Analyzing tissue samples using quantitative phase contrast microscopy

Abstract

Disclosed herein are methods of analyzing a tissue sample using quantitative phase contrast microscopy, as well as corresponding microfluidic systems and corresponding devices. The methods include providing the tissue sample in a sample volume of a microfluidic system, wherein the tissue sample includes a plurality of biological cells forming a continuous tissue material. At least a portion of the tissue sample is lysed into single cells and / or cell aggregates in a carrier fluid in the sample volume. A carrier fluid flow is generated from the sample volume to a measurement volume of the microfluidic system, and a first phase shift image of the single cells and / or cell aggregates in the measurement volume is acquired with a quantitative phase contrast microscope.

Description

[0001] Invention Field

[0002] This invention belongs to the fields of biomedical research and clinical diagnosis. In particular, this invention relates to a method for analyzing tissue samples using quantitative phase contrast microscopy, as well as the corresponding microfluidic system and apparatus.

[0003] background

[0004] Digital holographic microscopy uses the interference between the imaging beam and the reference beam to obtain information about the phase and amplitude of light transmitted through the sample, and, for example, allows for the reconstruction of quantitative phase-shift images of the sample, see EP1524491A1 and EP2357539A1. In recent years, digital holographic microscopy has been successfully used in biomedical applications, such as live-cell imaging. Phase-shift images of cells can be used for morphological parameter-based analysis and / or to reliably identify cell types using machine learning classifiers. Combined with microfluidic systems, this allows for high-throughput label-free blood sample analysis, such as blood cell counting, see, for example, US2019 / 0195774A1, facilitating the detection of diseases such as malaria, leukemia, and myeloproliferative neoplasms, see, for example, M. Ugele et al. Adv.Sci.1800761 (2018) WO2019 / 063548A1 and M. Ugele et al. Proc. SPIE 11060, Optical Methods for Inspection, Characterization, and Imaging of Biomaterials IV, 110600V (2019) .

[0005] While digital holographic microscopy can be used to study cells in liquid samples or cell cultures, including two-dimensional or three-dimensional tumor models, it is not well-suited for studying larger samples such as biopsy specimens. For many diseases, such as pancreatic cancer, which constitutes an important diagnostic tool, tissue samples can be extracted, for example, via endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) biopsy. These samples are typically analyzed using various cytopathological and histopathological techniques to obtain a diagnosis. However, these analyses are slow and expensive, and in some cases, such as up to 25% of pancreatic cancer cases, may not yield definitive results. Summary of the Invention

[0006] Therefore, one object of the present invention is to provide a rapid and reliable method for analyzing tissue samples using digital holographic microscopy.

[0007] This objective is achieved by using the quantitative phase contrast microscope, the microfluidic system, and the apparatus described in this invention for analyzing tissue samples. The embodiments of this invention are described in detail below.

[0008] The method for analyzing tissue samples using quantitative phase-contrast microscopy according to the present invention includes providing a tissue sample in a sample volume of a microfluidic system, wherein the tissue sample comprises a plurality of biological cells forming a continuous tissue material. At least a portion of the tissue sample is dissolved into single cells and / or cell aggregates in a carrier fluid within the sample volume. A carrier fluid flow is generated from the sample volume of the microfluidic system to a measurement volume, and a first phase-shift image of the single cells and / or cell aggregates in the measurement volume is acquired using quantitative phase-contrast microscopy.

[0009] For example, the tissue sample can be a biopsy sample already extracted from the patient, as detailed below. The tissue sample can, for example, have a size of 0.2 mm to 2 mm in each direction and / or 0.01 mm. 3 Up to 10 mm 2 The volume, and can contain, for example, approximately 10 6 Up to 10 9 Individual cells. Cells form continuous tissue material, which may, for example, contain cells and an extracellular matrix that connects cells to each other to form tightly connected structures.

[0010] Tissue samples can be provided, for example, by placing the tissue sample in a sample volume of a microfluidic system, such as through an insertion opening in the sample volume. In some embodiments, the tissue sample can be placed in the sample volume immediately after tissue sample extraction, for example, by inserting a biopsy needle containing the tissue sample into the sample volume and releasing the tissue sample from the biopsy needle. In some instances, providing a tissue sample may include rinsing the tissue sample, for example, to remove non-tissue cells, such as blood cells, before or after providing the tissue sample in the sample volume. After the tissue sample is provided into the sample volume, the sample volume can be closed, for example, by sealing the insertion opening with a capping member.

[0011] Microfluidic systems can be, for example, microfluidic chips having a substrate in which a sample volume and a measurement volume are formed, such as a delivery channel connecting the sample volume and the measurement volume. In other instances, a microfluidic system may include multiple elements, such as a sample chamber enclosing the sample volume, a measurement chamber enclosing the measurement volume, and a tube or channel connecting the sample chamber to the measurement chamber. In some instances, the microfluidic system may be a microfluidic system according to one of the embodiments of the invention described below.

[0012] Subsequently, the tissue sample in the sample volume is at least partially dissolved in the carrier fluid, thereby separating multiple single cells and / or cell aggregates from the tissue sample. The cell aggregates may be, for example, small clusters of cells adhering to each other, such as clusters containing 2 to 20 cells, while the single cells no longer adhere to any other cells from the tissue sample. The carrier fluid may be, for example, an aqueous solution, such as an isotonic solution, and may be provided in the sample volume before, simultaneously with, or after the tissue sample is provided. The carrier fluid may contain oxygen and / or cellular nutrients for the cells in the tissue sample. Additionally or optionally, the carrier fluid may include imaging markers for labeling the cells, such as those detailed below.

[0013] In a preferred embodiment, the tissue sample is dissolved by enzymatic digestion. This is achieved, for example, by adding a dissolving fluid or substance comprising one or more enzymes to a carrier fluid, which may include, for example, one or more enzymes for the enzymatic digestion of the tissue sample. The dissolving fluid or substance may be added to the carrier fluid before or after the sample volume is provided. In other instances, the dissolving fluid or substance may be provided in the sample volume independently of the carrier fluid, for example, before the carrier fluid is provided. The one or more enzymes may comprise one or more digestive enzymes configured to break down macromolecules, such as one or more proteases. The one or more enzymes may, for example, comprise one or more collagenases, such as type II collagenase, and / or trypsin.

[0014] Alternatively or additionally, the tissue sample can be dissolved, for example, by applying ultrasound to the tissue sample. The ultrasound can be applied, for example, via an ultrasound generator (sonotrode) that is in contact with or mechanically coupled to the microfluidic system, particularly applied to the sidewalls of the sample volume or a capping member for inserting an opening. Alternatively or additionally, the ultrasound can be generated, for example, by an ultrasound transmitter included in the microfluidic system. The frequency of the ultrasound can be, for example, from 10 kHz to 10 MHz.

[0015] A carrier fluid flow is generated from the sample volume of the microfluidic system to the measurement volume to deliver single cells and / or cell aggregates separated from a tissue sample to the measurement volume. The carrier fluid flow can be generated during and / or after dissolving the tissue sample and can be a continuous or pulsed flow. The pulsed flow may, for example, include alternating sequences of periods in which the carrier fluid flow is generated and maintained; and periods in which the flow is stopped, for example, to further dissolve the tissue sample before delivering the single cells and / or cell aggregates to the measurement volume.

[0016] A first phase-shift image of a single cell and / or cell aggregate in a measurement volume is acquired using quantitative phase-contrast microscopy. As used herein, the phase-shift image can encode the phase shift of light at one or more wavelengths as a function of position, such as the phase shift of light reflected or propagating through the sample as a function of position in the sample. The first phase-shift image can, for example, quantify the phase shift of light propagating through the measurement volume along the optical axis of the microscope. In some embodiments, the method may include acquiring multiple phase-shift images, for example, as detailed below. In one example, a continuous video stream of phase-shift images may be recorded.

[0017] Preferably, the quantitative phase contrast microscope is configured to determine the absolute value of the phase shift. In other instances, the microscope may simply be configured to determine the phase shift modulus 2π. For example, the microscope may be configured to obtain the phase shift, for example, by interference of light between a probe or imaging beam and a reference beam. In a preferred embodiment, the microscope is a digital holographic microscope, for example, as detailed below.

[0018] By dissolving tissue samples into single cells and / or cell aggregates in a carrier fluid, the method according to the invention provides the composition of tissue samples in a form suitable for analysis using quantitative phase-contrast microscopy, particularly digital holographic microscopy, thereby allowing, for example, rapid and reliable determination of cell type and / or morphology in the tissue sample. Because no additional sample preparation and / or analytical steps such as staining or DNA sequencing are required, this can be advantageous, for example, for diagnosing diseases such as pancreatic cancer. Furthermore, compared to cytological or histopathological methods, more reliable and comprehensive information about the disease state, such as the cellular composition of the tissue sample or the degree of differentiation of the cancer, can be obtained. Moreover, by performing the corresponding steps within the same microfluidic system used for microscopic imaging, the method can be used for automated high-throughput analysis—in contrast to conventional sample preparation techniques, which have been used, for example, as in F. Baharom et al. J. Vis. Exp. (119), e55222 (2017) Flow cytometry analysis of endobronchial biopsy samples is performed, involving multiple sample preparation and transfer steps across multiple devices. Furthermore, time-resolved measurements can be performed as detailed below, which allows for spatial resolution analysis of tissue samples.

[0019] In some embodiments, the method may include moving the tissue sample within a sample volume while dissolving the tissue sample. Specifically, the tissue sample may be moved relative to an outlet of the sample volume in fluid communication with the measurement volume. In this way, cells originating from different portions of the tissue sample, such as portions of the tissue sample adjacent to the outlet, may be selectively delivered to the measurement volume and analyzed using quantitative phase-contrast microscopy. In a preferred embodiment, the tissue sample is moved at an angle relative to the flow direction of the carrier fluid in the sample volume, for example, at 45° to 135°, and in one instance perpendicular to the flow direction of the carrier fluid in the sample volume. For example, a carrier fluid flow may be generated from an input channel via the sample volume to a delivery channel connecting the sample volume to the measurement volume. The tissue sample may be moved relative to openings in the input and delivery channels within the sample volume, such that different portions of the tissue sample are exposed to the carrier fluid flow.

[0020] For example, when the tissue sample is in a first position relative to the outlet of the sample volume, a first phase shift image of single cells and / or cell aggregates in the measurement volume can be acquired. Therefore, the carrier fluid flow to the measurement volume may primarily contain cells from a portion of the tissue sample adjacent to the outlet of the sample volume and / or exposed to the carrier fluid flow when the tissue sample is in the first position. The method may further include acquiring additional phase shift images of single cells and / or cell aggregates in the measurement volume when the tissue sample is in different positions relative to the outlet of the sample volume. For example, when the tissue sample is in a second position relative to the outlet of the sample volume, a second phase shift image of single cells and / or cell aggregates in the measurement volume is acquired, the second position being different from the first position. Preferably, multiple phase shift images of single cells and / or cell aggregates in the measurement volume are acquired, wherein for each phase shift image, the tissue sample is in a corresponding position relative to the outlet of the sample volume, for example, by moving the sample sequentially in a predetermined sequence. Thus, spatial resolving analysis of the tissue sample along the direction of movement can be performed, for example, along a longitudinal range of the tissue sample. In some embodiments, the sample volume and / or measurement volume may be flushed between the first and second phase shift images.

[0021] Alternatively or concurrently, the method may include acquiring multiple phase-shift images at different time points while the tissue sample is in the same location. For example, a first phase-shift image may be acquired at a first time point while the tissue sample is being dissolved, and the method may further include acquiring a third phase-shift image of single cells and / or cell aggregates in the measurement volume at a second time point while the tissue sample is being dissolved. When acquiring the first and third phase-shift images, the tissue sample may be in the same location, for example, in the first location, i.e., the tissue sample may not move between the first and second time points. When the tissue sample is dissolved sequentially between the first and second time points, cells contained in the sample fluid flow at the first time point may originate from the outer portion or layer of the tissue sample, such as the outer surface of the initially provided tissue sample, while cells contained in the sample fluid flow at the second time point may originate from the inner portion or layer of the tissue sample, such as the portion initially covered by the outer surface of the tissue sample. This may, for example, allow spatial resolution analysis of the tissue sample along a radial range (e.g., from the outer surface of the tissue sample to the core of the tissue sample). In some embodiments, the sample volume and / or measurement volume may be flushed between the first and third phase-shift images. In some implementations, multiple phase-shift images of single cells and / or cell aggregates within a measurement volume are acquired without moving the tissue sample, e.g., until the tissue sample is completely dissolved. In some instances, the method may also include moving the sample and acquiring one or more phase-shift images as the sample is in different locations, as described above. In one instance, the sample is repeatedly moved between two or more locations (e.g., first and second locations) while being dissolved. A phase-shift image can be acquired whenever the sample is in one of the two or more locations. This allows spatially resolved information along both the longitudinal and radial extents of the tissue sample to be obtained.

[0022] In some embodiments, the method may further include providing one or more imaging biomarkers, particularly one or more cell type-specific imaging biomarkers, in a carrier fluid for labeling single cells and / or cell aggregates. For example, each imaging biomarker may be configured to selectively bind to one or more cell types and may contain, for example, cell type-specific antibodies. The one or more imaging biomarkers may be configured to facilitate the detection and / or identification of the corresponding cells. For example, an imaging biomarker comprising a biomarker object for phase contrast microscopy may be provided, wherein the biomarker object may, for example, have a predetermined size and / or optical thickness, and may be, for example, a metallic cluster. Alternatively or additionally, an imaging biomarker comprising a biomarker object for fluorescence microscopy may be provided, wherein the biomarker object may, for example, have a characteristic absorption and / or emission spectrum, and may be, for example, a fluorophore.

[0023] In some embodiments, the method may further include performing one or more spectroscopic measurements on single cells and / or cell aggregates. Specifically, the method may include, for example, performing molecular spectroscopic analysis on single cells and / or cell aggregates in a measurement volume. The molecular spectroscopic analysis may include recording one or more spectra of the single cells and / or cell aggregates, such as absorption and / or emission spectra in one or more wavelength ranges. The method may also include determining features in one or more spectra, such as absorption and / or emission lines, for example, to detect the presence, absence, and / or concentration of specific molecules. Performing molecular spectroscopic analysis may, for example, include Raman spectroscopy and / or multiphoton spectroscopy on single cells and / or cell aggregates.

[0024] In a preferred embodiment, the method further includes determining the cell type of one or more cells from the phase-shifted image, particularly from a first phase-shifted image. For example, this may include determining one or more morphological parameters of the cells, and determining the cell type based on said one or more morphological parameters, such as thresholds and / or ranges of the morphological parameters and / or using one or more neural network-based classifiers for the morphological parameters. Alternatively or additionally, computer vision techniques, such as one or more neural network-based classifiers for the phase-shifted image, may be used to determine the cell type of one or more cells. In some embodiments, cell type-specific imaging markers may be used to determine the cell type of one or more cells, for example, by determining whether one or more cells are labeled with imaging markers.

[0025] In some instances, the method may include hydrodynamic focusing and / or viscoelastic focusing of single cells and / or cell aggregates in a measurement volume. For example, this can allow for precise localization of single cells and / or cell aggregates relative to a reference point of the microscope, such as within or adjacent to the focal plane of the microscope and / or at or near the focal point of the microscope. The hydrodynamic focusing may include generating one or more sheath flows, particularly laminar sheath flows, around a carrier fluid flow in the measurement volume, for example, to confine the carrier fluid flow to a small volume along one or more directions, such as at the center of the measurement volume or near the sidewalls of the measurement volume. Viscoelastic focusing may be achieved, for example, by providing a viscoelastic carrier fluid, such as a carrier fluid containing polymers such as polyethylene glycol or hyaluronic acid. The viscoelastic fluid can apply hydrodynamic forces to the single cells and / or cell aggregates, thereby causing controlled motion perpendicular to the flow direction. Preferably, single cells and / or cell aggregates are focused such that they propagate in the same plane or along the same path, for example, such that the center of the single cell and / or cell aggregate is displaced from its respective plane or path by less than 10 μm, less than 5 μm in some instances, and less than 2 μm in one instance.

[0026] The tissue sample may be, for example, a biopsy sample, particularly a fine-needle aspiration biopsy sample. The biopsy sample may be obtained directly from the patient, for example, through an excisional biopsy such as endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), or it may be obtained from tissue previously removed from the patient, for example, through an excisional biopsy or other surgical procedure. For example, the tissue sample may be known or suspected cancerous tissue, particularly pancreatic cancer tissue.

[0027] The present invention also provides a microfluidic system for analyzing tissue samples comprising a plurality of biological cells forming a continuous tissue material, using a method according to any embodiment described herein. The microfluidic system includes a sample volume configured to receive the tissue sample through an insertion opening and an input port in fluid communication with the sample volume and configured to receive a carrier fluid. The microfluidic system further includes a measurement volume in fluid communication with the sample volume via a microfluidic delivery channel and a detection window for acquiring phase-shift images of the cells in the measurement volume using quantitative phase-contrast microscopy, particularly digital holographic microscopy.

[0028] Microfluidic systems can be, for example, microfluidic chips having a substrate in which a sample volume, input port, delivery channel, measurement volume, and / or detection window are formed. The substrate may include one or more layers, each layer of which may comprise, for example, glass, plastic, metal, or a combination thereof, or a combination thereof. In particular, the substrate may include one or more layers comprising a transparent thermoplastic, such as polymethyl methacrylate (PMMA), or a combination thereof. In some instances, the microfluidic system may include multiple separate elements, for example, a sample chamber enclosing a sample volume and a measurement chamber enclosing a measurement volume, wherein the delivery channel may be formed, for example, by a tube connecting the sample chamber to the measurement chamber.

[0029] The sample volume can be enclosed by multiple sidewalls and can, for example, have a cuboid or cylindrical shape. The sample volume can, for example, have dimensions of 0.5 mm to 5 mm in each direction and / or 0.2 mm. 3 Up to 100 mm 2 The volume. The insertion opening may be an opening in one of the sidewalls, wherein the opening may extend on a portion of or throughout the respective sidewall. Preferably, the microfluidic system includes a cap member configured to be placed on or in the insertion opening, for example to close or seal the sample volume.

[0030] The input port may be disposed, for example, in the sidewall of the sample volume and may include an opening leading to the sample volume. In other instances, the input port may be connected to the sample volume via an input channel (e.g., a microfluidic channel in a microfluidic chip having an opening leading to the sample volume). The input port may be configured to receive a fluid connector, for example, connecting the input port to a fluid supply system via a tube. In some instances, the input port may also form an insertion opening, or may be disposed in a cover member for an insertion opening.

[0031] The measurement volume can also be enclosed by multiple sidewalls and can, for example, have a cuboid or cylindrical shape. The measurement volume is connected to the sample volume via a delivery channel, wherein the cross-sectional area of ​​the delivery channel is large enough to allow single cells and / or cell aggregates to pass through the delivery channel while preventing tissue samples from entering the delivery channel. The cross-sectional area of ​​the delivery channel can be, for example, 500 μm. 2 Up to 1 mm 2 In some instances, the measurement volume and the delivery channel can be a segment of the same channel, such as a channel connecting the sample volume to the output port of a microfluidic system or a reservoir; that is, the measurement volume can have the same cross-sectional area as the delivery channel. In other instances, the measurement volume can have a different cross-sectional area than the delivery channel, such as a larger cross-sectional area.

[0032] The detection window can be, for example, a transparent sidewall or a portion thereof of the measurement volume, or a transparent window disposed within the sidewall of the measurement volume. Preferably, the detection window for phase shift measurement is optimized. For example, the transmission wavefront error of the detection window can be less than [a certain value]. / 2, preferably less than / 4, less than in one instance / 8. The detection window may, for example, comprise or consist of transparent thermoplastic, borosilicate glass, and / or fused silica, or a combination thereof. The microfluidic system may also include an illumination window for illuminating cells in the measurement volume, wherein the illumination window may, for example, be arranged on the side of the measurement volume opposite to the detection window. The illumination window may, for example, be a transparent sidewall of the measurement volume facing the detection window, or a transparent window arranged in the sidewall facing the detection window. Preferably, the illumination window is parallel to the detection window and is also optimized for phase shift measurement.

[0033] In a preferred embodiment, the microfluidic system includes a movable element configured to move within a sample volume to move a tissue sample within the sample volume. The movable element may, for example, be configured to contact the tissue sample to push or pull the tissue sample along the sample volume. The movable element may be configured to slide along one or more sidewalls of the sample volume, in one instance, along a guide rail disposed on the respective sidewall. In a preferred embodiment, the movable element is a piston including a cap member configured to seal an insertion opening and slide along the sample volume to move the tissue sample along the sample volume. For example, the cap member may be a plate having a shape corresponding to the cross-section of the sample volume, wherein in some instances, the plate may include sealing elements along its edges. In another instance, the movable element may be disposed in a sidewall of the sample volume or in a cap member for the insertion opening, and may be configured to extend from the sidewall and cap member, respectively. For example, the movable element may be a movable rod disposed in an opening in the sidewall or cap member. The movable element may be configured to be mechanically coupled to an actuator, such as an electric motor, piezoelectric actuator, or hydraulic source. For example, the movable element may come into contact with elements such as rods actuated by an electric motor or hydraulic source.

[0034] As described above, a microfluidic system may include an input channel connecting an input port to a sample volume. Preferably, the openings of the input channel and the delivery channel are arranged on opposite sidewalls of the sample volume and aligned with each other along the flow direction of the input channel, for example, such that fluid flowing from the input channel to the delivery channel flows through the sample volume along a straight or substantially straight path. For example, this can facilitate the generation of a carrier fluid flow through the sample volume, which is confined to a portion of the sample volume, such as the portion of the sample volume extending between the openings of the input channel and the delivery channel. In one example, the input channel, and therefore the flow direction of the input channel at its opening, is perpendicular to the sidewall of the sample volume, and the opening of the input channel is arranged on the sidewall of the sample volume. The opening of the delivery channel may be arranged directly opposite the opening of the input channel on the opposite sidewall, wherein the delivery channel may also extend, for example, perpendicular to the respective sidewall. In another example, the flow direction of the input channel may extend at an angle relative to the respective sidewall, and the opening of the delivery channel on the opposite sidewall may be displaced relative to the opening of the input channel.

[0035] In a preferred embodiment, the microfluidic system includes one or more sheath flow channels in fluid communication with the measurement volume. The one or more sheath flow channels can be configured to generate one or more sheath flows to hydrodynamically focus the carrier fluid in the measurement volume. Each sheath flow channel may terminate, for example, at a corresponding opening in the sidewall of the measurement volume or delivery channel. The flow direction in the sheath flow channels may be angled relative to the flow direction of the delivery channel, for example, to generate a sheath flow surrounding the carrier fluid flow in the measurement volume. The sheath flow may restrict the carrier fluid flow in one or both directions, for example, by restricting it to a narrow sheet, which may have a thickness of, for example, 5 μm to 20 μm; or a narrow tube or filament, which may have a thickness of, for example, 5 μm to 20 μm in two orthogonal directions.

[0036] In some embodiments, the microfluidic system may include a cell sorter configured to sort single cells. The cell sorter may be arranged, for example, along a delivery channel or within a measurement volume. Preferably, the cell sorter is arranged along an output channel in fluid communication with the measurement volume and, for example, connects the measurement volume to one or more output ports and / or one or more storage devices of the microfluidic system. The cell sorter may be configured to selectively guide single cells to one of a plurality of channels or outlets. The cell sorter may, for example, include an active cell sorter configured to sort cells based on a control signal, which may be provided, for example, by an external controller; and / or a passive cell sorter configured to sort cells based on the electro-, magnetic, and / or hydrodynamic properties of the cells. The cell sorter may include one or more electrodes to generate an electric field for sorting cells, and / or one or more inductive elements to generate a magnetic field for sorting cells. Thus, the control signal may, for example, be an electrical control signal, such as voltage or current. Alternatively or additionally, the cell sorter may include one or more optical elements and / or a light source to generate an optical potential for sorting cells, and the control signal may be, for example, a light pulse. In some instances, the cell sorter may include one or more microfluidic elements, such as valves and / or channels, for example, channels that extend at an angle and particularly perpendicular to the output channel to generate a deflecting flow, or include one or more curved channels for hydrodynamic cell sorting.

[0037] The present invention also provides an apparatus for analyzing tissue samples using a method according to any embodiment described herein, the tissue sample comprising a plurality of biological cells forming a continuous tissue material. The apparatus includes a mount configured to receive a microfluidic system, wherein the microfluidic system includes a sample volume configured to receive the tissue sample and a measurement volume in fluid communication with the sample volume. The apparatus further includes a quantitative phase-contrast microscope and a controller, the quantitative phase-contrast microscope being configured to acquire phase-shifted images of cells in the measurement volume. The controller is configured to dissolve at least a portion of the tissue sample into single cells and / or cell aggregates in a carrier fluid within the sample volume and to provide control for generating a carrier fluid flow from the sample volume to the measurement volume.

[0038] The bracket can be configured to hold the microfluidic system in a fixed reference position, for example, relative to a microscope. Preferably, the bracket is configured to position the microfluidic system relative to the microscope, for example, by moving the microfluidic system along one or more directions and / or tilting the microfluidic system about one or more axes. In some instances, the bracket may also include one or more fluid connectors for connecting ports of the microfluidic system, such as input and output ports of the microfluidic system. For example, the microfluidic system may be a microfluidic system according to an embodiment of the invention described herein.

[0039] The quantitative phase contrast microscope is configured to acquire a phase-shift image, that is, an image encoding the phase shift of light at one or more wavelengths as a function of position. Preferably, the quantitative phase contrast microscope is configured to determine the absolute value of the phase shift. In other instances, the microscope may be configured simply to determine the phase shift modulus 2π. For example, the microscope may be configured to obtain the phase shift by, for example, interference of light between a probe or imaging beam and a reference beam. The microscope may be configured to acquire one or more interference images and reconstruct the phase-shift image from one or more interference images. In some instances, the microscope may also be configured to acquire an amplitude or intensity image, wherein the intensity image can encode light intensity as a function of position, for example, the intensity of light reflected or transmitted through the sample as a function of position in the sample. In a preferred embodiment, the microscope is a digital holographic microscope, such as those detailed below. In other instances, the microscope may be a ptychographic microscope.

[0040] The controller can be implemented in hardware, software, or a combination thereof. The controller may, for example, include a processing device and a memory storing instructions executed by the processing device to provide the functions described herein. The controller may be configured, for example, to generate control signals, such as digital and / or analog control signals, to control tools used for dissolving tissue samples and for generating carrier fluid flows. In some instances, the controller may be configured to perform methods for analyzing tissue samples, at least in part, according to one of the embodiments described herein.

[0041] The tool for dissolving tissue samples may, for example, at least partially include one or more microfluidic elements, such as valves and / or pumps, which may be configured, for example, to supply a dissolving fluid to the sample volume and / or to mix the dissolving fluid with a carrier fluid, wherein the dissolving fluid may, for example, include one or more enzymes for enzymatic digestion of the tissue sample, as detailed below. The controller may, for example, be configured to generate corresponding control signals for opening and closing the valve and / or for turning the pump on and off, for example, to initiate or stop the dissolution of the tissue sample. Additionally or alternatively, the tool for dissolving tissue samples may, for example, at least partially include an ultrasound source configured to apply ultrasound to the sample volume. The controller may, for example, be configured to generate corresponding control signals for turning the ultrasound source on and off, for example, to initiate or stop the dissolution of the tissue sample.

[0042] The tool used to generate the carrier fluid flow may also include one or more microfluidic elements, such as valves and / or pumps, which in some instances may also serve as tools for dissolving tissue samples, for example, if the carrier fluid contains dissolving fluids or dissolving substances. The controller may, for example, be configured to generate corresponding control signals for opening and closing valves and / or for turning pumps on and off, for example, to start or stop the carrier fluid flow.

[0043] In some embodiments, the device includes an ultrasonic source configured to apply ultrasound to a sample volume of the microfluidic system. A controller may be configured to control the ultrasonic source, for example, by providing a corresponding control signal, to dissolve at least a portion of the tissue sample by applying ultrasound to the tissue sample in the sample volume. The ultrasonic source may, for example, include an ultrasonic generator configured to contact or mechanically couple with a bracket and / or a cap member of the microfluidic system, particularly a sidewall of the sample volume, for an insertion opening into the sample volume. Additionally or alternatively, the ultrasonic source may, for example, include an ultrasonic transmitter integrated into the bracket. The ultrasonic source may, for example, be configured to generate vibrations at a frequency of 20 kHz to 40 kHz.

[0044] In a preferred embodiment, the device includes a microfluidic unit configured to supply one or more fluids to a microfluidic system. The microfluidic unit may include one or more reservoirs for storing fluid and / or one or more pumps and / or one or more valves for supplying fluid to the microfluidic system. The microfluidic unit may include one or more output ports configured to connect to corresponding input ports of a bracket and / or the microfluidic system. The controller may be configured to control the microfluidic unit to supply fluid to the microfluidic system.

[0045] The microfluidic unit may be specifically configured to supply a dissolving fluid to a sample volume, wherein the dissolving fluid includes an enzyme for the enzymatic digestion of the tissue sample, for example, as detailed above with respect to the method according to the invention. The microfluidic unit may include a reservoir for storing the dissolving fluid and a pump and / or one or more valves for supplying the dissolving fluid to the microfluidic system. The controller may be configured, for example, to control the microfluidic unit by providing corresponding control signals to dissolve at least a portion of the tissue sample by supplying the dissolving fluid to the sample volume. The controller may, for example, be configured to control the microfluidic unit to supply a predetermined amount of dissolving fluid to the sample volume and / or to generate a flow of dissolving fluid through the sample volume over a predetermined period of time.

[0046] Preferably, the microfluidic unit is further configured to generate a carrier fluid flow from the sample volume to the measurement volume, for example, in response to a corresponding control signal provided by a controller. The microfluidic unit may, for example, include a reservoir for storing the carrier fluid and a pump and / or one or more valves for supplying the carrier fluid to the microfluidic system. The controller may be configured to control the microfluidic unit to regulate the flow rate, velocity, and / or duration of the carrier fluid. In some instances, the carrier fluid may correspond to or may contain a dissolved fluid. The controller may, for example, be configured to control the microfluidic unit to mix a dissolved fluid or dissolved substance into the carrier fluid.

[0047] In some instances, the microfluidic unit is also configured to supply sheath fluid to the microfluidic system for generating one or more sheath flows in the measurement volume. The microfluidic unit may, for example, include a reservoir and a pump and / or one or more valves for storing the sheath fluid in the microfluidic system. In some instances, the sheath fluid may have the same composition as the carrier fluid and may, for example, be stored in the same reservoir. The controller may be configured to control the microfluidic unit to hydrodynamically focus the carrier fluid in the measurement volume by supplying sheath fluid to the microfluidic system, for example, to one or more sheath flow channels of the microfluidic system. The controller may be configured to control the microfluidic unit to regulate the flow rate, velocity, and / or duration of the sheath fluid.

[0048] In a preferred embodiment, the device further includes an actuator configured to move a movable element within a sample volume, wherein a controller is configured to control the actuator to move the movable element within the sample volume to move a tissue sample within the sample volume. The actuator may, for example, include an electric motor, a piezoelectric actuator, and / or a hydraulic source, and may be mechanically coupled to and / or configured to be mechanically coupled to the movable element. The movable element may be included in the device or may be part of a microfluidic system, such as as described above. The movable element may, for example, be a piston with a cap member configured to be inserted into the sample volume. For example, the cap member may be configured to seal an opening in the sample volume, such as an insertion opening, and slide along the sample volume, for example, when in contact with one or more sidewalls of the sample volume. The controller may be configured to control the actuator to move the piston along the sample volume for moving the tissue sample along the sample volume, for example, by advancing the piston from the insertion opening into the sample volume.

[0049] In some instances, the controller is configured to determine the cell type of one or more cells from a phase-shifted image, for example, as described above with respect to the method according to the invention. Specifically, the controller may be configured to determine one or more morphological parameters of the cells and determine the cell type based on said one or more morphological parameters. Alternatively or additionally, the controller may be configured to use computer vision techniques for analyzing phase-shifted images to determine the cell type.

[0050] In some embodiments, the microfluidic system includes a cell sorter configured to sort single cells based on control signals, such as those described above. The controller may be configured to control the cell sorter to sort cells, for example, by generating corresponding control signals. The controller may be configured, for example, to apply a voltage to one or more electrodes of the cell sorter and / or to apply a current to one or more inductive elements of the cell sorter, for example, to selectively guide cells to one of a plurality of channels or outlets. Additionally or alternatively, the controller may be configured to generate an optical potential for cells in the cell sorter by controlling one or more light sources and / or to generate a deflecting flow in the cell sorter, for example, in a direction perpendicular to the carrier fluid flow.

[0051] Preferably, the controller is configured to control a cell sorter to sort one or more cells whose cell types are determined from a phase-shifted image based on the determined cell types of one or more cells. For example, the controller may be configured to control the cell sorter to separate one or more predetermined cell types from other cell types. In one example, the controller is configured to control the cell sorter to guide cells of a first group of cell types (e.g., cells of a first type) into a first channel and to guide cells that are not of the first group of cell types into a second channel. In another example, the controller is configured to control the cell sorter to guide cells of a first group of cell types into a first channel, to guide cells of a second group of cell types (e.g., cells of a second type) into a second channel, and to guide cells that are neither of the first group of cell types nor of the second group of cell types into a third channel.

[0052] In a preferred embodiment, the quantitative phase contrast microscope is a digital holographic microscope configured to acquire phase shift images and amplitude or intensity images of the sample. The digital holographic microscope may, for example, be configured to interfere an image of the sample (e.g., an imaging beam transmitted through the sample) with a reference beam, wherein the reference beam may or may not pass through the sample. The digital holographic microscope may be configured to extract or reconstruct phase shift and intensity images from one or more interference images, for example, by reconstructing the wavefront of light transmitted through or reflected from the sample. The digital holographic microscope may be a coaxial digital holographic microscope, wherein the imaging beam and the reference beam propagate along the same axis during interference. Preferably, the digital holographic microscope is an off-axis digital holographic microscope, wherein the imaging beam and the reference beam interfere at an angle, and may be configured to extract a phase shift image from a single interference image of the sample. Such digital holographic microscopes are known, for example, from EP1524491A1 and EP2357539A1.

[0053] In some instances, the device may include one or more microfluidic systems according to one of the embodiments of the invention described herein.

[0054] The methods, microfluidic systems, and apparatus according to the invention can also be adapted for use with other detection or measurement techniques, or as a supplement to or alternative to quantitative phase-contrast microscopy. For example, different microscopy techniques can be used, such as bright-field microscopy, fluorescence microscopy, and / or super-resolution microscopy. Additionally or alternatively, any other type of measurement, such as another optical or electrical measurement, can be performed on single cells and / or cell aggregates in the measurement volume; that is, measurements can be performed in lieu of obtaining phase-shifted images in any embodiment of this disclosure, or measurements can be performed in addition to obtaining phase-shifted images in any embodiment of this disclosure.

[0055] Therefore, the present invention also provides a method for analyzing tissue samples using a microfluidic system, the method comprising: (1) providing a tissue sample in a sample volume of the microfluidic system, wherein the tissue sample comprises a plurality of biological cells forming a continuous tissue material; (2) dissolving at least a portion of the tissue sample into single cells and / or cell aggregates in a carrier fluid in the sample volume; (3) generating a carrier fluid flow from the sample volume of the microfluidic system to a measurement volume; and (4) performing a first measurement on the single cells and / or cell aggregates in the measurement volume. The foregoing numbering is for clarity only and does not imply a particular order of execution of the methods.

[0056] In some implementations, a microscope, such as a bright-field microscope or a fluorescence microscope, is used to analyze tissue samples. Therefore, performing the first measurement may include, or correspond to, acquiring a first microscopic image of a single cell and / or cell aggregate in the measurement volume using a microscope. In some instances, the method may include acquiring multiple microscopic images, for example, as described above for phase-shifted images.

[0057] The present invention also provides a microfluidic system for analyzing tissue samples comprising multiple biological cells forming continuous tissue material using a method according to the foregoing embodiments, the microfluidic system comprising: (1) a sample volume configured to receive the tissue sample through an insertion opening; (2) an input port fluidly connected to the sample volume and configured to receive carrier fluid; (3) a measurement volume fluidly connected to the sample volume via a microfluidic delivery channel; and (4) a detection window for acquiring microscopic images of cells in the measurement volume.

[0058] The present invention also provides an apparatus for analyzing a tissue sample comprising multiple biological cells forming continuous tissue material using a method according to the foregoing embodiments, the apparatus comprising: (1) a holder configured to receive a microfluidic system including a sample volume configured to receive the tissue sample and a measurement volume in fluid communication with the sample volume; and (2) a controller configured to dissolve at least a portion of the tissue sample into single cells and / or cell aggregates in a carrier fluid in the sample volume and to control a flow of the carrier fluid from the sample volume to the measurement volume. In other words, in some embodiments, the apparatus according to the invention may not include a quantitative phase-contrast microscope, but may instead include, for example, different measuring devices, such as different types of microscopes and / or electro-measuring devices. In some embodiments, the apparatus may not include any measuring device, but may instead be configured, for example, for use with measuring devices such as microscopes or electro-measuring devices.

[0059] List of Attachments

[0060] The invention and its exemplary embodiments are described in detail below with reference to the accompanying drawings. The drawings show the following schematic diagrams.

[0061] Figure 1 A microfluidic system for analyzing tissue samples according to an exemplary embodiment of the present invention;

[0062] Figure 2 A microfluidic system comprising movable elements and a cell sorter according to an exemplary embodiment of the present invention;

[0063] Figure 3 An apparatus for analyzing tissue samples according to an exemplary embodiment of the present invention;

[0064] Figure 4 According to an exemplary embodiment of the present invention Figure 3 The device's quantitative phase contrast microscope; and

[0065] Figure 5 A flowchart of a method for analyzing tissue samples using quantitative phase contrast microscopy according to an exemplary embodiment of the present invention.

[0066] Description of preferred implementation scheme

[0067] Figure 1 A top view illustrates a schematic diagram of a microfluidic system 100 for analyzing a tissue sample 102 according to an exemplary embodiment of the present invention. The tissue sample 102 comprises a plurality of biological cells forming a continuous tissue material, wherein the cells are interconnected by an extracellular matrix. The tissue sample 102 may, for example, be a needle aspiration biopsy sample previously extracted from a patient.

[0068] The microfluidic system 100 includes a microfluidic chip having a substrate 104, which may include, for example, one or more layers of a transparent thermoplastic, such as polymethyl methacrylate (PMMA), or composed of the above. The substrate 104 may include, for example, one or more structured layers in which some or all of the structures described below are formed; and a pair of capping layers sandwiched between the structured layers to seal the structures therein.

[0069] A substrate 104 surrounds a sample volume 106, which is configured to receive a tissue sample 102 through an insertion opening 106A. The insertion opening 106A may, for example, be configured to receive the tip of a biopsy needle containing the tissue sample 102, so as to release the tissue sample 102 from the biopsy needle within the sample volume 106. Figure 1 In one example, the insertion opening 106A is a lateral insertion opening 106A disposed on the side surface of the substrate 104. In other examples, the insertion opening 106A may be disposed, for example, on the top surface of the substrate 104, for example perpendicular to... Figure 1 The surface in the viewing direction. The sample volume 106 can, for example, have a diameter of 1 mm. 3 Up to 50 mm 3 The sample volume 106 may, for example, have a cuboid or cylindrical shape, with a length of 2 mm to 5 mm along its longitudinal axis, wherein the longitudinal axis may, for example, be perpendicular to the sample volume. Figure 1 Alignment in the X direction. The width and height or diameter of the sample volume 106 perpendicular to the longitudinal axis can be, for example, 0.5 mm to 2 mm. The microfluidic system 100 also includes a cap member 108 configured to be removably placed over the insertion opening 106A of the sample volume 106, for example, to seal the sample volume 106 to the environment of the microfluidic system 100 after the tissue sample 102 has been placed therein.

[0070] Microfluidic system 100 includes, for example, in such Figure 1 An input port 110 on the top surface of the substrate is shown, which is in fluid communication with the sample volume 106 via an input channel 112A. The input port 110 is configured to receive fluid, such as a carrier fluid, which can be supplied to the sample volume 106 via the input channel 112A. The input port 110 is configured, for example, to receive a fluid connector, such as a fluid connector connected to a tube, to connect the microfluidic system 100 to a fluid source, such as a microfluidic unit.

[0071] The microfluidic system 100 also includes a measurement volume 114 formed in a substrate 104 and in fluid communication with a sample volume 106 via a microfluidic delivery channel 112B in the substrate 104. The cross-sectional area of ​​the delivery channel 112B is significantly smaller than that of the sample volume 106, preventing the tissue sample 102 from entering the delivery channel 112B. The delivery channel 112B may, for example, have a diameter of 500 μm perpendicular to the flow direction in the delivery channel. 2 Up to 1mm 2 The cross-sectional area, in one example, is 0.001 mm. 2 up to 0.05 mm 2 .exist Figure 1 In one example, the measuring volume 114 and the conveying channel 112B are adjacent segments of the same channel, meaning the measuring volume 114 has the same cross-sectional area as the conveying channel 112B. In other examples, the measuring volume 114 may have a different cross-sectional area than the conveying channel 112B, for example, a larger cross-sectional area than the conveying channel 112B.

[0072] The measurement volume 114 is in fluid communication with the output port 116 of the microfluidic system 100 via the output channel 112C, wherein the output channel 112C may also be connected to... Figure 1 In this example, the delivery channel 112B and the measurement volume 114 are segments of the same channel. The output port 116 is disposed on the top surface of the substrate 104 and configured to receive a fluid connector, for example, to extract fluid from the microfluidic system 100. In other examples, the microfluidic system 100 may additionally or optionally include a reservoir (not shown) connected to the measurement volume 114 and configured to receive fluid from the measurement volume 114.

[0073] A detection window 118 is formed on the top surface of the substrate 104, above the measurement volume 114. The detection window 118 is adapted to acquire phase-shifted images using a quantitative phase-contrast microscope. For example, the transmission wavefront error of the detection window can be less than [value missing]. / 2, preferably less than / 4, less than in one instance / 8. The detection window 118 may be, for example, part of a capping layer of the substrate 104, or may be disposed in or on the capping layer. The detection window 118 and / or the capping layer may, for example, comprise transparent thermoplastic, borosilicate glass, and / or fused silica, or a combination thereof. The detection window 118 may cover the entire measurement volume 114 or a portion thereof, and may, for example, have dimensions from 2 mm × 2 mm to 25 mm × 25 mm. In some instances, the microfluidic system 100 may also include an illumination window (not shown) disposed on one side of the measurement volume 114 opposite the detection window 118, for example disposed in or on the bottom surface of the substrate 114, as shown below. Figure 4 As detailed above. The illumination window may be similar to or the same as the detection window 118.

[0074] The microfluidic system 100 also includes a plurality of sheath flow channels 120 in fluid communication with the measurement volume 114. Each sheath flow channel 120 connects its respective port to a portion of a delivery channel 112B in front of the measurement volume 114. Each sheath flow channel 120 may terminate at an outlet disposed in a sidewall of the delivery channel 112B, wherein the flow direction in each sheath flow channel 120, and thus the corresponding flow direction in the sheath flow channel 120, extends at an angle relative to the flow direction in the delivery channel 112B, and thus the corresponding flow direction in the delivery channel 112B. Thus, a plurality of laminar sheath flows 124B can be generated in the measurement volume 114 for hydrodynamic focusing, as detailed below.

[0075] The microfluidic system 100 can be used, for example, with the following reference. Figure 5The described method 500 analyzes a tissue sample 102 using quantitative phase-contrast microscopy. Briefly, the tissue sample 102 in sample volume 106 can be embedded in a carrier fluid supplied to sample volume 106 and can be at least partially dissolved into single cells 122 and / or cell aggregates (not shown), said cell aggregates comprising a small number of cells adhered to each other, for example by an enzyme contained in the carrier fluid or a dissolving fluid or substance added to the carrier fluid. For example, a carrier fluid flow 124A can be generated from sample volume 106 to measurement volume 114 by supplying carrier fluid via input port 110 and extracting carrier fluid via output port 116. The carrier fluid flow 124A can transport single cells 122 and / or cell aggregates separated from tissue sample 102 from sample volume 106 to measurement volume 114. Sheath fluid can be supplied to sheath flow channel 120 to generate sheath flow 124B for hydrodynamically focusing the carrier fluid flow 124A. The sheath flow 124B surrounds the carrier fluid flow 124A and confines the carrier fluid flow 124A, for example, to a plane or filament extending through the microscope focal point in one or two directions perpendicular to the flow direction of the carrier fluid flow 124A. The carrier fluid flow 124A can be concentrated, for example, to 20 μm. 2 Up to 500 μm 2 The cross-sectional area, for example, located at or near the center of the measurement volume 114 or near the detection window 118.

[0076] exist Figure 1 In one example, the microfluidic system 100 further includes an ultrasonic transmitter 126 configured to apply ultrasound to a sample volume 106, particularly a tissue sample 102 disposed therein. The ultrasonic transmitter 126 may be, for example, a sidewall or a portion thereof of the sample volume 106, configured to transmit ultrasound applied to the microfluidic system 100, such as to the outer surface of a substrate 104, into the interior of the sample volume 106. The shape, thickness, and / or material of the ultrasonic transmitter 126 may be adapted to increase the transmission efficiency into the sample volume 106. For example, the resonant frequency of the ultrasonic transmitter 126 may be adapted to the frequency of the ultrasound applied to the microfluidic system 100, and may be, for example, 20 kHz to 40 kHz. In other examples, the ultrasonic transmitter 126 may additionally or optionally include an ultrasonic transmitter configured to convert an electrical signal applied to the ultrasonic transmitter 126 into ultrasound. The ultrasonic transmitter may, for example, include one or more piezoelectric elements and / or one or more capacitive elements configured to convert alternating current (AC) electrical signals into ultrasound.

[0077] Figure 2 A schematic diagram of a microfluidic system 200 for analyzing a tissue sample 102 according to another exemplary embodiment of the present invention is depicted in top view. The microfluidic system 200 is similar to... Figure 1The microfluidic system 100, wherein the corresponding components use the same Figure 1 The same reference numerals are used throughout. Specifically, the microfluidic system 200 also includes a substrate 104, in which a sample volume 106 and a measurement volume 114 configured to receive a tissue sample 102 are formed. The sample volume 106 is connected to an input port 110 on a side of the substrate 104 via an input channel 112A, and to the measurement volume 114 via a delivery channel 112B.

[0078] The input channel 112A and delivery channel 112B of the microfluidic system 200 are aligned with each other such that the opening of the delivery channel 112B is arranged on the sidewall of the sample volume 106 opposite to and directly facing the opening of the input channel 112A. The flow path of the fluid flowing along the input channel 112A extends through the sample volume 106 into the opening of the delivery channel 112B, thereby promoting the generation of a carrier fluid flow 124A, which is confined to the central portion of the sample volume 106 and therefore flows, for example, only around a portion of the tissue sample 102. The flow path of the fluid flowing along the input channel 112A may extend, for example, perpendicular to the sidewall of the sample volume 106, on which the openings of the input and delivery channels 112A, 112B are arranged, as shown below. Figure 2 As shown.

[0079] The microfluidic system 200 also includes a movable element 202 configured to move within the sample volume 106 for moving the tissue sample 102 within the sample volume 106, such as... Figure 2 As indicated by the double-headed arrows. In this way, different portions of the tissue sample 102 can be exposed to the carrier fluid flow 124A and / or the outlet of the adjacent sample volume 106 can be positioned to the delivery channel 112B. Figure 2 In this example, the movable element 202 is a piston including a cap member 202A configured to seal the insertion opening 106A of the sample volume 106. The cap member 202A is configured to slide along the sample volume 106 to move the tissue sample 102 along the sample volume 106. The cap member 202A may, for example, comprise plastic, particularly thermoplastic, or be composed of the above, and may include a sealing member (not shown) along its edge, the sealing member being configured to contact the sidewall of the sample volume 106 to provide a tight seal while moving the cap member 202A along the sample volume 106. The piston 202 also includes a rod or shaft connected to the back of the cap member 202A and configured to be mechanically coupled to an actuator (not shown), such as an electric motor or piezoelectric element, for moving the piston 202 along the sample volume 106.

[0080] The microfluidic system 200 also includes a cell sorter 204 arranged along an output channel 112C, which connects the measurement volume 114 to a pair of output ports 116A, 116B on a side of a substrate 104. The cell sorter 204 includes a bifurcation or junction where the output channel 112C splits into a pair of channels, each channel connecting to a corresponding one of the output ports 116A, 116B. Between the junction and the measurement volume 114, a pair of electrodes 206 are arranged within or on the substrate on opposite sides of the output channel 112C, for example, on or adjacent to opposite sidewalls of the output channel 112C. Each electrode 206 is electrically coupled to a corresponding electrical connector, which may be connected, for example, to a voltage source to apply a voltage to the electrode 206. The voltage applied to electrode 206 generates an electric field on output channel 112C, which, according to the signal of the applied voltage, can be used, for example, to deflect cells in carrier fluid flow 124A in output channel 112C by electrophoresis toward one of the channels separated at the junction and thus toward output ports 116A, 116B.

[0081] Figure 3 A side view schematically illustrates an apparatus 300 for analyzing tissue sample 102 according to an exemplary embodiment of the present invention. The apparatus 300 includes a quantitative phase-contrast microscope 400 configured to acquire phase-shifted images, which in... Figure 4 The apparatus 300 is shown in more detail below. It can be used, for example, to implement a method for analyzing tissue samples according to one of the embodiments described herein, as shown below with reference to... Figure 5 Method 500 is described.

[0082] The device 300 includes a holder 302 configured to receive a microfluidic system 304. The microfluidic system 304 includes a sample volume 106 configured to receive a tissue sample 102 and a measurement volume 114 in fluid communication with the sample volume 106. The microfluidic system 304 may be similar to one of the microfluidic systems 100 and 200 described above, wherein the corresponding elements are used in conjunction with… Figure 1 and 2 The same reference numerals are used to denote these systems. Specifically, the microfluidic system 304 also includes an input port 110 and an output port 116. The input port 110 is disposed on the top surface of the microfluidic system 304 and is in fluid communication with the sample volume 106, while the output port 116 is disposed on the side surface of the microfluidic system 304 and is in fluid communication with the measurement volume 114. The holder 302 can also be configured to receive other microfluidic systems, particularly those according to an embodiment of the invention described herein, such as microfluidic system 100 and / or microfluidic system 200.

[0083] The bracket 302 is configured to hold the microfluidic system 304 in place, for example, to prevent the microfluidic system 304 from moving at least along... Figure 3 The Z-axis, preferably, moves relative to the focal point of the microscope 400 along all three axes. The bracket 302 may, for example, include one or more fasteners (not shown) for securing the microfluidic system 304 to the bracket 302. Preferably, the bracket 302 is configured, for example, to move along the Z-axis, preferably along all three axes. Figure 3 The position and / or orientation of the microfluidic system 304 can be adjusted by moving the microfluidic system 304 along the X, Y, and / or Z axes and / or by tilting the microfluidic system 304 about the X, Y, and / or Z axes, wherein the Y axis is parallel to the Z axis. Figure 3 The view orientation is aligned.

[0084] The apparatus 300 also includes a controller 306 configured to control the dissolution of at least a portion of the tissue sample 102 into single cells (not shown) and / or cell aggregates (not shown) in a carrier fluid within a sample volume 106 of the microfluidic system 304, and to generate a carrier fluid flow 124A from the sample volume 106 to the measurement volume 114. The controller 306 may be implemented in hardware, software, or a combination thereof, and may include, for example, a processing device and a memory storing instructions executed by the processing device to provide the functionality described herein. The controller 306 may, for example, include a central processing unit (CPU), a graphics processing unit (GPU), a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), and / or a microcontroller. Preferably, the controller 306 is configured to at least partially perform the method 500 for analyzing the tissue sample, such as some or all of steps 504 to 512.

[0085] exist Figure 3 In this example, controller 306 is configured to control microfluidic unit 308, which may be part of device 300 or provided as a stand-alone unit. Microfluidic unit 308 includes a reservoir 308A configured to store fluid, such as carrier fluid and / or dissolving fluid, and is connected to input port 110 of microfluidic system 304. Microfluidic unit 308 may also include a pump (not shown) and / or one or more valves (not shown) for supplying fluid stored in reservoir 308A to input port 110. Controller 306 may be configured to generate control signals for the pump and / or one or more actuators, configured to open and close the valves to start and stop fluid flow to measurement volume 106, for example, generating a carrier fluid flow from sample volume 106 to measurement volume 114 and / or supplying a dissolving fluid containing one or more enzymes for enzymatic digestion of tissue sample 102.

[0086] In some embodiments, the microfluidic unit 308 may also be configured to supply sheath fluid to the microfluidic system 304 to generate sheath flow in the measurement volume, for example, as referenced above. Figure 1 The microfluidic unit 308 may, for example, include an additional reservoir (not shown) and a pump (not shown) and / or one or more valves (not shown) for storing sheath fluid, which supply sheath fluid to a corresponding port of the microfluidic system 304. In other instances, the carrier fluid stored in the reservoir 308A may also be used as sheath fluid, for example, by connecting the reservoir 308A to a corresponding port of the microfluidic system 304. The controller 306 may be configured to control the microfluidic unit 308 to hydrodynamically focus the carrier fluid in the measurement volume 114 by supplying sheath fluid to the microfluidic system 304.

[0087] The controller 306 is also configured to control an ultrasonic source 310, which may be part of the device 300 or may be provided as a separate unit. The ultrasonic source 310 is configured to apply ultrasound to the sample volume 106 and may, for example, include an ultrasonic generator having one or more ultrasonic transmitters configured to contact the holder 302 and / or the microfluidic system 304. Figure 3 In one example, the ultrasonic source 310 is configured to apply ultrasound to the sample volume 106 in a targeted manner, for example by positioning the ultrasonic generator against the sidewalls of the sample volume 106 and / or the ultrasonic transmitter of the microfluidic system 304 (e.g., Figure 1 The ultrasonic source 310 contacts the ultrasonic transmitter 126 of the microfluidic system 100. In other instances, the ultrasonic source 310 may be configured to apply ultrasound to the entire microfluidic system 304, for example, via the bracket 302. In one instance, the ultrasonic source 310 or a combination thereof, such as an ultrasonic generator and / or one or more ultrasonic transmitters, may be integrated into the bracket 302.

[0088] The device 300 further includes an actuator 312 configured to move a movable element 202 within the sample volume 106, wherein the movable element 202 may be provided as part of the microfluidic system 304 or the device 300. The movable element 202 may, for example, be a piston with a cap member configured to seal the insertion opening 106A of the sample volume 106 and advanced into the sample volume 106 by the actuator 312, such as... Figure 3As shown, for example, the cap member slides along the sidewall of the sample volume 106 while sealing a corresponding portion of the sample volume 106. The actuator 312 may, for example, include an electric motor (not shown) configured to be mechanically coupled to the movable element 202. The electric motor may, for example, move a rod or shaft in contact with the movable element 202. In another example, the actuator 312 may include a piezoelectric actuator that is in contact with or mechanically coupled to the movable element 202. In yet another example, the actuator 312 may include a hydraulic pressure source configured to apply positive or negative pressure to the movable element 202 via hydraulic fluid to move the movable element 202. The controller 306 is configured to control the actuator 312 by generating corresponding control signals to move the movable element within the sample volume 106, particularly for moving the tissue sample 102 to a designated location within the sample volume 106.

[0089] The controller 306 can also be configured to provide other functions, such as controlling the bracket 302 and / or elements of the microfluidic system 304, such as a cell sorter (not shown) and / or one or more valves. The controller 306 can be specifically configured to control the quantitative phase contrast microscope 400 and / or acquire and analyze phase-shifted images acquired by the quantitative phase contrast microscope 400, for example, as shown in the following reference. Figure 4 Detailed explanation.

[0090] Figure 4 An exemplary embodiment of the present invention is depicted. Figure 3 A schematic diagram of a quantitative phase contrast microscope 400 of the apparatus 300. The quantitative phase contrast microscope 400 is a digital holographic microscope configured to acquire phase-shift images and amplitude or intensity images of a sample (particularly single cells 122 and / or cell aggregates in the measurement volume 114 of the microfluidic system 304).

[0091] The digital holographic microscope 400 includes an imaging system 402 having an objective lens 404, a holographic imaging system 406, and an imaging lens 408. The imaging system 402 is configured to image its focal plane onto a lens 410, for example, through a detection window 118 in the top surface of a microfluidic system 304. The lens may be, for example, a CCD or CMOS lens. The focal plane of the imaging system 402 is located within a measurement volume 114, preferably in the plane along which the carrier fluid flow 124A flows. The microscope 400 also includes an illumination source 412 for illuminating the measurement volume 114 and a controller 414 configured to acquire an image from the lens 410. The controller 414 may also be configured to control the holographic imaging system 406, the lens 410, and / or the illumination source 412.

[0092] Objective 404 may be, for example, a high NA objective with a numerical aperture greater than 0.4, and in some instances, greater than 0.5. The depth of field of objective 404 may be less than 10 μm, preferably less than 5 μm, wherein the depth of field may be defined, for example, as the minimum Rayleigh length of the laser beam focused by objective 404 at a wavelength of 1064 nm. This allows for precise focusing on objects such as cells within measurement volume 114, for example in a hydrodynamically focused carrier fluid flow 124A, and can provide sufficient spatial resolution to resolve the morphological features of the cells.

[0093] The holographic imaging system 406 is configured to create an interference image on the lens 410, for example, by interfering the imaging beam with a reference beam on the lens 410. The imaging beam may, for example, be a beam that passes through the measurement volume 114 and propagates from the focal plane of the imaging system 402 to the lens 410 along a first optical path through the holographic imaging system 406. The reference beam may, for example, be a beam that propagates to the lens 410 along a second optical path through the holographic imaging system 406. In some instances, the reference beam may be separated from the imaging beam, for example, using a beam splitter or diffraction grating; that is, the reference beam may have already passed through the measurement volume 114 and can propagate from the focal plane of the imaging system 402 to the lens 410 along the second optical path. In other instances, the reference beam may not pass through the measurement volume 114 and may, for example, be separated from the imaging beam in front of the measurement volume 114.

[0094] The digital holographic microscope 400 can be a coaxial digital holographic microscope, where the imaging beam and the reference beam propagate along the same axis during interference, i.e., interfere at a 0° angle. The controller 414 can, for example, be configured to extract or reconstruct a phase-shifted image and an intensity image of the sample from multiple interference images by using the holographic imaging system 406 to change the phase shift between the reference beam and the imaging beam. Preferably, the microscope 400 is an off-axis digital holographic microscope, where the imaging beam and the reference beam interfere at an angle. In this case, the controller 414 can be configured to extract or reconstruct a phase-shifted image and an intensity image of the sample from a single interference image.

[0095] Illumination source 412 is configured to illuminate measurement volume 114, for example, through an illumination window in the bottom surface of microfluidic system 304 opposite to detection window 118. Illumination source 412 is configured to illuminate measurement volume 114 with spatially and / or temporally coherent light, wherein the coherence length of the illumination light can be, for example, greater than the field of view of microscope 400, and the coherence time of the illumination light can be, for example, greater than the time delay between the image and the reference beam, so that an interference pattern can be observed on lens 410. Illumination source 412 may, for example, include a laser or a light-emitting diode, and can be configured to emit monochromatic light, for example, at wavelengths in the range of 500 nm to 1100 nm.

[0096] Controller 414 may be implemented in hardware, software, or a combination thereof. Controller 414 may be configured to provide phase-shift and intensity images to another device (e.g., controller 306 of device 300). In some instances, controller 414 or a portion thereof may be integrated into controller 306. In addition to reconstructing phase-shift and intensity images, controller 414 may also be configured to analyze phase-shift and / or intensity images. Controller 414 may, for example, be configured to determine the cell type of one or more cells in an image by determining one or more morphological parameters of cells from the phase-shift and / or intensity images and determining the cell type based on the morphological parameters. Controller 414 may be configured to analyze phase-shift and / or intensity images using computer vision techniques and may, for example, be configured to determine the cell type of one or more cells from the phase-shift images using a neural network-based classifier.

[0097] In some instances, microscope 400 may also be configured to acquire images, particularly intensity images, of single cells 122 and / or cell aggregates in measurement volume 114 using other imaging techniques, such as bright-field microscopy, fluorescence microscopy, and / or super-resolution microscopy. Optionally, apparatus 300 may also include a second microscope (not shown) configured to acquire such images.

[0098] In some embodiments, the controller 306 of the device 300 is configured to control the cell sorter of the microfluidic system 304 based on the determined cell type of one or more cells, for example, to selectively separate a certain type of cell from other cells in the carrier fluid flow 124A. The controller 306 may, for example, be configured to determine whether a corresponding type of cell is included in the phase-shifted image. The controller 306 may be configured to determine the arrival time of the corresponding cell in the cell sorter, for example, based on the cell's position in the phase-shifted image and the flow rate of the carrier fluid flow 124. The controller 306 may be configured to provide a control signal, such as a voltage, to the cell sorter at the arrival time, for example, to deflect the cell to one of a plurality of channels.

[0099] Figure 5 A flowchart of a method 500 for analyzing tissue samples using quantitative phase-contrast microscopy according to an exemplary embodiment of the present invention is shown. For example, the method 500 can be used... Figure 3 Device 300 and Figure 2 The method 500 is implemented using a microfluidic system 200, which is used below as an example for illustrative purposes. However, this is not intended to be limiting in any way, and the method 500 can also be implemented using different devices with quantitative phase contrast microscopy and / or using different microfluidic systems, such as... Figure 1 microfluidic systems 100 or Figure 3 The microfluidic system 304. Furthermore, method 500 is not limited to... Figure 5 The execution order is shown in the flowchart. Method 500 can be executed in any order, provided it is technically feasible, and parts of it can be executed at least partially simultaneously.

[0100] In step 502, a tissue sample 102 is provided in a sample volume 106 of the microfluidic system 200. The tissue sample 102 comprises a plurality of biological cells forming a continuous tissue material, wherein the cells are interconnected by an extracellular matrix. The tissue sample 102 may, for example, be a biopsy sample previously extracted from a patient, for example, by incision or excision biopsy. Preferably, the tissue sample is a needle aspiration biopsy sample, which may be extracted directly from the patient or from a larger tissue mass already removed from the patient. The tissue sample 102 may be provided in the sample volume 106, for example, by inserting the tip of a biopsy needle containing the tissue sample 102 into the sample volume 106 through an insertion opening 106A and releasing the tissue sample 102 from the biopsy needle. Subsequently, the sample volume 106 may be sealed by placing a capping member 202A into the insertion opening 106A. In some instances, step 502 may include flushing the tissue sample 102 before or after providing the tissue sample 102 to the sample volume 106, for example, to remove blood from the tissue sample 102.

[0101] In step 504, at least a portion of the tissue sample 102 is dissolved into single cells 122 and / or cell aggregates, wherein the cell aggregates may be, for example, small clusters each containing 2 to 20 cells. For this purpose, a carrier fluid (e.g., an isotonic fluid and / or a buffer solution, such as phosphate-buffered saline (PBS) or a minimal basal medium such as Dulbecco's modified Eagle's medium (DMEM)) is supplied to the sample volume 106, for example, through the input port 110 and / or the insertion opening 106A. The carrier fluid may be supplied, for example, by the controller 306 using a microfluidic unit 308. The fluid may fill the entire sample volume 102 and may completely surround the tissue sample 102.

[0102] The tissue sample 102 can be dissolved, for example, by enzymatic digestion. For this purpose, the dissolved fluid can be supplied to the sample volume 106, for example, via a controller 306 using a microfluidic unit 308. The dissolving fluid includes one or more enzymes for the enzymatic digestion of the tissue sample 102. The one or more enzymes may, for example, comprise one or more collagenases, such as type II collagenase, and / or trypsin. In some instances, the carrier fluid may include one or more enzymes, i.e., also be used as the dissolving fluid, or the dissolving fluid may be supplied to a reservoir 308A or connected to a channel of the input port 110 to mix the dissolving fluid into the carrier fluid before it is supplied to the sample volume 106. The tissue sample 102 can be exposed to one or more enzymes for a predetermined amount of time, for example, 5 to 60 minutes, or 20 to 40 minutes in one instance. For example, the exposure time can be selected based on the size of the tissue sample 102, the type of tissue in the tissue sample 102, and / or one or more enzymes. In some embodiments, one or more enzymes are removed from the sample volume 106 before proceeding to step 506. In other instances, tissue sample 102 may be continuously exposed to one or more enzymes during some or all of steps 506 to 512, for example until tissue sample 102 is completely dissolved.

[0103] In some instances, one or more enzymes may be supplied to sample volume 106 at different time points and / or tissue sample 102 may be exposed to one or more enzymes at different time intervals. For example, a first enzyme may be supplied to sample volume 106 at a first time point, and a second enzyme may be supplied to sample volume 106 at a second time point later than the first time point, wherein the first enzyme may be removed from sample volume 106 before the second enzyme is supplied or may not be removed from sample volume 106 before the second enzyme is supplied. Alternatively or additionally, the tissue sample may be exposed to the first enzyme at a first exposure time and to the second enzyme at a second exposure time different from the first exposure time. For example, one enzyme may be type II collagenase and the other enzyme may be trypsin, wherein the exposure time to trypsin may be shorter than the exposure time to type II collagenase, for example, to avoid damage to cell surface proteins due to prolonged exposure of tissue sample 102 to trypsin.

[0104] Alternatively or additionally, tissue sample 102 can be dissolved by applying ultrasound to the tissue sample, for example using ultrasound source 310 via controller 306. The frequency of the ultrasound can be, for example, 10 kHz to 10 MHz, and in one example, 20 kHz to 100 kHz, and can be applied for, for example, a predetermined amount of time, such as 30 seconds to 60 minutes. The ultrasound can be applied continuously or as a pulse sequence, for example, during the predetermined amount of time. In some embodiments, tissue sample 102 can be permanently or repeatedly exposed to ultrasound during some or all of steps 506 to 512, for example, until tissue sample 102 is completely dissolved. For example, the application of ultrasound can be combined with enzymatic digestion by applying ultrasound before, during, and / or after the above-described enzymatic digestion.

[0105] In step 506, a carrier fluid flow 124A is generated from the sample volume 106 to the measurement volume 114. The controller 306 may, for example, control the pump and / or one or more valves of the microfluidic unit 308 to supply carrier fluid from the reservoir 308A to the input port 110, thereby generating a carrier fluid flow 124A through the microfluidic system 200 from the input port 110 to the output ports 116A, 116B. The carrier fluid flow 124A may transport single cells 122 and / or cell aggregates separated from the tissue sample 102 from the sample volume 106 to the measurement volume 114. In some instances, the carrier fluid flow 124A may be a continuous flow, for example, which is maintained for a predetermined amount of time while, for example, dissolving the tissue sample 102. In other instances, the carrier fluid flow 124A may be a pulsed flow. For example, the pulsed flow may include alternating sequences of periods in which a carrier fluid flow 124A is generated and maintained to deliver single cells and / or cell aggregates to the measurement volume 114; and periods in which the carrier fluid flow 124A is stopped, for example, to further dissolve the tissue sample 102 before delivering single cells and / or cell aggregates to the measurement volume 114 again. In one example, the carrier fluid flow 124A may be maintained for a period of 1 second to 60 seconds before stopping for a period of 1 minute to 30 minutes. As mentioned above, the carrier fluid may also be or include a dissolving fluid for enzymatic digestion of the tissue sample, i.e., it may include one or more enzymes.

[0106] In step 508, a first set of phase-shifted images of single cells 122 and / or cell aggregates in measurement volume 114 is acquired using a quantitative phase-contrast microscope 400. The first set of phase-shifted images may be, for example, a single image or multiple images. Specifically, the first set of phase-shifted images may be, for example, a continuous video stream of images with a repetition rate of 15 Hz to 240 Hz, which may be recorded simultaneously with the single cells 122 and / or cell aggregates being transported through measurement volume 114 via carrier fluid flow 124A.

[0107] Subsequently, in step 510, tissue sample 102 is moved relative to the outlet of sample volume 102, which is in fluid communication with measurement volume 114, for example, relative to the opening of delivery channel 112B in the sidewall of sample volume 106. Tissue sample 102 can be moved, for example, from a first position in sample volume 102 (e.g., the position of tissue sample 102 in steps 506 and / or 508) to a second position. To move tissue sample 102, piston 202 having movable element 202A can be advanced from insertion opening 106A into sample volume 106, thereby further pushing tissue sample 102 into sample volume 106. Tissue sample 102 can be moved, for example, between the first and second positions by 50 μm to 1 mm. In one example, the front of tissue sample 102 may be adjacent to the opening of delivery channel 112B and exposed to carrier fluid flow 124A when tissue sample 102 is in the first position. Accordingly, different portions of the tissue sample 102, such as the portion adjacent to the front, or the central portion, may be adjacent to the opening of the delivery channel 112B and exposed to the carrier fluid flow 124A when the tissue sample 102 is in the second position. After the tissue sample has been moved to the second position, dissolution of the tissue sample may continue, for example, by means of an enzyme contained in the carrier fluid, by providing a dissolving fluid, and / or by applying ultrasound.

[0108] In step 512, while the tissue sample 102 is in the second position, a second set of phase-shifted images of single cells 122 and / or cell aggregates in the measurement volume 114 is acquired using a quantitative phase-contrast microscope 400. Similar to the first set of phase-shifted images, the second set of phase-shifted images may be, for example, a single image or multiple images, particularly a continuous video stream of images.

[0109] In some embodiments, steps 510 and 512 can be repeated sequentially, for example, by moving tissue sample 102 sequentially through the outlet of sample volume 106. Tissue sample 102 can be moved sequentially, for example, in isometric steps of 50 μm to 500 μm. By exposing different portions of tissue sample 102 to the carrier fluid flow 124A, information about the cellular composition of tissue sample 102 can be obtained and spatially resolved along the longitudinal direction of tissue sample 102 (corresponding to the direction in which tissue sample 102 moves within sample volume 106). Tissue sample 102 can be moved sequentially until its end is adjacent to the outlet of sample volume 106, acquiring a set of phase-shifted images at each location.

[0110] In one instance, tissue sample 102 can then be moved back to the first position, and sequential scanning can be repeated. Due to the dissolution of tissue sample 102, the outer layer of tissue sample 102 may have been removed, thereby exposing the inner layer of tissue sample 102. Therefore, the carrier fluid flow 124A may primarily comprise single cells 122 and / or cell aggregates originating from the inner layer, which can be imaged by acquiring another set of phase-shifted images. This allows information about the cellular composition of tissue sample 102 to be obtained, spatially resolved along the longitudinal direction of tissue sample 102 and in the radial direction from the outer surface of tissue sample 102 to the core of tissue sample 102. In some embodiments, tissue sample 102 may not be moved during the execution of method 500, but multiple sets of phase-shifted images can be acquired at different time points while dissolving tissue sample 102.

[0111] Method 500 may also include additional steps. Method 500 may, for example, include generating a sheath flow 124B by supplying a sheath fluid, such as a carrier fluid or a fluid having a different composition from the carrier fluid, to the sheath flow channel 120, so as to hydrodynamically focus the carrier fluid flow 124B onto, for example, the focal plane or focal point of a microscope 400. Additionally or alternatively, the carrier fluid flow 124A may be viscoelastically focused, for example, by providing a viscoelastic carrier fluid containing a polymer such as polyethylene glycol or hyaluronic acid.

[0112] Method 500 may also include, for example, determining the cell type of one or more cells from the phase-shift image based on morphological characteristics of the cells and / or using a machine learning classifier. In one embodiment, cell type-specific imaging markers, such as metal clusters attached to cell type-specific antibodies, may be provided to, for example, a sample volume 106 and / or a measurement volume 114 in a carrier fluid, and the imaging markers may be used to determine the cell type.

[0113] Method 500 may further include sorting single cells 122 and / or cell aggregates using a cell sorter 204, for example by selectively applying a voltage to electrode 206 to deflect one or more cells 122 and / or cell aggregates in the carrier fluid flow 124A into channels respectively connected to output ports 116A and 116B. For example, a voltage may be applied such that single cells 122 and / or cell aggregates of a first cell type are deflected toward output port 116A, while all other cells 122 and / or cell aggregates are deflected toward output port 116B.

[0114] The embodiments of the invention disclosed herein constitute only specific examples for illustrative purposes. The invention can be implemented in various ways and with many modifications that do not alter its underlying fundamental characteristics. Therefore, the invention is limited only by the stated claims.

[0115] Reference Symbol List

[0116] 100-Microfluidic System

[0117] 102-Tissue Sample

[0118] 104-substrate

[0119] 106 - Sample volume

[0120] 106A-Insert Opening

[0121] 108-Cover Components

[0122] 110-Input Port

[0123] 112A-Input Channel

[0124] 112B - Conveyor Channel

[0125] 112C - Output Channel

[0126] 114 - Measuring Volume

[0127] 116, 116A, 116B - Output Ports

[0128] 118-Detection Window

[0129] 120-Sheath Flow Channel

[0130] 122-Single Cell

[0131] 124A-Carrier Fluid Flow

[0132] 124B-Sheath Flow

[0133] 126-Ultrasonic transmitter

[0134] 200-Microfluidic System

[0135] 202-Modible Components / Piston

[0136] 202A-Cover Components

[0137] 204-Cell Sorter

[0138] 206-electrode

[0139] 300-device

[0140] 302-Bracket

[0141] 304-Microfluidic Systems

[0142] 306-Controller

[0143] 308-Microfluidic Unit

[0144] 308A - Storage

[0145] 310-Ultrasound Source

[0146] 312-Actuator

[0147] 400-Quantitative Phase Contrast Microscope

[0148] 402 Imaging System

[0149] 404 Objective Lens

[0150] 406-Holographic Imaging System

[0151] 408-Imaging Lens

[0152] 410-lens

[0153] 412-Lighting Source

[0154] 500 - Methods for analyzing tissue samples

[0155] 502 - Steps for providing tissue samples

[0156] 504 - Steps for at least partially dissolving tissue samples

[0157] 506 - Steps for generating carrier fluid flow

[0158] 508 - Steps for obtaining the first set of phase-shifted images

[0159] 510 - Procedure for moving tissue samples within a sample volume

[0160] 512 - Steps to obtain the second set of phase-shifted images

Claims

1. A method (500) for analyzing tissue samples (102) using a microfluidic system (100, 200, 304), the method (500) comprising: The tissue sample (102) is provided in the sample volume (106) of the microfluidic system (100, 200, 304), wherein the tissue sample (102) comprises a plurality of biological cells forming a continuous tissue material; At least a portion of the tissue sample (102) is dissolved into single cells (122) and / or cell aggregates in a carrier fluid in the sample volume (106); A carrier fluid flow (124A) is generated from the sample volume (106) of the microfluidic system (100, 200, 304) to the measurement volume (114); and A first measurement is performed on single cells (122) and / or cell aggregates in the measurement volume (114). The method (500) further includes, while dissolving the tissue sample (102), moving the tissue sample (102) relative to the outlet of a sample volume (106) in fluid communication with the measurement volume (114), wherein the tissue sample (102) is moved at an angle relative to the flow direction of the carrier fluid in the sample volume (106); and / or The first measurement was performed at the first time point while the tissue sample (102) was being dissolved; and The method (500) further includes performing a second measurement on single cells (122) and / or cell aggregates in the measurement volume (114) at a second time point while dissolving the tissue sample (102). The single cells (122) and / or cell aggregates in the measurement volume (114) at the first time point originate from the external portion of the tissue sample (102), and the single cells (122) and / or cell aggregates in the measurement volume (114) at the second time point originate from the internal portion of the tissue sample (102).

2. The method (500) according to claim 1, wherein the tissue sample (102) is dissolved by enzymatic digestion and / or by applying ultrasound to the tissue sample (102).

3. The method (500) according to claim 1 or 2, wherein the tissue sample (102) moves perpendicular to the flow direction of the carrier fluid in the sample volume (106).

4. The method (500) according to claim 1, wherein: When the tissue sample (102) is in a first position relative to the outlet of the sample volume (106), a first measurement is performed on single cells (122) and / or cell aggregates in the measurement volume (114); and The method (500) further includes performing a third measurement on single cells (122) and / or cell aggregates in the measurement volume (114) when the tissue sample (102) is in a second position relative to the outlet of the sample volume (106).

5. The method (500) according to claim 1, wherein performing the first measurement includes or corresponds to acquiring a first microscopic image of a single cell (122) and / or cell aggregate in the measurement volume (114).

6. The method (500) according to claim 5, wherein performing the first measurement includes or corresponds to acquiring a first phase-shifted image of a single cell (122) and / or cell aggregate in the measurement volume (114) using a quantitative phase-contrast microscope (400).

7. The method (500) according to claim 6, wherein the quantitative phase contrast microscope (400) is a digital holographic microscope.

8. The method (500) according to claim 6 or 7, further comprising determining the cell type of one or more cells (122) from the first phase-shift image.

9. The method (500) according to claim 1, further comprising providing imaging markers in the carrier fluid for labeling the single cells (122) and / or cell aggregates.

10. The method (500) according to claim 9, wherein the imaging marker is a cell type-specific imaging marker.

11. The method (500) according to claim 1, further comprising performing molecular spectral analysis on the single cell (122) and / or cell aggregate.

12. The method (500) according to claim 1, further comprising hydrodynamic focusing and / or viscoelastic focusing of single cells (122) and / or cell aggregates in the measurement volume (114).

13. The method (500) according to claim 1, wherein the tissue sample (102) is a biopsy sample.

14. The method (500) according to claim 13, wherein the biopsy sample is a needle aspiration biopsy sample.

15. A microfluidic system (100, 200, 304) for analyzing a tissue sample (102) using the method (500) of any one of claims 1 to 14, said tissue sample (102) comprising a plurality of biological cells forming a continuous tissue material, said microfluidic system (100, 200, 304) comprising: A sample volume (106) is configured to receive the tissue sample (102) through an insertion opening (106A); An input port (110) is in fluid communication with the sample volume (106) and is configured to receive carrier fluid; The measurement volume (114) is in fluid communication with the sample volume (106) via a microfluidic delivery channel (112B); and A detection window (118) is used to acquire a microscopic image of cells (122) in the measurement volume (114), wherein the microfluidic system (200, 304) further includes a movable element (202) configured to move within the sample volume (106) to move the tissue sample (102) within the sample volume (106), wherein the tissue sample (102) moves at an angle relative to the flow direction of the carrier fluid in the sample volume (106).

16. The microfluidic system (200, 304) of claim 15, wherein the movable element (202) is a piston including a cap member (202A) configured to seal the insertion opening (106A) and slide along the sample volume (106) to move the tissue sample (102) along the sample volume (106).

17. The microfluidic system (100, 200, 304) according to claim 15 or 16, further comprising an input channel (112A) connecting the input port (110) to the sample volume (106), wherein the openings of the input channel and the delivery channels (112A, 112B) are arranged on opposite sidewalls of the sample volume (106) and aligned with each other along the flow direction of the input channel (112A).

18. The microfluidic system (100) of claim 15, further comprising one or more sheath flow channels (120) in fluid communication with the measurement volume (114), wherein the one or more sheath flow channels (120) are configured to generate one or more sheath flows (124B) to hydrodynamically focus the carrier fluid in the measurement volume (114).

19. The microfluidic system (200) of claim 15, further comprising a cell sorter (206) configured to sort the single cells (122) based on control signals and / or based on the electrical, magnetic and / or hydrodynamic properties of the cells.

20. An apparatus (300) for analyzing a tissue sample (102) using the method (500) of any one of claims 1 to 14, said tissue sample (102) comprising a plurality of biological cells forming a continuous tissue material, said apparatus (300) comprising: A bracket (302) is configured to receive a microfluidic system (100, 200, 304), the microfluidic system (100, 200, 304) including a sample volume (106) configured to receive the tissue sample (102) and a measurement volume (114) in fluid communication with the sample volume (106); A measuring device configured to measure cells (122) in the measured volume (114); as well as A controller (306) configured to dissolve at least a portion of the tissue sample (102) into single cells (122) and / or cell aggregates in a carrier fluid within the sample volume (106), and a control tool for generating a carrier fluid flow (124A) from the sample volume (106) to the measurement volume (114). The device (300) further includes an actuator (312) configured to move a movable element (202) within the sample volume (106), wherein the controller (306) is configured to control the actuator (312) to move the movable element (202) within the sample volume (106) for moving the tissue sample (102) within the sample volume (106), wherein the tissue sample (102) moves at an angle relative to the flow direction of the carrier fluid in the sample volume (106); and / or The controller (306) is configured to control the measuring device to perform a first measurement of cells (122) and / or cell aggregates in the measuring volume (114) at a first time point while dissolving the tissue sample (102), and to perform a second measurement of cells (122) and / or cell aggregates in the measuring volume (114) at a second time point while dissolving the tissue sample (102), wherein at the first time point the single cells (122) and / or cell aggregates in the measuring volume (114) originate from the external portion of the tissue sample (102), and at the second time point the single cells (122) and / or cell aggregates in the measuring volume (114) originate from the internal portion of the tissue sample (102).

21. The apparatus (300) of claim 20, further comprising an ultrasonic source (310) configured to apply ultrasound to a sample volume (106) of the microfluidic system (100, 200, 304), wherein the controller (306) is configured to control the ultrasonic source (310) to dissolve at least a portion of the tissue sample (102) by applying ultrasound to the tissue sample (102) in the sample volume (106).

22. The apparatus (300) according to claim 20 or 21, further comprising a microfluidic unit (308) configured to supply fluid to the sample volume (106), wherein the controller (306) is configured to control the microfluidic unit (308) to dissolve at least a portion of the tissue sample (102) by supplying a dissolving fluid to the sample volume (106), the dissolving fluid comprising an enzyme for enzymatic digestion of the tissue sample (102).

23. The apparatus (300) of claim 22, wherein the microfluidic unit (308) is further configured to supply sheath fluid to the microfluidic system (100, 200, 304) to generate a sheath flow in the measurement volume (114), wherein the controller (306) is configured to control the microfluidic unit (308) to hydrodynamically focus the carrier fluid in the measurement volume (114) by supplying the sheath fluid to the microfluidic system (100, 200, 304).

24. The apparatus (300) of claim 20, further comprising a piston having a cap member (202A), the piston being mechanically coupled to the actuator (312) and configured to be inserted into the sample volume (106), wherein the controller (306) is configured to control the actuator (312) to move the piston along the sample volume (106) for moving the tissue sample (102) along the sample volume (106).

25. The apparatus (300) of claim 20, wherein the measuring device is a quantitative phase contrast microscope (400) configured to acquire phase-shifted images of cells (122) in the measurement volume (114).

26. The apparatus (300) according to claim 25, wherein the quantitative phase contrast microscope (400) is a digital holographic microscope.

27. The apparatus (300) according to claim 25 or 26, wherein the controller (306) is configured to determine the cell type of one or more cells (122) based on the phase-shifted image.

28. The apparatus (300) according to claim 25, wherein: The microfluidic system (200) includes a cell sorter (206) configured to sort single cells (122) based on control signals; and The controller (306) is configured to control the cell sorter (206) to sort the one or more cells (122) based on the cell type of the one or more cells (122) determined according to the phase-shift image.

29. The apparatus (300) according to claim 20, further comprising the microfluidic system (100, 200, 304) according to any one of claims 15 to 19.

Images