of shorter laser pulses. For example, the preparation pulse sequence may be a predefined number of pulses identical to the analysis pulse and preceding the analysis pulse. Such a preparation pulse sequence may in particular remove liquid in a target tissue, wherein the target tissue is intended to be hit by the analysis pulse. Furthermore, the preparation pulse may provide a certain ablation depth so that the analysis pulse hits the target tissue at a lower layer.

[0064] Therefore, the time width of the preparation pulse sequence is preferably greater than the time width of the analysis pulse. The time width of the analysis pulse can be in the range of femtoseconds (fs) to nanoseconds (ns).

[0065] Preferably, the control unit is configured to operate the laser generator to provide the analysis pulse less than 1 millisecond, less than 1 microsecond, less than 100 nanoseconds, or less than 50 nanoseconds after the preparation pulse sequence. Such a small time gap allows preventing liquids such as blood and / or water from flowing to the spot where the analysis pulse hits the target tissue. In addition, such a time gap also allows to achieve that the effect of the preparation is still present when the analysis pulse is provided. Preferably, the control unit is configured to evaluate the spectral data provided by the spectrometer module, wherein the evaluation of the spectral data includes at least one of the following: (i) discarding spectra with a maximum signal below a predefined value; (ii) discarding spectra that saturate the spectrometer module; (iii) discarding spectra related to air by analyzing the emission peak intensity of N, O and / or H, which may be derived from the photon density breakdown before the sample surface; (iv) discarding spectra generated outside the predefined plasma electron temperature and / or plasma electron density; (v) filtering unsuitable spectra; and (vi) summing or integrating the peak areas of the emission lines.

[0066] Each of the evaluation steps listed can improve the analysis results of biological tissues. For example, by summing or integrating the peak areas, the sensitivity of the device can be increased or improved. Most preferably, a plurality or in particular all of the steps listed are combined in order to achieve the best results.

[0067] Preferably, the spectrometer module has a spectrometer board, wherein the control unit is embodied on the spectrometer board. The term "board" used in this context may particularly relate to a printed circuit board (PCB) or a printed wiring board (PWB), which is a laminated sandwich structure consisting of a conductive layer and an insulating layer. Typically, a PCB has two complementary functions. The first is to fix the electronic components in specified positions of the outer layer by welding. The second is to provide reliable electrical connections and, depending on the specific circumstances, also reliable open circuits between components in a controlled manner generally referred to as PCB design. This implementation of the spectrometer module on a board allows for a fairly high performance and fast communication between the control unit and the spectrometer module. In this way, the runtime analysis of the target tissue is particularly effective.

[0068] Preferably, the biological tissue analysis apparatus comprises an xyz-stage configured to support the biological tissue sample at a predetermined position and to move the sample in an x-direction, a y-direction perpendicular to the x-direction, and a z-direction perpendicular to the x-direction and the y-direction. Such a three-dimensionally movable xyz-stage can accurately position the tissue sample.

[0069] In particular, since biological tissues often have heterogeneous properties, such as the distribution of tumors in tissues, it is necessary or beneficial to detect not only the presence of specific cells, but also their xyz distribution in the tissue itself. Therefore, it may be important to have a single-shot sensitivity and the spectrum recorded each time has the same quality so that statistical methods can quantify the amount of biological cell regulation mainly through the electrolytes of the biological cells. Due to this single-shot sensitivity combined with the xyz information obtained by the xyz-stage, a tissue scan, such as a tumor distribution map, can be generated.

[0070] On the other hand, the present invention is a method for detecting a specific type of cells in a biological target tissue, comprising the following steps: a laser generator provides laser beam pulses to a target tissue according to the elemental composition of the target tissue, wherein at least one laser beam pulse is configured as an analysis pulse, and the analysis pulse generates an analysis plasma at the laser pulse time, and the analysis plasma includes a reference element in at least a first excited state and a second excited state; a spectrometer module collects emission light of the analysis plasma at a collection time and analyzes emission lines in the collected emission light; and an emission line relationship between a first emission line of the reference element of the analysis plasma at a first wavelength associated with the first excited state and a second emission line of the reference element of the analysis plasma at a second wavelength associated with the second excited state is maintained within a predefined reference emission line range.

[0071] The method according to the present invention and the preferred embodiments thereof described below allow achieving the effects and advantages of the biological tissue analysis apparatus and the preferred embodiments thereof described above.

[0072] In advantageous applications, they may in particular allow for the effective identification of cancerous tissues. More specifically, when healthy cells are transformed into tumor cells, the electrolyte and element systems (Mg, Fe, K, Na, Ca, CN, etc.) of the healthy cells change. This change is measured by indirect methods such as Raman and fluorescence microscopy systems. Many tumor cells show a significant increase in potassium. For example, in mandibular tumor cells, the calcium content decreases, while the potassium increases. In addition, other LIBS signals may also change during the transformation from a healthy state to a tumor state, such as CN. Such molecules may be produced by incomplete molecular decomposition of amino acids (proteins). For example, the amino acid sequence of collagen is based on 20% proline and hydroxyproline. For example, typical changes in collagen may change the content of proline and hydroxyproline, resulting in changes in the CN signal. It is worth noting that the biological tissue analysis device of the present invention can be constructed in a way that inhibits the formation of CN by reaction with ambient air.

[0073] Among other advantageous applications, they allow identification of the treated tissue. For example, in applications where laser ablation of tissue is performed, instant identification of the type of tissue ablated can be achieved. For example, when ablating bone tissue or cartilage, the type of tissue actually ablated can be monitored and appropriate actions can be triggered if other tissue is involved. In this way, it is possible, for example, to prevent other tissue from being ablated.

[0074] Preferably, the laser generator and / or the spectrometer module are adjusted so that the plasma temperature of the analysis pulse is within a predefined temperature range and the plasma density of the analysis pulse is within a predefined density range, while the emission line relationship is maintained within a predefined reference emission line range.

[0075] Preferably, the reference element is Ca. Preferably, when the reference element is Ca, the first wavelength is about 393 nanometers or about 396 nanometers, and the second wavelength is about 422 nanometers.

[0076] Thus, when the reference element is Ca, the predefined reference emission line range is the ratio between the sum of the emission lines at the first wavelength of about 393 nm or about 396 nm and the emission lines at the second wavelength of about 422 nm, wherein the ratio is preferably greater than 0.6 and less than 1.

[0077] Preferably, the shot line relationship is a ratio between the first shot line and the second shot line.

[0078] Preferably, the emission line relationship is kept within a predefined reference emission line range and / or backscatter is eliminated to allow quantitative analysis of reference elements by adapting the delay time (ie the difference between the acquisition time and the laser pulse time).

[0079] Therefore, the laser generator preferably provides a plurality of analysis pulses, wherein the emission line relation of each of the plurality of analysis pulses is evaluated and wherein the delay times of subsequent analysis pulses are adapted to keep the emission line relation within a predefined reference emission line range and / or to eliminate backscatter.

[0080] The delay time is preferably in the range of about 0.5 microseconds to about 25 microseconds, or in the range of about 2 microseconds to 10 microseconds.

[0081] The delay time is preferably adapted to a reference element and / or a clean baseline within the recorded spectrum to avoid backscattering effects.

[0082] Preferably, the emission line relationship is kept within a predefined reference emission line range by adapting the power of the analysis pulses and / or by controlling the laser or the delay time, since longer delay times will also reduce the strength of the signal.

[0083] Preferably, the emission line relationship is maintained within a predefined reference emission line range by adapting the integration time.

[0084] Preferably, the laser generator focuses a plurality of laser beam pulses.

[0085] Thus, the plurality of laser beam pulses preferably have a focal length of at least about 100 mm, at least about 200 mm, or in the range of about 150 mm to about 250 mm.

[0086] Preferably, the spot diameter of the beam pulse is in the range of about 0.1 mm to about 0.4 mm.

[0087] Preferably, the focal depth of the beam pulses is greater than 10 mm.

[0088] Preferably, the acoustic wave sensor records the acoustic shock waves of the analysis plasma generated by the analysis pulse provided to the target tissue.

[0089] Thereby, the method preferably comprises the step of keeping the recorded acoustic shock wave within the shock wave range by adapting the distance between the laser generator and the target tissue, adapting the focus of the laser generator, or adjusting the power of the laser generator.

[0090] The method preferably includes: positioning a laser generator and a target tissue at a plurality of distances relative to each other; activating the laser generator to provide at least one analysis pulse at each of the plurality of distances; an acoustic wave sensor recording acoustic shock waves at each of the plurality of distances; filtering inappropriate spectra; and positioning the laser generator and the target tissue relative to each other at one of the plurality of distances that meets a selection criterion.

[0091] Thereby, the selection criterion is preferably the height of the intensity of the recorded acoustic shock wave.

[0092] Preferably, the mass is determined by acoustic shock waves from an acoustic wave sensor.

[0093] Preferably, the laser generator comprises a high power Q-switched Nd:YAG laser.

[0094] Preferably, the spectrometer module comprises a gated spectrometer.

[0095] Preferably, at least one laser beam pulse is provided as a preparation pulse sequence.

[0096] Therefore, the time width of the preparation pulse sequence is preferably greater than the time width of the analysis pulse.

[0097] The analysis pulse is preferably provided within 10 ns after the preparation pulse sequence.

[0098] Preferably, the spectral data provided by the spectrometer module are evaluated, wherein the evaluation of the spectral data comprises at least one of the following: discarding spectra with a maximum signal below a predefined value; discarding spectra that saturate the spectrometer module; discarding spectra associated with air by analyzing the emission peak intensities of N, O and / or H; discarding spectra generated outside a predefined plasma electron temperature and / or plasma electron density; and summing or integrating the peak areas of emission lines.

[0099] Preferably, the specific cell type is cancer.

[0100] Preferably, the tissue is extracted tissue, so that the method is an in vitro method.

[0101] In another aspect, the present invention is a computer program comprising instructions which, when executed by a computer, cause the computer to perform the above method or any embodiment thereof.

[0102] The computer program may be a computer program product comprising computer code means which, when executed on a computer, are configured to control a processor of the computer to implement the computer-implemented method described above or below or any preferred embodiment thereof. In addition, a computer-readable medium may be provided, the medium comprising instructions which, when executed by a computer, cause the computer to perform the method described above or below or any preferred embodiment thereof. The medium may be a storage medium and, in order to allow for convenient distribution, may also be a mobile or portable storage medium. Alternatively, in order to allow for transmission via the Internet or similar means, or for other purposes, a data carrier signal may be provided which carries the computer program described hereinabove. A computer program may also be referred to as software or consist of software.

[0103] Such a computer program allows to effectively implement the method according to the invention or any preferred embodiment thereof, thereby achieving the effects and benefits involved. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] The present invention is described in more detail below by means of exemplary embodiments and with reference to the accompanying drawings, in which:

[0105] Figure 1 A schematic diagram showing an embodiment of a biological tissue analysis device according to the present invention implementing an embodiment of a method according to the present invention;

[0106] Figure 2 A schematic diagram showing the evolution of spectral emission line signals and continuous emission line signals over time;

[0107] Figure 3 A schematic diagram of an appropriate evaluation spectrum is shown;

[0108] Figure 4a schematic diagram showing an improperly evaluated spectrum; and

[0109] Figure 5 Schematic diagrams showing other inappropriately evaluated spectra. DETAILED DESCRIPTION

[0110] In the following description, the use of certain terms is for convenience and is not intended to limit the present invention. The terms "right", "left", "up", 'down', "below" and "above" refer to the directions in the figure. The terms include the terms and their derivatives and terms with similar meanings that are explicitly mentioned. In addition, spatial relative terms, such as "below", "below", "lower", "above", "upper", "proximal end", "distal end", etc., can be used to describe the relationship between one element or feature and another element or feature, as shown in the figure. In addition to the positions and orientations shown in the figures, these spatial relative terms are intended to also include different positions and orientations of the device in use or operation. For example, if the device in the figure is reversed, the element described as "below" or "below" will be "above" or "above" other elements or features. Therefore, the exemplary term "below" can include both above and below positions and orientations. The device can also be oriented in other ways (rotated 90 degrees or other orientations), and the spatial relative descriptors used here can also be interpreted accordingly. Similarly, the description of moving along various axes and around various axes also includes various special device positions and orientations.

[0111] In order to avoid repetition in the figures and descriptions of various aspects and illustrative embodiments, it should be understood that many features are common to many aspects and embodiments. Omitting an aspect in the description or figures does not mean that the embodiment containing the aspect lacks the aspect. On the contrary, omitting the aspect may be for clarity and to avoid lengthy description.

[0112] In this case, the following applies to the remainder of the description: If, for the purpose of clarifying the drawing, a figure contains reference signs which are not explained in the directly related description part, reference is made to the preceding or following description part.

[0113] Furthermore, for the sake of clarity, if in a figure not all features of a component are provided with reference symbols, reference is made to other figures showing the same component. The same numerals in two or more figures represent the same or similar elements.

[0114] In the table below, an overview of the general LIBS process is shown. The LIBS process is based on many different reactions which all affect the signal intensity.

[0115]

[0116]

[0117] The steps listed can be started sequentially or in parallel. An important question is how the laser energy is converted into temperature. This depends largely on the absorption conditions of the sample surface. Laser pulses usually generate high temperatures so that the plasma plume expands in a fast shock wave. Another obstacle is the inhomogeneous plasma plume, especially in biological tissue. Hot and cold regions influence each other, for example through the Stark effect. Emitted photons from the hot region will be absorbed by the same elements in the cold region. Such photons are lost during detection, resulting in strong peak broadening and peak splitting. This also leads to nonlinear calibration curves. All of this makes LIBS of biological tissues often unpredictable. To overcome these difficulties, hundreds of laser shots are sometimes averaged to reduce variability and distinguish different tissues. However, this approach only works if a homogeneous sample is available. But biological tissue is cell-based, and cells give the tissue a fine structure, and averaging may not be a suitable analysis method.

[0118] In common LIBS systems, it is difficult to control the application process when biological tissue or samples need to be analyzed. For improvement, the energy transfer from the photon energy to the rapid heating can be considered. The absorption of the light energy is based on the absorption coefficient of the molecules involved. But it also depends on the density of the material that will be ablated and generate the expanding plasma plume. Here, the photon density or photon flux that will be applied to the surface is an important rule. If the photon density is too high, N and O in the ambient air will also be analyzed. If the density is too low, there are not enough excited atoms to detect. Therefore, highly sensitive detector systems are required to collect always enough light for a complete spectrum. The next important step is a homogenous plasma plume. This can only be successful if the plasma plume has a certain size and an appropriate particle density, and if the delay time between the analysis laser pulse and the start of the gated spectrometer is long.

[0119] Some of the important items involved in LIBS analysis of biological tissues are: correct photon density, i.e. avoiding N and O emission peaks from ambient air, which requires a fairly low photon density, and light breakdown in the focused area should be prevented in clean air (this item can be solved by a long focus lens (i.e. long focal depth) and a laser that provides a uniform beam profile, low divergence and sufficient power to obtain a large spot size); large laser spot size to obtain sufficient signal and a uniform plasma plume (this item can also be solved by a long focus lens); high sensitivity collection of emission light within 0 to 50 μs after the initial analytical laser pulse, which requires a sensitive spectrometer, does not require the highest wavelength resolution, and optical light collection is performed from the top (with a long focus lens, the expanding plasma plume will more or less stay within the focal cone of the lens); precise timing without jitter to obtain the most constant conditions (fs to low ps accuracy); analysis of differences within the emission light patterns of a given element to acquire only patterns with the same or similar ratios. (Several ratios can be combined, where other elements can show similar effects. This depends mainly on the applied temperature); biological tissues or samples can be pre-analyzed by probing within a given delay range to obtain the best conditions for a specific sample); for quantification and tissue differentiation, the main emission wavelengths of a given element should be combined to obtain more stable results (Lorentzian fitting can be used to calculate the peak area. In this way, changes in the uniformity of the plasma plume can be reduced); a hardware unit as a control unit, which analyzes the acquired spectra in real time and is able to make decisions for the upcoming laser shots in real time within the 10 Hz frame of the laser device (the control unit can have multiple tasks: one is to use the acquired emission spectra for real-time quality control. This can be achieved with some Ca emission lines, which The emission lines must be within a given ratio range to be accepted. All spectra outside this range are discarded. Another task can be to decide if a cleaning, drying or drilling laser pulse needs to be applied before the next analytical LIBS laser pulse. The control unit can decide to stay on the current spot and measure under the same or different conditions, or go to the next spot, based on the currently acquired measurement spectrum); sample pre-evaluation, i.e. before tissue differentiation begins, the control unit evaluates the sample by measuring the emission spectra under a set of different settings or conditions; optimize the laser intensity, for example by flash lamp voltage and pulse length; determine the best delay time to achieve a homogeneous signal; determine if a cleaning or drying laser pulse is needed; and measure H, O, N, Ca and Na emission lines (which may involve peak fitting to receive typical peak parameters (Lorentzian)).

[0120] Figure 1An embodiment of a biological tissue analysis device according to the present invention is shown. The device comprises a laser generator having a Nd:YAG, 1064nm, 8ns, 70mJ flash lamp pumped laser source 1 and a laser controller 2. The laser generator is configured to provide laser beam pulses to an XYZ stage 4, which is designed to accommodate or support biological target tissue, also referred to as a sample.

[0121] The biological tissue analysis device also includes a spectrometer module having a gated spectrometer 7 configured to open and close in less than 1 ns, an emission light collection system 8 , and an optical fiber 12 connecting the spectrometer 7 and the emission light collection system 8 .

[0122] In addition, the biological tissue analysis device also includes a delay time generator 4, a fast photodiode trigger 9, a CCD camera 10, a microphone 11 as an acoustic wave sensor, and an optical system. The spectrometer 7, the laser controller 2, the delay time generator 3, the CCD camera 10 and the microphone 11 are connected to a computer 5 of a control unit, and the control unit also includes a plasma analysis or temperature controller 6. The optical system includes an emission or focusing lens 14 with a focal length of at least about 250 mm, a converging or collecting lens 15 as a collection optical device, and a silicon dioxide window 13 for generating light reflection for the fast photodiode trigger 9.

[0123] The computer 5 is configured to operate the laser controller 2 to provide at least one laser beam pulse as an analysis pulse, which is directed toward the XYZ stage 4 and focused by the focusing lens 14. During operation, when hitting the target tissue arranged on the XYZ stage 4, the analysis pulse generates an analysis plasma within the laser pulse time. The analysis plasma includes a reference element in at least a first excited state and a second excited state. For example, when the target tissue is or includes bone tissue, the reference element can be Ca, wherein the first and second excited states are two different ionizations of Ca.

[0124] The emission light collection system 8 is configured to collect the emission light of the analysis plasma through the collection lens 15 at the collection time, and provide the collected emission light to the spectrometer 7 through the optical fiber 12. The spectrometer 7 is configured to analyze the emission lines in the collected emission light and communicate with the computer 5. Based on the data received from the spectrometer 7, the computer 5 together with the plasma temperature control unit 6 is configured to maintain the reference emission line relationship between the first emission line of the reference element at the first wavelength (i.e., about 393 nm) associated with the first excited state and the second emission line of the reference element at the second wavelength (i.e., about 422 nm) associated with the second excited state within a predefined reference emission line range.

[0125] The plasma analysis or temperature controller 6 interacts with the computer 5 to analyze and / or maintain the emission line relationship within a predefined reference emission line range by adjusting the laser controller 2 and the delay time generator 3 so that the plasma temperature of the analysis pulse is within a predefined temperature range and the plasma density of the analysis pulse is within a predefined density range.

[0126] More specifically, this is achieved by selecting and discarding spectra that meet or do not meet predefined criteria. If background scattering is present, the delay time generator 3 adapts the delay time to the difference between the acquisition time and the laser pulse time. The delay time is set in the range of about 1 microsecond to 10 microseconds based on a flat baseline. The plasma temperature is corrected by Ca as a reference element. In addition, the laser controller 2 is adjusted to adapt the power of the analysis pulse.

[0127] The microphone 11 is configured to record the acoustic shock waves of the analysis pulses provided to the target tissue. In addition, the computer 5 is configured to receive the recorded acoustic shock waves from the microphone and keep the recorded acoustic shock waves within the shock wave range by adapting the distance between the focusing lens 14 and the XYZ stage 4 and / or by adjusting the laser power via the laser controller 2.

[0128] To set up the microphone 11, the computer is configured to position the focusing lens 14 and the XYZ stage relative to each other at a plurality of distances, activate the laser controller to induce the laser 1 to provide at least one analysis pulse at each of the plurality of distances, receive a recorded acoustic shock wave from the microphone 11 for each of the plurality of distances, and position the focusing lens 14 and the XYZ stage 4 relative to each other at one of the plurality of distances based on the intensity of the recorded acoustic shock wave.

[0129] In order to obtain clean and appropriate analysis results, the computer 5 is configured to operate the laser generator to provide a preparation pulse sequence of less than 1 millisecond before each analysis pulse. The preparation pulse sequence can be a relatively long laser pulse, or a series of shorter laser pulses. The time width of the preparation pulse sequence is greater than the time width of the analysis pulse.

[0130] When evaluating the spectral data received by the spectrometer, the computer discards spectra with maximum signals below a predefined value, discards spectra that saturate the spectrometer module, discards spectra related to air by analyzing the emission peak intensity of N, O and / or H, discards spectra generated outside a predefined plasma electron temperature and / or plasma electron density, and integrates the peak areas of the emission lines.

[0131] The computer 5 and the plasma temperature control unit 6 are configured to perform spectral control by applying the following procedure through the plume configuration: In the first step, the peak of the defined Ca emission line is identified, and Lorentz peak fitting and peak area calculation are performed. In the second step, the measured ratio is compared with a quality standard (such as a sound signal recorded by microphone 11) by peak width and peak area. If the quality standard is met, the current measured spectrum is recorded and the first step is continued for a new analysis pulse. If the quality standard is not met, the reproducibility of the key parameters is checked in the third step to be within a given range of a large number of recorded spectra. It is then moved to a new point and a new analysis pulse is provided. In the fourth step, if a wet sample is identified in the current spectrum, the target tissue is dried by preparing a pulse sequence, a new analysis pulse is provided, and the first step is continued. In the fifth step, it is identified that the sample condition is poor, and the delay time, laser power and photon density are changed to generate improved conditions.

[0132] for Figure 1 As shown, the biological tissue analysis apparatus according to the present invention can consider the following configuration aspects.

[0133] Photon density and photon flux: The identification of the absorption behavior of a given target tissue is important for controlling the photon density required to initiate the spectroscopy or LIBS process. From a single recorded LIBS spectrum, full and poor spectra can be identified. This can be achieved by the presence of closely spaced N and O emission lines and / or the ratio of Ca or Na emission lines. The system can be adjusted for laser photon fluxes that are too low to perform LIBS in ambient air. Using a long focal length offers the possibility to illuminate a larger spot in a uniform manner, so that the sample reaches nearly the same temperature over the entire spot area.

[0134] Emission collection: The rapid heating of the sample spot area leads to ablation of material in a rapidly expanding plume. This can be detected by acoustic shock waves. The resulting plasma plume is still expanding as electronic excitation and temperature-induced breakdown of atoms occur. Therefore, the optical collection system may need to have the potential to track a moving plasma plume. Using a collection lens with a long focal point allows the plasma plume to remain in focus for a considerable time. Furthermore, every emission beam within the collection cone of the lens can be recorded. For a spectrometer, sensitivity is more important than wavelength resolution because it is needed when the plasma plume is more expanded and homogeneous, but with fewer excited atoms.

[0135] Laser spot size on the target: Each laser shot can ablate and analyze a given volume. This volume is defined by the laser beam diameter and the target material itself. Here, material density and absorption coefficient may play a major role. Larger spot sizes offer the advantage of taking an average value over a given sample area. All cells and all extracellular fluid within this spot can be analyzed. Small spot sizes may vary too much due to the heterogeneity of biological samples. In addition, larger plasma plumes stay longer and have more options to become more humified over time. Vice versa, a small spot size may have a shorter plasma lifetime and the emitted light can only be recorded for a short time.

[0136] Timing: During the first microsecond, continuous emission can occur precisely due to electron acceleration. During the next 2 to 30 microseconds, the emitted light can be measured from the expanding plasma plume. Because conditions within the plasma plume change rapidly, it is important to always measure at the exact same time after the initial laser pulse.

[0137] Emission line pattern: The pattern of light emitted by a given element can be determined by the number of excited states and the number of potential ground states. All allowed transitions will produce emission lines. There is a temperature-dependent Boltzmann distribution that fills in the potential excited states for a given element. Therefore, when the plasma temperature changes, the specific emission line pattern of an element will also change.

[0138] Quantification: Using LIBS as a biomarker tool is only possible if the exact elemental amounts can be measured and compared. In general, biomarkers should identify the smallest differences in the study. This is only possible if the analytical method is reproducible, sensitive, and accurate. Since each emitted photon is associated with an atomic nucleus, all major emission lines for a given element must be measured. And as mentioned above, when the plasma temperature changes, the individual emission lines will also change. But the total number of emitted photons for a given element can remain constant. In addition, when high element concentrations are involved, the Stark effect (a self-broadening effect) can sometimes cause the peaks to be relatively broad. This effect can lead to an underestimation of dense peaks and an overestimation of low-abundance peaks. However, it is important to note that the number of atoms involved is always the same. Therefore, it is important to use a Lorentzian fit for the major emission lines (peaks) of a given element and then use the Lorentzian peak area to determine the elemental amount (combined peak area for a given element).

[0139] Control Unit: The control unit or System Smart Control Unit (SSC) analyzes the data quality (quality control (QC)) of all incoming spectra in real time. It also performs sample evaluation to determine the best settings for data collection and tissue resolution. In addition, the system decides when enough data points have been recorded and when the next point should be acquired. The SSC performs Lorentzian peak fitting of established emission lines and determines peak area and peak width as the main decision factors.

[0140] For the first setting (delay setting): a time scan will be performed with a varying delay time from 0.5 to 30 μs. The best setting is reached when there are almost no N, O and H emission lines. In addition, the total intensity and spectral complexity can also be a criterion. When the setup phase has determined the best setting, the measurement phase begins.

[0141] For spectral online quality control, the Ca emission line (e.g. 422nm) must be lower than both 393nm and 396nm lines, or within a given range (ratio). Only if the criteria are met, the current spectrum is acquired and recorded. Elements C, H, N, O should be low or absent or have a constant signal. The ratio of the Ca line (393+396) / 422 should be greater than 0.6 and less than 1. The specific value can be determined in the pre-analysis stage.

[0142] Furthermore, other combinations can be used. From the acquired emission spectra, the following elements can be evaluated, for example: H 656nm; Ca 393+396+422nm; Na 589+819nm; C 193nm. This is further confirmed by the strong Na line at 819nm. During the automatic measurement, the SSC unit analyzes each acquired emission spectrum to meet the above quality criteria.

[0143] After each laser shot, the system can analyze the reproducibility of the spectrum using the above criteria, which should be within the given range. This also depends on the use of cleaning, drying or drilling laser pulses. If the criteria are met, the system starts with a new spot.

[0144] Figure 2 The time evolution of the emission lines of possible reference elements and continuous emission lines (e.g. caused by ambient gas or air) in the plasma generated by the analysis pulse is schematically shown. As can be seen, the continuous emission line is relatively high or strong shortly after the analysis pulse is provided. At the same time, the reference element emission line is relatively low. The older the continuous emission line is, the lower or weaker its intensity is, until its intensity is approximately zero after about 1650ns. Therefore, at a delay time of about 1650ns, the difference between the time when the analysis pulse is generated (i.e., the laser pulse time) and the time when the spectrometer collects the emission light (i.e., the acquisition time), the continuous emission line is essentially undetectable.

[0145] In contrast, the reference element emission line first increases and then decreases more slowly than the continuous emission line, so that when the continuous emission light is already approximately zero, there is still a signal of the reference element emission light.

[0146] Therefore, at a delay time of about 1'650ns, the spectrum consists more or less only of the emission from the reference element. Although the signal at this stage is not at its maximum, there is essentially no backscattering or similar. Therefore, by using a sufficiently sensitive spectrometer, the delay time can be adjusted to exclude any backscattering or similar.

[0147] exist Figure 3 In the figure, the fit of the Ca emission lines in a cortical sample as target tissue is shown. The calculation for Ca was performed at Te = 2 eV and Ne = 2.0e+21 cm-3. One line shows the minimum intensity and the other line shows the maximum intensity of 20 analysis pulses (also called shots). This shows that by adjusting the electron temperature and density, the calculation can fit a given LIBS spectrum. In this fit, the plasma temperature was set to 23'200K (Te = 2 eV). Only with this setting do the Ca I emission lines at 430.21 and 445.56 nm have the correct intensity. Therefore, Figure 3 The spectrum described in meets the requirements, as the ratio of the CaI emission line to the Ca II emission line is within the predefined range.

[0148] Figure 4 and Figure 5 In particular, Figure 4 In the case of , only CaII emission lines can be detected at about 422 nm, so that the ratio of CaI emission line to CaII emission line is not within the predefined range. Figure 5 In the experiment, only Ca I emission lines at about 393 nm and about 396 nm can be detected, so that the ratio of Ca I emission line to Ca II emission line is also not within the predefined range. Figure 4 and Figure 5 From the spectrum of , it can be concluded that the temperature and / or density of the plasma is not suitable and must be adjusted, for example by adapting the power of the laser beam generator. Figure 4 and Figure 5 The spectral ratios are identified as inappropriate and thus they can be discarded from further evaluation or from consideration.

[0149] This specification and drawings, which illustrate various aspects and embodiments of the invention, should not be construed as limiting the claims defining the invention to be protected. In other words, although the invention has been described and illustrated in detail in the drawings and the foregoing description, such description and illustration should be construed as illustrative or exemplary rather than restrictive. Various mechanical, compositional, structural, electrical and operational modifications may be made to the invention without departing from the spirit and scope of the present specification and claims. In some cases, well-known circuits, structures and techniques are not shown in detail in order not to obscure the invention. Therefore, it will be understood that changes and modifications may be made by a person of ordinary skill within the scope and spirit of the following claims. In particular, the present invention encompasses further embodiments having any combination of features of the different embodiments described above and below.

[0150] The invention also covers all further features shown individually in the figures, even though these may not be described in the preceding or following description. In addition, single alternatives of the embodiments described in the figures and description and single alternatives of their features may be exempted from the subject matter of the invention or the subject matter disclosed. The present disclosure includes subject matter consisting of the features defined in the claims or exemplary embodiments as well as subject matter comprising said features.

[0151] Furthermore, in the claims, the word "comprising" does not exclude other elements or steps, nor does the indefinite article "a" or "an" exclude a plurality. A single unit or step may fulfill the functions of several features recited in the claims. The fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. The terms "substantially", 'approximately', "approximately" and the like relating to attributes or values ​​also specifically define the exact attribute or exact value, respectively. In the case of a given value or range, the term "approximately" refers to a value or range, for example, within 20%, 10%, 5% or 2% of the given value or range. Components described as coupled or connected may be directly coupled electrically or mechanically, or they may be indirectly coupled through one or more intermediate components. Any reference signs in the claims should not be understood as limiting the scope.

[0152] The computer program may be stored / distributed on a suitable medium, such as an optical storage medium or solid-state medium provided with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless communication systems. In particular, for example, the computer program may be a computer program product stored on a computer-readable medium, which may have a computer executable program code suitable for being executed to implement a specific method, such as the method according to the present invention. In addition, the computer program may also be a data structure product or a signal for embodying a specific method, such as the method according to the present invention.

Claims

1. A biological tissue analysis device for analyzing biological tissue by laser induced breakdown spectroscopy, comprising: a laser generator (1, 2) configured to provide laser beam pulses to a biological target tissue; Spectrometer module (7, 8, 12); as well as a control unit (5, 6) connected to the laser generator (1, 2) and the spectrometer module (7, 8, 12), wherein the control unit (5, 6) is configured to operate the laser generator (1, 2) to provide at least one of the laser beam pulses as an analysis pulse, the analysis pulse generating an analysis plasma at the laser pulse time, the analysis plasma comprising a reference element in at least a first excited state and a second excited state, and The spectrometer module (7, 8, 12) is configured to collect the emission light of the analytical plasma during the acquisition time and analyze the emission lines in the collected emission light. Features: The control unit (5, 6) is configured to maintain an emission line relationship between a first emission line of the reference element of the analysis plasma at a first wavelength associated with the first excited state and a second emission line of the reference element of the analysis plasma at a second wavelength associated with the second excited state within a predefined reference emission line range.

2. The biological tissue analysis apparatus according to claim 1, wherein: The control unit (5, 6) is configured to maintain the emission line relationship within the predefined reference emission line range by adjusting the laser generator (1, 2) and / or the spectrometer module (7, 8, 12) so that the plasma temperature of the analysis plasma is within a predefined temperature range and the plasma density of the analysis plasma is within a predefined density range.

3. The biological tissue analysis device according to claim 1 or 2, wherein: The reference element is Ca.

4. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: When the reference element is Ca, the first wavelength is in the range of about 393 nm to about 397 nm, in particular about 393 nm or about 396 nm, and the second wavelength is in the range of about 422 nm to about 424 nm, in particular about 423 nm.

5. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The shot line relationship is a ratio between the first shot line and the second shot line.

6. The biological tissue analysis apparatus according to claims 4 and 5, wherein: When the reference element is Ca, the ratio between the first emission line and the second emission line is the ratio between the sum of the emission lines at the first wavelength of about 393 nanometers and / or about 396 nanometers and the emission line at the second wavelength of about 422 nanometers, wherein the ratio is preferably greater than 0.6 and less than 1.

7. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The control unit (5, 6) is configured to keep the shot line relationship within the predefined reference shot line range and / or eliminate backscatter by adapting a delay time, the delay time being the difference between the acquisition time and the laser pulse time.

8. The biological tissue analysis apparatus according to claim 7, wherein: The laser generator (1, 2) is configured to provide a plurality of analysis pulses, and wherein the control unit (5, 6) is configured to evaluate the emission line relationship of each analysis pulse of the plurality of analysis pulses and to adapt the delay time of subsequent analysis pulses to keep the emission line relationship within the predefined reference emission line range and / or eliminate backscatter.

9. The biological tissue analysis apparatus according to claim 7 or 8, wherein: The delay time is in a range of about 0.5 microseconds to about 25 microseconds, or in a range of about 2 microseconds to 10 microseconds.

10. The biological tissue analysis apparatus according to any one of claims 7 to 9, wherein: The control unit (5, 6) is configured to adapt the delay time depending on the reference element and / or the backscatter.

11. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The control unit (5, 6) is configured to keep the shot line relationship within the predefined reference shot line range by adapting the power of the analysis pulses.

12. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The control unit (5, 6) is configured to keep the shot line relationship within the predefined reference shot line range by adapting the integration time.

13. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The laser generator (1, 2) has beam guiding optics configured to focus the plurality of laser beam pulses.

14. The biological tissue analysis apparatus according to claim 13, wherein: The beam directing optics of the laser generator (1, 2) has an emission lens (14) having a focal length of at least about 50 mm, at least about 400 mm, or in the range of about 150 mm to about 250 mm.

15. The biological tissue analysis apparatus according to claim 13 or 14, wherein: The beam directing optics are configured to generate a laser beam having a spot diameter in a range of about 0.1 mm to about 0.4 mm.

16. The biological tissue analysis apparatus according to any one of claims 13 to 15, wherein: The beam directing optics are configured to produce a laser beam having a focal depth greater than 10 millimeters.

17. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The spectrometer module has a converging lens, wherein the analysis pulse has a focus, and wherein the converging lens of the spectrometer module is arranged in the projection of a cone, the vertex of the cone is located at the focus of the analysis pulse, and the cone angle of the cone is less than about 60°, preferably about 30° or about 11°.

18. A biological tissue analysis device according to any one of the preceding claims, comprising an acoustic wave sensor (11) configured to record acoustic shock waves of the analysis pulse provided to the target tissue, wherein The acoustic wave sensor is connected to the control unit (5, 6).

19. The biological tissue analysis apparatus according to any one of claims 13 or 17 and 18, in, The control unit (5, 6) is configured to receive the recorded acoustic shock waves from the acoustic wave sensor (11), and The control unit (5, 6) is configured to keep the recorded acoustic shock wave within the shock wave range by adapting the distance between the beam guiding optics and the target tissue, by adapting the focus of the beam guiding optics, and / or by adjusting the power of the laser generator (1, 2).

20. The biological tissue analysis device according to claim 18 or 19, in, The control unit (5, 6) is configured to position the beam guiding optics and the target tissue at a plurality of distances relative to each other, activate the laser generator (1, 2) to provide at least one analysis pulse at each of the plurality of distances, receive recorded acoustic shock waves from the acoustic wave sensor (11) for each of the plurality of distances, and position the beam guiding optics and the target tissue at one of the plurality of distances that meets a selection criterion.

21. The biological tissue analysis apparatus according to claim 20, wherein: The selection criterion is the height of the intensity of the recorded acoustic shock wave.

22. The biological tissue analysis apparatus according to any one of claims 18 to 21, wherein: The control unit (5, 6) is configured to determine the mass by means of the acoustic shock waves from the acoustic wave sensor (11).

23. The biological tissue analysis apparatus according to any of the preceding claims, comprising light collection optics configured to direct light towards the spectrometer module (7, 8, 12).

24. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The spectrometer modules (7, 8, 12) are configured to turn on in one nanosecond or less and turn off in one nanosecond or less.

25. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The laser generator (1, 2) comprises a high-power Q-switched Nd:YAG laser.

26. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The spectrometer module (7, 8, 12) comprises a gated spectrometer.

27. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The control unit (5, 6) is configured to operate the laser generator (1, 2) to provide at least one of the laser beam pulses as a preparation pulse sequence.

28. The biological tissue analysis apparatus according to claim 27, wherein: The time width of the preparation pulse sequence is greater than the time width of the analysis pulse.

29. The biological tissue analysis apparatus according to claim 27 or 28, wherein: The control unit (5, 6) is configured to operate the laser generator (1, 2) to provide the analysis pulse less than 1 millisecond, less than 1 microsecond, less than 100 nanoseconds, or less than 50 nanoseconds after the preparation pulse sequence.

30. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The control unit (5, 6) is configured to evaluate spectral data provided by the spectrometer module, wherein evaluating the spectral data comprises at least one of the following: Spectra with maximum signals below a predefined value were discarded; discarding spectra that saturate the spectrometer module (7, 8, 12); Spectra associated with air were discarded by analyzing the emission peak intensities of N, O, and / or H; discarding spectra generated outside of a predefined plasma electron temperature and / or plasma electron density; Filtering out inappropriate spectra; and The peak areas of the emission lines are summed or integrated.

31. A biological tissue analysis apparatus according to any one of the preceding claims, wherein: The spectrometer module (7, 8, 12) has a spectrometer board and wherein the control unit (5, 6) is implemented on the spectrometer board.

32. Biological tissue analysis apparatus according to any of the preceding claims, comprising an xyz-stage, wherein: The xyz-stage is configured to support a sample of biological tissue at a predefined position and to move the sample in an x-direction, a y-direction perpendicular to the x-direction, and a z-direction perpendicular to the x-direction and the y-direction.

33. A method for detecting a specific type of cell in a biological target tissue, comprising: a laser generator (1, 2) which provides laser beam pulses to the target tissue, wherein at least one of the laser beam pulses is configured as an analysis pulse, the analysis pulse generates an analysis plasma at the laser pulse time, the analysis plasma comprising a reference element in at least a first excited state and a second excited state; and a spectrometer module (7, 8, 12) which collects the emission light of the analysis plasma during a collection time and analyzes the emission lines in the collected emission light; Features: An emission line relationship between a first emission line of the reference element of the analysis plasma at a first wavelength associated with the first excited state and a second emission line of the reference element of the analysis plasma at a second wavelength associated with the second excited state is maintained within a predefined reference emission line range.

34. The method of claim 33, wherein: The emission line relationship is maintained within the predefined reference emission line range by adjusting the laser generator (1, 2) and / or the spectrometer module (7, 8, 12) so that the plasma temperature of the analysis plasma is within a predefined temperature range and the plasma density of the analysis plasma is within a predefined density range.

35. The method according to claim 33 or 34, wherein: The reference element is Ca.

36. A method according to any one of claims 33 to 35, wherein: When the reference element is Ca, the first wavelength is about 393 nanometers or about 396 nanometers, and the second wavelength is about 423 nanometers.

37. The method of claim 36, wherein: When the reference element is Ca, the predefined reference emission line range is the ratio between the sum of the emission lines at the first wavelength of about 393 nanometers and the emission lines at the second wavelength of about 423 nanometers, wherein the ratio is preferably greater than 0.6 and less than 1.

38. A method according to any one of claims 33 to 37, wherein: The shot line relationship is a ratio between the first shot line and the second shot line.

39. The method according to any one of claims 33 to 38, wherein: By adapting a delay time, which is the difference between the acquisition time and the laser pulse time, the shot line relationship is kept within the predefined reference shot line range and / or backscatter is eliminated.

40. The method of claim 39, wherein: The laser generator (1, 2) provides a plurality of analysis pulses, wherein the emission line relationship of each analysis pulse of the plurality of analysis pulses is evaluated and wherein the delay time of subsequent analysis pulses is adapted to keep the emission line relationship within the predefined reference emission line range and / or to eliminate backscatter.

41. The method according to claim 39 or 40, wherein: The delay time is in a range of about 0.5 microseconds to about 25 microseconds, or in a range of about 2 microseconds to 10 microseconds.

42. The method according to any one of claims 39 to 41, wherein: The delay time is adapted according to the reference element and / or backscattering.

43. The method according to any one of claims 33 to 42, wherein: By adapting the power of the analysis pulses, the emission line relationship is kept within the predefined reference emission line range.

44. The method according to any one of claims 33 to 43, wherein: By adapting the integration time, the shot line relationship is kept within the predefined reference shot line range.

45. The method according to any one of claims 33 to 44, wherein: The laser generator (1, 2) focuses the plurality of laser beam pulses.

46. ​​The method of claim 45, wherein: The focal length of the plurality of laser beam pulses is at least about 50 mm, at least about 400 mm, or in a range of about 150 mm to about 250 mm.

47. The method according to any one of claims 33 or 46, wherein: The spot diameter of the beam pulse is in a range of about 0.1 mm to about 0.4 mm.

48. The method of any one of claims 33 to 47, wherein: The focal depth of the beam pulse is greater than 10 mm.

49. The method according to any one of claims 33 to 48, wherein: An acoustic wave sensor (11) records the acoustic shock waves of the analysis plasma generated by the analysis pulses supplied to the target tissue.

50. The method according to any one of claims 45 to 48 and claim 49, comprising: The recorded acoustic shock wave is kept within the shock wave range by adapting the distance between the laser generator (1, 2) and the target tissue, by adapting the focus of the laser generator (1, 2), or by adjusting the power of the laser generator (1, 2).

51. The method of claim 47 or 48, comprising: The laser generator (1, 2) and the target tissue are positioned relative to each other at a plurality of distances, the laser generator (1, 2) is activated to provide at least one analysis pulse at each of the plurality of distances, the acoustic wave sensor (11) records an acoustic shock wave for each of the plurality of distances, and the laser generator (1, 2) and the target tissue are positioned relative to each other at a distance of the plurality of distances that meets a selection criterion.

52. The method of claim 51, wherein: The selection criterion is the height of the intensity of the recorded acoustic shock wave.

53. A method according to any one of claims 49 to 52, wherein: The mass is determined by the acoustic shock wave from the acoustic wave sensor (11).

54. The method of any one of claims 33 to 53, wherein: The laser generator (1, 2) comprises a high-power Q-switched Nd:YAG laser.

55. The method of any one of claims 33 to 54, wherein: The spectrometer module (7, 8, 12) comprises a gated spectrometer.

56. The method of any one of claims 33 to 55, wherein: At least one of the laser beam pulses is provided as a preparation pulse sequence.

57. The method of claim 56, wherein: The time width of the preparation pulse sequence is greater than the time width of the analysis pulse.

58. The method of claim 56 or 57, wherein: The analysis pulse is provided less than 10 nanoseconds after the preparation pulse sequence.

59. The method of any one of claims 33 to 58, wherein: Spectral data provided by the spectrometer module is evaluated, wherein evaluating the spectral data comprises at least one of: Spectra with maximum signals below a predefined value were discarded; discarding spectra that saturate the spectrometer module (7, 8, 12); discarding spectra associated with air by analyzing the emission peak intensities of N, O, and / or H; and discarding spectra generated outside of a predefined plasma electron temperature and / or plasma electron density; Filtering out inappropriate spectra; and The peak areas of the emission lines are summed or integrated.

60. The method of any one of claims 33 to 59, wherein: The particular cell type is cancer.

61. The method of any one of claims 33 to 60, wherein: The tissue is an extracted tissue, such that the method is an in vitro method.

62. A computer program comprising instructions which, when executed by a computer, cause the computer to perform the method according to any one of claims 33 to 61.

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