Tissue fixation and imaging

Abstract

A method for intraoperative assessment of a tissue sample obtained from a patient is comprising the steps of: (a) fixing the tissue sample by subjecting it to a flow of a dehydrating liquid caused by a pressure difference between the tissue sample and the dehydrating liquid; (b) subjecting the fixed tissue sample from step (a) to X-ray microimaging to generate multiple X-ray projections; (c) obtaining a 3D image of the fixed tissue sample by X-ray microtomography using the X-ray projections obtained in step (b).

Description

[0001] TISSUE FIXATION AND IMAGING

[0002] Technical field of the invention

[0003] The present invention relates to a method for intraoperative assessment of a tissue sample obtained from a patient. art

[0004] Surgery is the most common way to treat various injuries and diseases.

[0005] Surgeons often send dissected tissue samples for further analysis, such as classical histopathology, especially if the dissected tissue samples are suspected to comprise tumor cells. This analysis is particularly important in patients undergoing surgical removal of a primary tumor. The surgeon needs to be completely sure that the entire tumor has been removed during the procedure, since it will require additional treatment otherwise. One way to do this is to evaluate the dissected tissue samples from the patient, particularly the tissue samples comprising the resection margins, i.e., the margin of presumed non-tumorous tissue surrounding the tumor that has been removed (tumor resection margin assessment), using classical histology. In the classical histopathology, tumor-positive cells are found in the resection margin in 10- 35% of the cases involving the most common solid cancers. When including also vulvar and oral cancer, the number increase to 40-60%. Typically, this does not lead to renewed surgery, due to risk and patient discomfort. Instead, the post-surgery treatment plan is adjusted, including more chemotherapy and / or radiation therapy, leading to patient discomfort and higher cost.

[0006] Therefore, it is beneficial to use intraoperative (during surgery) feedback to evaluate any dissected tissue sample, especially if the tissue sample is suspected to comprise tumor cells. Furthermore, it is beneficial to use intraoperative (during surgery) feedback to evaluate any dissected tissue sample obtained from patients undergoing surgery for removal of solid tumor(s). It is particularly beneficial to use intraoperative (during surgery) feedback to evaluate any dissected tissues sample comprising the tumor resection margin.

[0007] Classical histology provides great detail and pathological insight via elaborate staining methods in combination with high 2D resolution microscopy. Routine histology involves fixation with formalin, multi-step dehydration with ethanol, infiltration, embedding, sectioning, rehydration and staining to allow for a lightmicroscopy evaluation. Particularly time-consuming step in classical histology is fixation with formalin as formalin penetrates tissues slowly. Therefore, dissected tissue samples are sliced, typically to slices of 3-5 mm thickness, to allow adequate permeation of fixative such as formalin. However, even on the sliced tissue samples fixation in formalin takes between 12 and 48 hours. As a result, it is time consuming and together with the dehydration steps it usually takes several days to get the result, which is not suitable for intraoperative assessment. It also requires large amount of manual labor, and does not provide 3D data.

[0008] In the field of light microscopy, an example of a time consuming method is presented in Wisse et al. ("Fixation methods for electron microscopy of human and other liver" World journal of gastroenterology, 2010, p. 2851-2866). It discusses, inter alia, use of perfusion-fixation methods on liver tissue samples. These methods use glutaraldehyde as a fixative under isotonic conditions, and comprise several washing, dehydrating and embedding steps. The disclosed methods take at least overnight to prepare a tissue sample for analysis and are very labor intensive.

[0009] An alternative method is frozen section analysis (FSA), wherein the tissue sample is quickly frozen and then analyzed using histology by a pathologist. The freezing of the sample takes about 10 minutes, and the total time of receiving the results takes about 30-60 minutes, which is an acceptable time for intraoperative assessment. Problems with this method are that only 2D images of small tissue biopsies can be acquired, and that the image quality is below the quality given by classical histology. It is very labor intensive.

[0010] Lagerqvist F. ("Rapid preparation of Soft Tissue for X-ray Virtual Histology" KTH, Aug 2022, p. 1-60.) aimed to develop a method for rapid fixation of tissues which may be suitable for intraoperative feedback to the surgeons by immersing the tissues in acetone. However, Lagerqvist F. discloses that only immersion in acetone for 24 hours provides fixed tissue samples.

[0011] With improvements to the fixation time of soft tissue, a X-ray microimaging and a high-resolution X-ray tomography, such as X-ray microtomography, could allow 3D intraoperative feedback to the surgeons.

[0012] The high recurrence rates are disturbing both from a patient outcome perspective and from health economy perspective. Accordingly, there is a need for an improved method for intraoperative assessment of a tissue sample obtained from a patient.

[0013] Summary of the invention

[0014] It is an object to provide an improved method for intraoperative assessment of a tissue sample which allows for rapid fixation of said tissue sample, which enables intraoperative feedback and therefore improved surgical outcome. Particularly, it is an object to provide an improved method for intraoperative assessment of a relatively thick tissue sample which allows for rapid fixation of said thick tissue sample, which enables intraoperative feedback and therefore improved surgical outcome. Moreover, it is an object to provide an improved method for intraoperative assessment of a tissue sample which allows for a rapid, single-step and labor efficient chemical fixation process of preparing the tissue sample for said intraoperative assessment.

[0015] It is a further object to provide an improved method for intraoperative assessment of a tissue sample which allows for rapid image analysis, which additionally enables improved intraoperative feedback and therefore improved surgical outcome.

[0016] It is an object to provide an improved method for intraoperative assessment of a tissue sample obtained from a patient, which achieves improved contrast and / or quality in the images obtained.

[0017] It is also an object to provide an improved method for intraoperative assessment of a tissue sample obtained from a patient, which achieves improved differentiation between healthy and cancerous tissue in the images obtained.

[0018] It is an object of the present invention to provide an improved method for intraoperative assessment of a tissue sample obtained from a patient, such as any dissected tissue sample from the patient, especially if the dissected tissue sample is suspected to comprise tumor cells, if the dissected tissue sample is obtained from a patient undergoing surgery for removal of a solid tumor, and / or if the dissected tissue sample comprises a tumor resection margin.

[0019] For these and other objects which will be evident from this disclosure, the present invention provides a method for intraoperative assessment of a tissue sample obtained from a patient comprising the steps of:

[0020] (a) fixing the tissue sample by subjecting it to a flow of a dehydrating liquid caused by a pressure difference between the tissue sample and the dehydrating liquid;

[0021] (b) subjecting the fixed tissue sample from step (a) to X-ray microimaging to generate multiple X-ray projections;

[0022] (c) obtaining a 3D image of the fixed tissue sample by X-ray microtomography using the X-ray projections obtained in step (b).

[0023] The inventors have realized and demonstrated that instead of application of classical histopathology for evaluation of dissected tissue samples, the method as described above achieves • a rapid, single-step and labor efficient chemical fixation of relatively thick tissue samples, which in turn enables intraoperative feedback from X-ray microtomography and therefore improved surgical outcome,

[0024] • generation of contrast without additional agents, such as fluorophores, radionuclides or other biomarkers,

[0025] • improved contrast in the 3D images obtained from the X-ray microtomography, and

[0026] • improved differentiation between healthy and cancerous tissue in the 3D images obtained from the X-ray microtomography.

[0027] The inventors have also realized and demonstrated that instead of the classical immersion fixation, subjecting a tissue sample to a flow of a dehydrating liquid caused by a pressure difference between the tissues sample and the dehydrating liquid is surprisingly useful in the intraoperative assessment method as a fixation step prior to the X-ray microimaging, because it enables a rapid fixation of tissue samples of any thickness, and thereby slicing of the thick tissue samples can be avoided. The fixation step of the present invention is typically a single-step chemical process of preparing tissue samples for the X-ray microimaging, which does not require further dehydration, washing or embedding steps before imaging of the tissue samples. Furthermore, said rapid fixation of the tissue samples is also compatible with classical histology and, as X-ray imaging is a nondestructive technique, the tissue samples can be analyzed with classical histology after said X- ray imaging, i.e., after X-ray microimaging.

[0028] In one embodiment, the patient is undergoing surgery for removal of a solid tumor. In certain embodiments, the tissue resection margin of the tissue sample in step (a) is assessed. The method is particularly advantageous when applied on a dissected tissue sample obtained from the patient undergoing surgery for removal of the solid tumor, and more particularly on the dissected tissue sample which comprises a tumor resection margin.

[0029] In an embodiment, the tissue sample in step (a) is a non-sliced tissue sample, which contributes to fixation and analysis of the whole dissected tissue sample. Furthermore, it saves time and prevents any uncertainties connected with slicing of a tissue sample into thinner pieces. In certain embodiments, the thickness of the tissues sample in step (a) is at least 3 mm. In certain embodiments, the thickness of the tissues sample in step (a) is at least 10 mm. In some embodiments, the thickness of the tissues sample in step (a) is at least 12 mm, such as at least 15 mm.

[0030] In an embodiment, the fixed tissue samples from step (a) is not sliced before subjecting it to step (b), which additionally contributes to analysis of the whole dissected tissue sample, saves time, and prevents any uncertainties connected with slicing of a tissue sample into thinner pieces.

[0031] In an embodiment, the fixed tissue samples from step (a) is sliced before subjecting it to step (b). In certain embodiments, the fixed tissues samples from step (a) are sliced to 1-9 mm thickness, such as 3-5 mm thickness.

[0032] In an embodiment, the fixing in step (a) is up to 30 minutes long, such as up to 10 minutes long, which contributes to a fast intraoperative assessment of a tissue sample obtained from a patient.

[0033] In an embodiment, the dehydrating liquid used in step (a) comprises at least one protein-precipitating organic solvent. In an embodiment, the dehydration liquid used in step (a) comprises acetone, methanol, or ethanol, or any combination thereof. The dehydrating liquid used in step (a) thus comprises one or more organic solvents which are strong dehydrants and have ability to cause precipitation of cellular proteins, disruption of cell membranes and cell lysis in the tissue sample. An advantage of using such liquid is that the tissue sample is both fixed and dehydrated at the same time, leading to a single-step and time-efficient fixation process.

[0034] In an embodiment, the dehydration liquid used in step (a) comprises at least 50 vol% of acetone, methanol, or ethanol, or any combination thereof. In an embodiment, the dehydration liquid used in step (a) comprises at least 70 vol%, such as at least 75 vol%, such as at least 85%, such as at least 90%, such as at least 95% of acetone, methanol, or ethanol, or any combination thereof. In certain embodiments, the liquid used in step (a) is acetone, methanol, or ethanol, or any combination thereof.

[0035] In an embodiment, the dehydration liquid used in step (a) comprises at least 50 vol% of acetone. In an embodiment, the dehydration liquid used in step (a) comprises at least 70 vol% acetone, such as at least 75 vol% acetone. In certain embodiments, the liquid used in step (a) comprises at least 80%, such as at least 85 vol% acetone. In some embodiments, the liquid used in step (a) comprises at least 90%, such as at least 95 vol% acetone. In certain embodiments, the liquid used in step (a) is acetone. An increased acetone content increases the speed of the fixing, which contributes to achieving the objective of an intraoperative assessment.

[0036] In certain embodiments, the flow in step (a) is achieved by injecting the dehydration liquid into the tissue sample to achieve a locally increased pressure of the dehydration liquid at the injection point.

[0037] In certain embodiments, injecting the dehydration liquid in the tissue sample is performed using needles. In some embodiments, injecting the dehydration liquid in the tissues sample is performed using needles with gauge number of 25 or larger, such as with gauge number 30 or larger.

[0038] In some embodiments, injecting the dehydration liquid in the tissue sample is performed by a single injection or by multiple parallel injections. In certain embodiments, injecting the dehydrating liquid in the tissue sample is performed by multiple parallel injections. In some embodiments, injecting the dehydration liquid in the tissue sample is performed by multiple parallel injections with a constant separation between injection points.

[0039] In some embodiments, injecting the dehydration liquid in the tissue sample is performed by multiple parallel injections with needles of different lengths.

[0040] In certain embodiments, injecting the dehydration liquid in the tissue sample is performed using needles comprising a single side hole or multiple side holes on each needle.

[0041] In certain embodiments, injecting the dehydration liquid in the tissue sample is performed using needles, wherein each needle is connected to a flow actuator and optionally a flow sensor.

[0042] In one embodiment, the method is further comprising a step:

[0043] (d) analyzing the 3D images to assess whether the tissue sample contains cancerous tissue or not.

[0044] In an embodiment, the X-ray microimaging of step (b) is a phase-contrast X-ray imaging. In certain embodiments, the phase-contrast X-ray imaging is propagationbased phase-contrast X-ray imaging (PBI ) .

[0045] In an embodiment, the X-ray microtomography of step (c) is a phase-contrast computed tomography (PC-CT).

[0046] Preferred embodiments and further aspects are defined in the appended claims, listed embodiments and throughout the application text.

[0047] Brief description of the drawings

[0048] The above and other aspects of the present invention will now be described in more detail, with reference to the appended figures. The figures are not necessarily to scale, and generally only show parts that are necessary in order to elucidate the inventive concept, wherein other parts may be omitted or merely suggested.

[0049] Fig. 1 is a flow chart illustrating a method according to the present disclosure.

[0050] Fig. 2 illustrates the general concept of fixing a tissue sample according to the present disclosure. Fig. 3 illustrates different needles for performing the method according to the present disclosure. Fig. 3A-3C show needles with a hole on the tip of each needle, and optionally one or more side holes. Fig. 3D show different needles with the closed tip on each needle and one or more side holes on each needle.

[0051] Fig. 4 illustrates different multi-needle systems for performing the method according to the present disclosure. Fig. 4A shows linear (top) and circular (bottom) multi-needle systems. Fig. 4B shows a linear multi-needle system wherein each needle is independently controlled. Fig. 4C shows a linear multi-needle system comprising needles of different lengths.

[0052] Fig. 5 illustrates a fixation experiment performed on a tissue sample. Fig. 5A shows injecting a dehydrating liquid by multiple injections. Fig. 5B illustrates cutting of the tissue sample along the multiple injection points. Fig. 5C shows the cut tissue sample.

[0053] Fig. 6 shows an image of a kidney tissue after injecting acetone and cutting.

[0054] Fig. 7 shows images of a tenderloin tissue. Fig. 7A shows an image of the tenderloin tissue before injecting acetone. Fig. 7B shows an image of the tenderloin tissue after injecting acetone. Fig. 7C shows an image of the tenderloin tissue after injecting acetone and cutting.

[0055] Detailed description

[0056] Surgery is an essential part of the curative plan for many patients. Dissected tissue samples are often sent to post-surgical analysis, especially if any of said dissected tissue samples is suspected to comprise tumor cells. This routine is particularly applied for cancer patients, such as the patients affected with solid tumors. The outcome of surgical removal of a solid tumor (recurrence rates and patient survival) depends on several factors where the resection margin is key indicator. The resection margin is the rim of apparently nontumorous tissue around a tumor that has been surgically removed. For a tumor to be considered completely removed, the resection margin should be free of tumorous cells and small satellite tumors.

[0057] All surgical oncology is followed by a histopathological assessment of any tissue sample obtained from a patient undergoing a surgery, including the tissue samples comprising the resection margin, seeking for tumorous cells and / or small satellite tumors inside the resection margin. This classical process is presently timeconsuming, it typically takes several days before the surgeon receives the pathology report. It is estimated that tumor-positive margins occur in 5% - 35% of the cases, depending on cancer type. Furthermore, due to the present process of slicing, the pathologist risks missing small groups of tumorous cells hidden within the slices. The time, the inadequate margin assessment, and the hidden tumor cells all risk compromising patient outcome.

[0058] The present invention provides a method for intraoperative assessment of a tissue sample obtained from a patient, wherein the tissue sample may be a relatively thick tissue sample. The method also provides a rapid fixation of a tissue sample obtained from a patient, wherein the tissue sample may be a relatively thick tissue sample, such as a tissue sample with a thickness of at least 3 mm. Advantageously, this method enable fixation of thick tissue samples and thereby prevents any uncertainties connected to slicing of said thick tissue samples. Furthermore, the method enables a 3D intraoperative assessment of the tissue sample to determine if the tissue sample comprises any tumor cells. The 3D assessment may be done on fixed tissue samples of any thickness. Therefore, the present invention enables improved intraoperative feedback and therefore improved surgical outcome for patients. Moreover, the present invention provides a rapid, single-step and labor efficient chemical fixation process of preparing a tissue sample for the intraoperative 3D assessment.

[0059] The terminology used herein is for the purpose of describing particular aspects of the disclosure only, and is not intended to limit the disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0060] As used herein, an intraoperative assessment refers to an assessment which occurs or is performed during the course of a surgical operation. The intraoperative assessment of the present invention is performed on a dissected tissue sample from a patient undergoing a surgery and does not encompasses in its scope a surgical step of obtaining said dissected tissue sample. The surgical operation may be associated with any disease, such as removal of a solid tumor, partly or preferably completely. However, any surgical operation may be in need of an assessment if tumor cells are present in a tissue sample obtained from a patient, especially if the surgeon suspects that the tissue sample comprises tumor cells. The assessment is an objective histopathological determination of the status of a removed tissue sample, in particular to distinguish between nontumorous tissue and tumorous tissue in the tissue sample.

[0061] As used herein, a tissue sample refers to a sample of tissue which has been surgically removed from a patient. The tissue sample may be suspected to contain tumor cells. For patients with solid tumors, the tissue sample may be any tissue sample, the tissue sample may contain the whole solid tumor or a part of the solid tumor which has been surgically removed, or the tissue sample may comprise the tumor resection margin. The tissue sample may also be prepared from the whole solid tumor which has been surgically removed. In particular, the tissue sample encompasses the tumor resection margin, i.e., an assumed outer boundary of the solid tumor which has been surgically removed.

[0062] As used herein, the tumor resection margin refers to the rim of apparently nontumorous tissue around a solid tumor that has been surgically removed. For a solid tumor to be considered completely removed, the resection margin should be free of tumorous cells and small satellite tumors inside the resection margin. Often it is preferable to keep the margin as small as possible, e.g., for cosmetic reasons (e.g. breast, skin) or to preserve organ function (e.g. liver, kidney).

[0063] As used herein, a solid tumor refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can occur in several places, including bones, muscles and organs. Solid tumors may be benign or malignant. Different types of solid tumors are named for the type of cells that form them, e.g. sarcomas, carcinomas, and lymphoma. Preferably, this method is applicable on solid tumors including liver, pancreas, colon, kidney, bone, prostate, breast and skin cancers, but the method is considered as applicable for any solid tumor, including soft tissue tumors. As exemplified herein, the method is applicable to, inter alia, liver, pancreas, kidney and colon cancers.

[0064] As used herein, a flow of a liquid, such as a dehydrating liquid, refers to the motion of a liquid that is subjected to different unbalanced forces. Applying a force on an area will create an increased pressure therein. The liquid will move from an area with a higher pressure to an area with a lower pressure. In this case, the flow of the dehydrating liquid is caused by a pressure difference between the tissue sample and the dehydrating liquid.

[0065] Using the method as defined herein, the surgeon can potentially get correct and immediate intraoperative feedback from the pathologist while still having the patient available on the operating table. Such fast intra-operative feedback allows immediate corrective surgery for a more complete surgical tumor removal.

[0066] The first method step defines (a) fixing the tissue sample by subjecting it to a flow of a dehydrating liquid caused by a pressure difference between the tissue sample and the dehydrating liquid. The first method step is typically a single-step chemical fixation process of preparing said tissue sample for the intraoperative 3D assessment, and does not necessarily comprise any additional chemical steps, such as washing, dehydrating or embedding steps, before the assessment. In an embodiment, the first method step may optionally comprise a post-fixation water rinse of the fixed tissue sample from step (a) to remove the excess of the dehydrating liquid and thereby protect the X-ray imaging equipment from the dehydrating liquid vapors. The water rinse may be performed by submerging the fixed tissue sample in water, and optionally applying a slow-moving water stream, for up to 5 minutes, such as up to 3 minutes. However, the water rinse step is not mandatory and the X-ray microimaging may be performed on the fixed tissue sample from step (a) as such.

[0067] The tissue sample in step (a) may be obtained from a patient undergoing surgery of a solid tumor. The tissue sample in step (a) may comprise a tumor resection margin. The tumor resection margin of the tissue sample in step (a) may be assessed. The tissue sample in step (a) may be a non-sliced tissue sample. The thickness of the tissue sample in step (a) may be at least 3 mm. Preferably, the thickness of the tissue sample in step (a) may be at least 10 mm, such as at least 12 mm, such as least 15 mm.

[0068] In certain embodiments, the tissue sample may be divided / split into smaller pieces before subjecting it to fixation in step (a). As used herein, the term "divided" or "split" refers to cutting a tissue sample into smaller pieces. This is particularly relevant for dissected tissue samples with a size of 40 mm or larger. These dissected tissue sample may be divided into two or more smaller pieces. The size of the resulting pieces may be up to 35 mm, such as up to 30 mm, up to 25 mm or up to 20 mm. Typically, the thickness of the resulting pieces is at least 10 mm, such as at least 12 mm, such as least 15 mm.

[0069] In certain embodiments, the tissue sample may be sliced before subjecting it to fixation in step (a). As used herein, the term "sliced" refers to cutting a tissue sample into thin pieces, i.e., into thin slices. The slices typically have two essentially parallel surfaces where the tissue sample has been cut. The tissue sample may be sliced to a thickness of 1-9 mm, such as 1-7, 1-5, 1-4, 1-3 mm or 2-7, 2-5, 2-4, 2-3 mm, e.g. about 3 mm or about 4 mm or about 5 mm, to form sliced tissue samples. It is also possible to slice the tissue samples during the actual fixing, i.e. at the same time as the dehydration occurs in step (a).

[0070] Preferably, the fixing is a single step dehydration for less than 30 minutes, such as less than 20 minute, preferably less than 15 minutes or less than 10 minutes. In this step, the tissue sample, which may be a non-sliced tissue sample, a divided tissue sample or a sliced tissue sample, is subjected to a flow of a dehydrating liquid for less than 30 minutes, such as less than 10 minutes, under a pressure difference between the tissue sample and the dehydrating liquid. Fixation of tissue samples is essential for pathological examination thereof. Typically fixation of a tissue sample is done by submerging a tissue sample in a fixative. "Immersion fixation" or "drop fixation" are also used to refer to this process.

[0071] The goal with fixating tissue is traditionally to preserve morphological features in the tissue. The fixation process therefore allows for dissection of the tissue without movement. By eliminating these movements, examination of the tissue at hand can be performed without much structural differences in the tissue. Structural differences are here referred to as changes when comparing the tissue in its natural environment and the tissue when it has been resected.

[0072] The tissue sample should remain in a fixative until the fixative has completely penetrated said tissue sample. In the prior art, the most common fixatives comprise formalin (formaldehyde), such as neutral buffered formalin (NBF; 10% formaldehyde phosphate-buffer solution) and paraformaldehyde solution (PFA; 4% PFA solution). The obtained results are usually very similar. However, the rate of diffusion of formalin through a tissue sample is up to 1 mm / h and the rate of diffusion decreases from the outer to the inner areas of the tissue sample. It is thus recommended to slice the tissue sample, such as to slices of 3-5 mm thickness. Still, even fixation for the sliced tissues samples takes long times, such as between 12 and 48 hours. As an example, a fixation time of 24 h is recommended for tissues samples exposed to NBF solutions.

[0073] The inventors have realized that instead of classical fixation, subjecting a tissue sample a flow of a dehydrating liquid under a pressure difference between the tissue sample and the dehydrating liquid provides a quick fixation prior to X-ray microimaging. Compared to the classical fixation, the advantage is that a rapid fixation of relatively thick tissue samples is possible, which in turn can contribute to avoiding of any slicing of such thick tissue samples. However, if necessary or preferred, a tissue sample can be sliced before, during, or after the fixing in step (a).

[0074] Moreover, said quick fixation of tissue samples is also compatible with classical histology. Fixed tissue samples according to the present method can be also analyzed with classical histology, even after they are analyzed with X-ray imaging techniques, as these imaging technique do not affect the fixed tissue samples.

[0075] Furthermore, the present inventors have realized that a rapid fixation using a flow of a dehydration liquid will advantageously result in a differential shrinkage of the treated tissue. In traditional histology, differential shrinkage has been highly undesirable, and complicated methods have been developed to avoid differential shrinkage so as to preserve morphological features in the tissue. Without wishing to be limited to any particular theory, it is considered that for the present purpose of rapidly distinguishing between healthy and cancerous tissue (rather than providing a morphologically optimal picture), the differential shrinkage, e.g. between healthy and cancerous tissue, obtained with a rapid fixation with a dehydrating liquid provides a sufficient contrast in the X-ray microimaging, such as phase-contrast X- ray imaging.

[0076] For the purpose of rapidly distinguishing between healthy and cancerous tissue in any tissue sample obtained from a patient, in order to evaluate if the tumor cells are present therein and / or assess / determine the tumor resection margin of the tissue sample, the present method involving rapid fixation with a dehydrating liquid provides improved contrast and / or differentiation, both compared to rapid fixation without the dehydrating liquid and compared to light microscopy pictures using classical histology. Compared to classical histology, an improved differentiation is achieved with the present method due to the creation of a 3D image.

[0077] To achieve a rapid dehydration, the fixing in step (a) is performed by subjecting it to a flow of a dehydrating liquid caused by a pressure difference between the tissue sample and the dehydrating liquid. The dehydration liquid used in step (a) may comprise acetone, methanol, or ethanol, or any combination thereof. The dehydrating liquid used in step (a) thus comprises one or more organic solvents, which can cause significant damage to the cells of the tissue sample, such as disruption of cell membranes, cell lysis, precipitation of cellular proteins, and dehydration. For example, the above-mentioned dehydrating liquids can dissolve lipids of cell membranes, leading to disruption of the membrane integrity, increased cell membrane permeability and cell lysis. Moreover, these dehydrating liquids are highly effective at dehydrating cells by extracting water from the cellular environment, and at precipitating cellular proteins. Additionally, the dehydrating liquid of the present invention cannot be considered as an isotonic liquid to the cells of the tissue sample. The effect of the dehydrating liquid of the present invention is thus different from the effect of aldehyde fixatives applied under isotonic conditions in the prior art. Preferred examples of protein-precipitating organic solvents include acetone, methanol and ethanol, and combinations thereof.

[0078] In an embodiment, the dehydration liquid used in step (a) comprises at least 50 vol% (vol / vol) of acetone, methanol, or ethanol, or any combination thereof. In an embodiment, the dehydration liquid used in step (a) comprises at least 50% of acetone. Preferably, the dehydration liquid of step (a) is comprising at least 70 vol% (vol / vol) acetone, such at least 75 vol% acetone, such as at least 80 vol% acetone, such as at least 85 vol% acetone, such as at least 90 vol% acetone, such as at least 95 vol% acetone, or even at least 99 vol% acetone. The remainder of the dehydrating liquid may be water and / or any other suitable liquid, e.g. ethanol, methanol, acetic acid, trichloroacetic acid, picric acid, trinitrophenol, zinc chloride, zinc acetate, chloral hydrate, glyoxal, or xylene, or combinations thereof. Preferably, the remainder of the dehydrating liquid may be ethanol, or methanol, of any combination thereof.

[0079] Examples of compositions which are useful as dehydrating liquids include, without limitation thereto:

[0080] 70 - 99 vol% acetone, 1 - 30 vol% ethanol;

[0081] 80 - 99 vol% acetone, 1 - 20 vol% ethanol;

[0082] 90 - 99 vol% acetone, 1 - 20 vol% ethanol;

[0083] 70 - 99 vol% acetone, 1 - 30 vol% methanol;

[0084] 80 - 99 vol% acetone, 1 - 20 vol% methanol;

[0085] 90 - 99 vol% acetone, 1 - 10 vol% methanol;

[0086] 70 - 99 vol% acetone, 1 - 30 vol% acetic acid;

[0087] 80 - 99 vol% acetone, 1 - 20 vol% acetic acid; and

[0088] 90 - 99 vol% acetone, 1 - 10 vol% acetic acid.

[0089] The dehydrating liquid may also comprise aldehydes, such as formaldehyde or glutaraldehyde. In certain embodiments, aldehydes, such as formaldehyde and glutaraldehyde are excluded from the fixing liquid.

[0090] Preferably, the dehydrating liquid does not contain any water.

[0091] In a preferred embodiment, the dehydrating liquid of step (a) is acetone, i.e. 100 vol% acetone.

[0092] The tissue sample may be treated with the dehydrating liquid at room temperature or above room temperature. A higher temperature provides a more rapid fixation step, and it is therefore preferred that the fixation temperature is 20-60 °C, preferably 30-50 °C, such as 40-50 °C.

[0093] Advantageously, the flat fixed tissue samples obtained from step (a) do not require any further embedding step, e.g. in paraffin. If desired, it is possible to include a further embedding step, e.g. a paraffin embedding step, between steps (a) and (b).

[0094] The fixation in step (a) is achieved using a flow of a dehydrating liquid caused by a pressure difference between the tissue sample and the dehydrating liquid. Thereby, the dehydrating liquid is "pushed" or "forced" through the tissue sample in order to fix said tissue sample. However, the present method should not be confused with "perfusion fixation", which typically describes fixation methods which use existing (and intact) vascular systems in order to distribute a fixative throughout an organ or organism. The present method is applied on dissected tissue samples from a patient undergoing a surgery and achieves the flow of the dehydrating liquid also through any extracellular space present in the tissue samples. Thereby, the present method does not rally on the vascular system potentially present in the tissue samples.

[0095] Preferably, the flow of the dehydrating liquid in step (a) is achieved by injecting the dehydration liquid into the tissue sample to achieve a locally increased pressure of the dehydration liquid at the injection point.

[0096] Injecting of the dehydration liquid in the tissue sample is performed using injection needles, which contribute to fast and precise injecting of the dehydrating liquid. The needles may be relatively fine needles, i.e., with gauge number 25 or higher, such as with gauge number 26 or higher, with gauge number 27 or higher, with gauge number 28 or higher, with gauge number 29 or higher, or with gauge number 30 or higher. In a preferred embodiment, the needles may have gauge number 30 or higher.

[0097] Injecting the dehydrating liquid in the tissue sample may be performed by a single injection or by multiple parallel injections. Preferably, injecting is performed by the multiple parallel injections. The multiple parallel injections may have a predetermined separation between injection points. In an embodiment, the predetermined separation between the injection points may typically be 1-5 mm, such as about 1 mm, or about 2 mm, or about 3 mm, or about 4 mm, or about 5 mm. In some embodiments, injecting of the dehydrating liquid is performed by the multiple parallel injections with a non-constant separation between injection points, i.e., with a non-constant separation between adjacent injection points. Each separation between two adjacent injection points in the non-constant separation between injection points may be 1-5 mm and may be independently selected from about 1 mm, about 2 mm, about 3 mm, about 4 mm, and about 5 mm. In a preferred embodiment, injecting of the dehydrating liquid is performed by the multiple parallel injections with a constant separation between injection points, i.e., with a constant separation between adjacent injection points. In an embodiment, the constant separation between the injection points may be 1-5 mm, such as about 1 mm, or about 2 mm, or about 3 mm, or about 4 mm, or about 5 mm.

[0098] Injecting the dehydration liquid in the tissue sample may be performed by multiple parallel injections with needles of different lengths. In an embodiment, the needles of different lengths may comprise one or more needles with lengths of 1-20 mm. The length of each needle of the one or more needles may be independently selected from 1-20 mm, such as independently selected from about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, and about 20 mm.

[0099] Injecting the dehydration liquid in the tissue sample may be performed by using needles comprising one or more exit points on each needle. The exit points are often referred to as exit orifices, orifices, or holes.

[0100] Injecting the dehydration liquid in the tissue sample may be performed by using needles comprising a single side hole or multiple side holes on each needle. These needles may contain side hole(s) on each needle in addition to a hole on the tip of each needle. These needles may also contain only side hole(s) on each needle. Alternatively, the needles may contain only a hole of the tip of each needle, and no side hole(s).

[0101] Injecting the dehydration liquid in the tissue sample may be performed using needles, wherein each needle is connected to a flow actuator and optionally a flow sensor. As used herein, a flow actuators refers to a device that receives an energy input and converts it into motion or force. The flow actuators may include any of the commercially available flow actuators, such as pneumatic, hydraulic, electric, magnetic, thermal, or mechanical actuators. The flow actuator initiates and controls a flow of a liquid in said needle, as it pushes the liquid through the needle by applying force / pressure on the liquid. As used herein, a flow sensor or a flow meter is an electronic device that measures and / or regulates the flow rate of a liquid within a needle. The flow sensor may include any of the commercially available flow sensors, such as vortex, mechanical, or ultrasonic flow sensors.

[0102] In an embodiment, each needle of the needles is connected to a flow actuator. The needles may be part of a micro-fluidic system, wherein the microfluidic system may comprise up to several hundred channels for a flow of a liquid. Each channel of the micro-fluidic system may be connected to one needle of the needles. Each needle of the needles may be connected to a flow actuator, which in turn controls the flow of the liquid in said needle. In some embodiments, each needle of the needles is connected to a flow actuator and a flow sensor. The flow actuator may initiate and maintain the flow of the liquid through the needle. The flow sensor may measure the flow of the liquid in / through said needle. The flow sensor and the flow actuator may be coupled together, to achieve desired flow through each needle of the needles. In such case, the flow sensor may provide a feedback to the flow actuator, wherein the feedback may be the flow rate of the liquid through the needle and / or the difference between the flow rate and the desired flow rate of the liquid through the needle. Based on the feedback, the force / pressure applied on the liquid by the flow actuator may be adjusted to achieve the desired flow rate of the liquid through the needle.

[0103] The second method step defines (b) subjecting the fixed tissue samples from step (a) to X-ray microimaging. Thereby, multiple X-ray projections are generated. X- ray microimaging is a type of X-ray imaging and therefore it is a nondestructive technique that generates an image of the internal features of an object by measuring the attenuation of photons. As X-rays have the advantage that they have a short wavelength and can penetrate through thick samples. The image contrast is determined by the difference in absorption of X-rays by the different components in the object. X-ray microimaging has the spatial resolution in the micrometer range and requires a high brilliance monochromatic X-ray beam to provide such high- resolution. This technique is since many years performed at synchrotrons, since they can provide such high brilliance monochromatic X-ray beam. In recent decades, the optics-based X-ray microimaging based on laboratory sources are commercially available, which often use high-power rotating-anode sources. Achieving significantly higher brightness and smaller spot sizes can create very brilliant and small beams enable a major increase of the image acquisition speed with maintained resolution in an X-ray imaging application or measurement throughput improvement combined with better precision and accuracy in an analytical application. Thus, X-ray microimaging enables rapid high-resolution 3D imaging of the fixed tissue sample from step (a) for an intraoperative assessment of said tissue sample according to the present invention. It is particularly surprising that a dehydrating liquid itself, such as acetone, methanol, or ethanol, or any combination thereof, generates sufficient contrast, and that no additional step and / or additional agent (e.g., a stain) is necessary to achieve the contrast needed for differentiation between non-cancerous and cancerous tissue in the X-ray images.

[0104] Moreover, the X-ray microimaging enables rapid high-resolution imaging of thick samples. Therefore, a thick tissue sample obtained from a patient can be subjected to both the fixating step and the imaging step as a whole sample, i.e., without any splitting or slicing. The tissue sample obtained from the patient may have a thickness of at least 3 mm, e.g. at least 10 mm, at least 12 mm, or at least 15 mm. Splitting of a tissue sample into smaller pieces with a size of up to 35 mm may be performed, typically to achieve a thickness of the resulting pieces of at least 10 mm, at least 12 mm, or at least 15 mm. Alternatively, slicing of a tissue sample into slices with a thickness of 1-9 mm, may be performed. In some embodiments, the fixed tissue sample obtained from step (a) is not sliced before subjecting it to step (b). In certain embodiments, the fixed tissue sample from step (a) may be sliced before subjecting it to step (b). The fixed tissue sample from step (a) may be sliced to a thickness of 1-9 mm, such as 1-7, 1-5, 1-4, 1- 3 mm or 2-7, 2-5, 2-4, 2-3 mm, e.g. about 3 mm or about 4 mm or about 5 mm, to form sliced fixed tissue samples that are further subjected to imaging in step (b) . Splitting of a tissue sample into smaller pieces with a size of up to 35 mm may be performed, typically to achieve a thickness of the resulting pieces of at least 10 mm, at least 12 mm, or at least 15 mm.

[0105] In an embodiment, the X-ray microimaging in step (b) is a phase-contrast X- ray imaging. As used herein, phase-contrast X-ray imaging or phase-sensitive X-ray imaging comprise technical methods that use information concerning changes in the phase of an X-ray beam that passes through an object in order to create images. The beam's phase shift caused by the sample is not measured directly, but is transformed into variations in intensity, which then can be recorded by a detector. Advantageously, several / all flat fixed tissue samples can be imaged simultaneously.

[0106] Phase contrast allows detailed X-ray imaging of soft tissues, where classical absorption contrast does not provide sufficient contrast. The 3D phase contrast images obtained include both absorption contrast and phase contrast. This has obvious clinical and pre-clinical value, as demonstrated for e.g. lungs, cartilage, and tumors. To date, most phase-contrast bioimaging aimed at high spatial resolution with cellular detail has been performed at synchrotron light sources since only this high-brightness source provides sufficient coherence and flux to allow phasecontrast imaging with adequate exposure times. Hemberg et al., Appl. Phys. Lett. 83: 1483 (2003) developed a liquid-metal-jet electron-impact microfocus source which operates at an order of magnitude higher brightness than competing laboratory sources and with potential for another order of magnitude. This small-spot and high- power X-ray tube enables phase-contrast X-ray imaging with high spatial resolution and adequate exposure times in the clinic.

[0107] Examples of useful phase-contrast X-ray imaging techniques include crystal interferometry, propagation-based imaging, analyzer-based imaging, edgeillumination and grating-based imaging. They are all based on the observation of interference patterns between diffracted and undiffracted waves.

[0108] In a preferred embodiment, the phase-contrast X-ray imaging in step (b) is propagation-based phase-contrast X-ray imaging (PBI). PBI typically detects the transverse Laplacian of the phase (V24>) with a single measurement and without extra optical elements. With a high-brightness and high-spatial-coherence liquid- metal-jet microfocus source, PBI provides a cellular spatial resolution with sufficient contrast at reasonable exposure times. Accurate marking of cellular details (like the resection margin or small satellite tumors inside the resection margin) is achieved with post-processing, including phase retrieval, artifact reduction and tomographic reconstruction. Compared to other methods, PBI results in the shortest exposure times but phase retrieval is necessary for quantitative interpretation. Exposure time and resolution are important parameters. As a comparison, for imaging gas-filled structures (like COz-fil led blood vessels or air-filled lung alveoli) the necessary exposure time for observing structures smaller than 50 pm may differ a factor ten in favor of PBI, see e.g. Zhou et al., Opt. Express. 21, 30183 (2013). The method is readily expandable to 3D imaging via tomographic methods, see e.g., U. Lundstrom et al., Phys Med. Biol. 57, 2603-2617 (2012).

[0109] Phase-contrast X-ray tomography allows cellular-resolution 3D imaging of the resection margin in tumors. Advantageously, the pathological assessment is done in full 3D (three dimensions) with cellular resolution, to capture all cellular-sized pathological features, not only those on top of the slice. Furthermore, phase contrast X-ray imaging is more sensitive to density variations in the sample than conventional transmission-based X-ray imaging. This leads to images with improved soft tissue contrast.

[0110] It is particularly surprising that a dehydrating liquid itself, such as acetone, methanol, or ethanol, or any combination thereof, generates sufficient contrast, and that no additional step and / or additional agent (e.g., a stain) is necessary to achieve the contrast needed for differentiation between non-cancerous and cancerous tissue in the X-ray images.

[0111] The third method step defines (c) obtaining a 3D image of the fixed tissue samples by X-ray microtomography using the X-ray projections obtained in step (b). As used herein, tomography refers to imaging by sections or sectioning that uses a penetrating wave, in this case X-rays. The method preferably utilizes a high- resolution X-ray tomography and more specifically X-ray microtomography, implying that the pixel sizes of the cross-sections are in the micrometer range. In practice, all tomography today is computed tomography. Consequently, the method step c) preferably utilizes micro-computed tomography (sometimes abbreviated micro-CT or pCT).

[0112] Micro-CT has applications both in medical imaging and in industrial computed tomography. In general, there are two types of scanner setups. In one setup, the X-ray source and detector are typically stationary during the scan while the sample / animal rotates. The second setup, much more like a clinical CT scanner, is gantry based where the animal / specimen is stationary in space while the X-ray tube and detector rotate around. These scanners are typically used for small animals (in vivo scanners), whereas for biomedical samples, foods, microfossils, and other studies for which minute detail is desired, the X-ray source and detector are kept stationary.

[0113] An exemplary system includes a metal-jet x-ray source (Excillum D2+, 250 W), a high- accuracy rotating stage, and a large high-resolution detector, all on an approximately 200x80 cm radiation-safe bench. The sample holder provides simple, fast, safe, and flexible sample handling. The key factors for observing the relevant low-contrast cellular-sized details at short exposure times is optimizing source parameters, distances and detector parameters. This system has demonstrated 1 pm resolution in 3D imaging. By exchanging the source to a 1 kW source, the acquisition times can be reduced a factor 4.

[0114] The power of the x-ray source is preferably at least 100 W, more preferably at least 200 W.

[0115] The software integrates hardware control and calibration, scan algorithms, tomographic reconstruction, and image processing algorithms. High-fidelity imaging relies on the synergy of hardware scan algorithms and post-data-acquisition image reconstruction. Key techniques involve phase retrieval, reduction of reconstruction artifacts, mathematical noise filtering, and observability analysis.

[0116] In an embodiment, the X-ray microtomography of step (c) is phase-contrast computed tomography (PC-CT). In that case, step (c) comprises obtaining a phasecontrast computed tomography (PC-CT) 3D image of the flat fixed tissue samples from the X-ray data obtained in step (b). PC-CT is an X-ray method that measures the shift in phase of the X-rays as they pass through the imaged material. This is different to regular X-ray imaging, which measures the attenuation of photons. The phase contrast provides a better definition of the edges between tumor and healthy tissue.

[0117] The phase contrast X-ray imaging data obtained in step b) is combined with tomographic techniques to obtain a 3D distribution of the real part of the refractive index of the sample. The X-rays are used to create cross-sections of a physical object that can be used to recreate a virtual model (3D model) without destroying the original object. As phase contrast X-ray imaging is more sensitive to density variations in the sample than conventional transmission-based X-ray imaging, images with improved soft tissue contrast can be obtained.

[0118] Compared to conventional histology, phase-contrast X-ray tomography on excised samples provide advantages including speed, true 3D imaging with thinner effective slicing, and less destructive and simplified sample preparation. For intraoperative resection margin assessment, high spatial resolution and short exposure times are essential. For the purposes of intraoperative assessment of a tumor resection margin in a tissue sample obtained from a patient undergoing surgery for removal of a solid tumor, it is realized that a short exposure time is required but at the same time a sufficiently high resolution to differentiate between non-cancerous and cancerous tissue. Previously, histology with similar resolution and exposure time as in the present resection margin project has only been observed at synchrotron radiation facilities, on rat brain specimen (Tbpperwien et al., Sci Rep. 7, 42847 (2017)) and human coronary arteries (Holme et al., Nature Prot. 9, 1401 (2014)). However, for our application, the local access close to the operating suite is a must, ruling out the synchrotron-source-based methods.

[0119] The method may further comprise a step (d) of analyzing the 3D image to assess whether the tissue sample contains cancerous tissue or not.

[0120] As a consequence of the assessment in step (d), it may be concluded that the surgery has been sufficient. Alternatively, it may be decided to resume surgery and excise more tissue from the patient, in which case the method for intraoperative assessment may be repeated.

[0121] The present invention will in the following be further illustrated by the following non-limiting examples.

[0122] The proposed method will now be described in more detail referring to Fig. 1. It should be appreciated that Fig. 1 comprises some operations which are illustrated with a solid border and some operations which are illustrated with a dashed border. The operations which are illustrated with solid border are operations which are comprised in the broadest example embodiment. The operations which are illustrated with dashed border are example embodiments which may be comprised in, or a part of, or are further embodiments which may be taken in addition to the operations of the broader example embodiments. It should be appreciated that the operations do not need to be performed in order. Furthermore, it should be appreciated that not all of the operations need to be performed.

[0123] Fig. 1 illustrates a method for intraoperative assessment of a tissue obtained from a patient, the method comprising: fixing the tissue sample by subjecting it to a flow of a dehydrating liquid caused by a pressure difference between the tissue sample and the dehydrating liquid; subjecting the fixed tissue sample to X-ray microimaging to generate multiple X-ray projections; obtaining a 3D image of the fixed tissue sample by X-ray microtomography using the X-ray projections. The method may further comprise a step of analyzing the 3D image to assess whether the tissue sample comprises cancerous tissue or not.

[0124] It has been shown that fixing the tissue sample by subjecting it to the flow of a dehydrating liquid caused by a pressure difference between the tissue sample and the dehydrating liquid enables both rapid fixation of the tissue sample, even when the tissue sample has thickness of at least 3 mm. Preferably, the method is applied on tissue sample with thickness of at least 10 mm, e.g. with thickness of at least 12 mm or at least 15 mm. The fixing step of the method is up to 30 minutes long, such as up to 10 minutes long, even for tissue samples that have thickness of at least 15 mm. The tissue sample may be subjected to the fixing step as a whole, without any slicing. Alternatively, the tissue sample may be split into smaller pieces with a size of up to 35 mm, or sliced into slices with a thickness of 1-9 mm, such as 3-5 mm.

[0125] The tissue sample is obtained from any patient undergoing surgery. Particularly, the tissue sample is suspected to comprise cancerous tissue by a surgeon performing the surgery, such as the surgery for removal of a solid tumor. More particularly, the tissue sample is obtained from a patient undergoing surgery for removal of a solid tumor, and the tumor resection margin of the tissue sample is assessed. As an example, the solid tumor can be removed from the patient by the surgeon and analyzed using the method according to the present invention while the patient is still in the surgery. The tissue sample comprising the solid tumor may be analyzed as a whole, without any slicing. Both fixing and analysis of the tissue sample can be performed within an hour, such within 45 minutes or within 30 minutes, from dissecting the tissue sample.

[0126] If the analysis shows that the tumor resection margin of the tissue sample comprising the solid tumor is free of cancerous tissue and small satellite tumors, the surgery can be finalized. If the analysis shows that the tumor resection margin of the tissue sample comprising the solid tumor has cancerous tissue and / or small satellite tumors, the surgery can be continued and the surgeon can remove additional tissue which can be also analyzed using the method according to the present invention. Once the tissue sample comprising the tumor resection margin is free of cancerous tissue and small satellite tumors inside the resection margin, the surgery can be finalized.

[0127] The dehydrating liquid used in the present method comprises acetone, methanol, ethanol, or any combination thereof, such as at least 50 vol% of acetone, methanol, or ethanol, or any combination thereof. Preferably, the dehydration liquid comprises at least 50% acetone, such as at least 70 vol% acetone, such as 80 vol% acetone. More preferably, the dehydrating liquid is acetone. Dehydrating liquids, such as acetone, provide enhanced contrast in X-ray microimaging and X-ray microtomography, and better differentiation between cancerous and non- cancerous / healthy parts of a tissue sample.

[0128] The tissue sample is fixed by subjecting it to the flow of the dehydrating liquid caused by a pressure difference between the tissue sample and the dehydrating liquid. The flow of the dehydrating liquid is achieved by injecting the dehydrating liquid into the tissue sample to achieve a locally increased pressure of the dehydrating liquid at the injection point, wherein injecting the dehydration liquid is performed using needles. Such needles typically have gauge number of 25 or larger, such as gauge number of 30 or larger.

[0129] Fig. 2 illustrates the general concept of fixing a tissue sample. As shown on Fig. 2, a tissue sample (200) is injected with a dehydrating liquid, e.g., with acetone, methanol, ethanol, or any combination thereof. For example, the tissue sample is injected four times to obtain four injection points (211, 212, 213, 214). Injecting is performed using one or more needles (220) connected to a syringe (230) filled with acetone. As an example, injecting is performed by multiple parallel injections using a linear multi-needle system connected to a syringe filled with acetone. Alternatively, injecting is performed by four single injections using a needle connected to a syringe fille with acetone.

[0130] Fig. 3 illustrates different needles that can be used to inject a dehydrating liquid into a tissue sample. As shown on Fig. 3, the needles can have one or more exit points / holes. The needles can contain only a hole on the tip (10) of each needle (Fig. 3A). The needles can contain one side hole (20) in addition to the hole on the tip (10) of each needle (Fig. 3B). The needles can also contain a plurality of side holes (20) in addition to the hole on the tip (10) of each needle (Fig. 3C). Alternatively, the needles can only contain side holes (20, 21; Fig. 3D). In this case, the tip of the needle is closed. The closed tip of the needle can have different shapes, such as pencil (30), bevel (40), round (50), and trocar (60) shape. Each needle on Fig. 3D has one side hole (20). Optionally, each of these needles can additionally have a plurality of side holes (21).

[0131] Injecting the dehydrating liquid is performed by any of the needles illustrated on Fig. 3, by a single injection or by multiple parallel injections. Preferably, injecting of the dehydrating liquid is performed by multiple parallel injections, as fast and precise injecting of the dehydrating liquid is achieved. The multiple parallel injections can be achieved using different multi-needle systems, i.e., multiple needle assemblies. Examples of such multi-needle systems with five needles are shown on Fig. 4. However, the number of needles in the multi-needle systems may be up to several hundreds. The multi-needle system for injecting the dehydrating liquid comprises a plurality of needles (100), wherein each needle of the plurality of needles can be independently selected from any of the needles shown on Fig. 3. The separation between the injection points in the multi-needle system is predetermined, wherein the separation may typically be 1-5 mm. Preferably, the separation between the injections points in the multi-needle system is constant, wherein the constant separation may typically be 1-5 mm.

[0132] Fig. 4A shows a linear multi-needle system (70) and a circular multi-needle system (80). The linear and circular multi-needle system comprise a plurality of needles (100), which are arranged in a straight / linear or round / circular way, respectively. Each needle of the plurality of needles in the linear or in the circular multi-needle system is connected to a holder (71, 81) via a needle hub (101). The holder (71, 81) comprises a fluid chamber (72, 82) and a connector (73, 83).

[0133] In the linear (70) and circular (80) multi-needle systems on Fig. 4A, the internal space of the fluid chamber (72, 82) is connected to the internal space of all needle hubs (101) and thereby to the internal space of all needles in the plurality of needles (100) in said linear (70) or circular (80) multi-needle system. The connector (73, 83), such as a Luer connector, is used to attach the multi-needle system to a fluid reservoir, such as a syringe, wherein the fluid reservoir contains a dehydrating liquid. The fluid reservoir can input the dehydrating liquid into the internal space of the fluid chamber (72, 82), and further to the internal space of all needle hubs (101) and the internal space of all needles in the plurality of needles (100). The output of the dehydrating liquid will be through one or more holes on each needle of the plurality of needles (100) in said linear (70) or circular (80) multi-needle system. Such flow of the dehydrating liquid through the multi-needle system is achieved by increasing pressure on the dehydrating liquid in the liquid reservoir and therefore in the multi-needle system. As the pressure on the dehydrating liquid is increased in the multi-needle system, the dehydration liquid will be pushed through the system until it reaches one or more exit holes on each needle in the plurality of needles. There will be also a locally increased pressure of the dehydrating liquid at the one or more exit holes on each needle, which can be in turn used to inject the dehydrating liquid in the tissue sample and enable the flow of the dehydrating liquid through said tissue sample. The tissue sample will be then fixed by pushing the dehydrating liquid through the tissue sample using the increased pressure on the dehydrating liquid in the liquid reservoir and in the multi-needle system. The increased pressure on the dehydrating liquid in the system can be achieved by applying pressure on the dehydrating liquid in the liquid reservoir. As an example, if the liquid reservoir is a syringe, increasing pressure on the dehydrating liquid can be achieved by pushing the barrel of said syringe manually (e.g., using a finger) or automatically (e.g., using a flow pump or a flow actuator). Thus, the flow of the dehydrating liquid is directly controlled by the level of applied pressure on the dehydrating liquid. The flow can thereby be controlled by e.g. a flow actuator. The flow actuator can achieve the desired flow of the dehydrating liquid by applying a specific and / or constant pressure on the dehydrating liquid. Optionally, the flow of the dehydrating liquid may be controlled by a flow actuator and a flow sensor. The flow sensor can measure the flow of the liquid in / through needles in the system. The desired flow of the dehydrating liquid can be achieved by applying a pressure on the dehydrating liquid through the flow actuator. However, the pressure applied by the flow actuator can be adjusted depending on readings of the flow sensor. If the flow through the needles in the system is higher than the desired flow, the pressure on the dehydrating liquid obtained by the flow actuator will be reduced. If the flow through the needles in the system is lower than the desired flow, the pressure on the dehydrating liquid obtained by the flow actuator will be increased.

[0134] In the multi-needle systems shown on Fig. 4A the liquid reservoir is connected to all needles of the plurality of needles (100). The total flow of the dehydration liquid through all needles of the plurality of needles in such systems is controlled by a flow actuator and optionally a flow sensor. Alternatively, the flow of the dehydrating liquid can be controlled for each needle of the plurality of needles in the multi-needle system. In that case, each needles of the plurality of needles will have an individual fluid chamber and an individual connector. As an example, Fig. 4B shows a linear multi-needle system (701) comprising a plurality of needles (100), a plurality of needle hubs (101), a plurality of fluid chambers (74), and a plurality of connectors (75), wherein each needle of the plurality of needles (100) is connected to a separate needle hub (101), fluid chamber (74), and connector (75). Each needle of the plurality of needles (100) is also connected to a separate flow actuator and optionally a separate flow sensor. The flow of the dehydrating liquid through each needle of the plurality of needles can be then controlled the flow actuator and optionally the flow sensor, in the same way as described above for Fig. 4A.

[0135] Any multi-needle systems previously described can have needles of same or different lengths. The length of each needle of the plurality of needles in the multineedle system can be independently selected. As an example, Fig. 4C shows a linear multi-needle system (702) comprising a plurality of needles (100), wherein one or more needles of the plurality of needles (100) have different lengths. Each needle of the plurality of needles may be independently selected from needles with lengths of 1-20 mm.

[0136] Once the tissue sample is fixed, the fixed tissue sample can be subjected to X- ray microimaging as a whole, without any slicing. Alternatively, the fixed tissue sample may be split into smaller pieces with a size of up to 35 mm, or sliced into slices with a thickness of 1-9 mm, such as 3-5 mm.

[0137] The X-ray microimaging, such as a phase-contrast X-ray imaging or a propagation-based phase-contrast X-ray imaging, of the fixed tissue sample will generate multiple X-ray projections. The multiple X-ray projections are used to obtain a 3D image of the fixed tissue sample by X-ray microtomography, such as phase-contrast computed tomography. As an example of an instrument which may be used for X-ray microimaging is the instrument which includes a metal-jet X-ray source (Excillum D2+, 250 W), a high accuracy rotating stage, and a large high- resolution detector, all on an approximately 200 x 80 cm radiation-safe bench. The sample holder provides simple, fast, safe, and flexible sample handling. The key factors for observing the relevant low-contrast cellular-sized details at short exposure times is optimizing source parameters, distances and detector parameters.

[0138] The software integrates hardware control and calibration, scan algorithms, tomographic reconstruction, and image processing algorithms in a user-friendly package. High-fidelity imaging relies on the synergy of hardware scan algorithms and post-data-acquisition image reconstruction. Key techniques involve phase retrieval, reduction of reconstruction artifacts, mathematical noise filtering, and observability analysis. The system, supplied by Exciscope AB, has demonstrated 1 pm resolution in 3D imaging.

[0139] Example - Fixation procedure

[0140] Fixation of animal meat, such as kidney or tenderloin meat, with acetone was performed and visually evaluated. Said kidney and tenderloin animal meat was obtained from a food store.

[0141] Pieces of tissue (4-5 cm long, 2 cm thick, 2-4 cm wide) were places in a beaker. A linear multi-needle system with five needles (Mesoram multiinjector) was connected to a syringe filled with 20-50 ml 100% acetone (VWR International S.A.S., Fontenay-sous-Bois, France). The acetone was injected during approximately 1.5-2 minutes, after which the tissue was cut along the injection points. Fig. 5A illustrates said sample preparation. As shown on Fig. 5A, a tissue sample (200) was injected with acetone, i.e., a dehydrating liquid. The tissue sample was injected using said linear multi-needle system to obtain several injection points (211, 212, 213, 214).

[0142] Results

[0143] Fixation of animal meat, such as kidney or tenderloin meat, with acetone was performed as described under "Sample preparation". Each tissue sample was visually evaluated after injecting a dehydrating liquid, i.e., acetone. To perform this evaluation, the tissue sample (200) was cut along the injection points (211, 212, 213, 214) using a scalpel (250). Cutting of the tissue sample was performed by following a virtual cutting line (300) connecting the injection points, as shown in Fig. 5B.

[0144] The cut tissue sample was visually evaluated by opening the tissue sample along the cutting line (300) using a set of tweezers. An illustration of the cut tissue sample is shown in Fig. 5C, wherein each injection point of the injection points is indicated with a dotted circle. Said opening of the tissue sample along the cutting line is indicated with arrows in Fig. 5C. As seen on Fig. 5C, cutting was performed at around middle of three injection points (212, 213, 214), such as that a portion of each of the three injection points was on both sides of the cutting line after the opening of the tissue sample along said cutting line. On the other hand, one injection point (211) was missed, and the whole injection point was only on one side of the cutting line after the opening of the tissue sample along said cutting line. The effect of acetone on tissue around the injection points and extent of fixed areas (400) around the injection points was visually evaluated in the cut tissue sample.

[0145] Fig. 6 shows an image of a kidney tissue after injecting acetone and cutting. The kidney tissue was injected with acetone using the linear multiple-needle system to obtain several injection points, which correspond to injection points 211, 212, 213, and 214 in Fig. 5B. Cutting was performed along these injections points, such as that three injection points were approximately cut in half, while one injection point was unintentionally missed. The kidney tissue was opened using a set of tweezers. Circles in Fig. 6 indicate a portion or a whole injection point of the injection points, after the opening of the kidney tissues using the set of tweezers. Rectangles in Fig. 6 indicate areas that were fixed with acetone, which correspond to fixed areas 400 in Fig. 5C. The fixed areas were observed as gray volumes around each injection point of the injection points.

[0146] Fig. 7 shows images of a tenderloin tissue. The tenderloin tissue before injecting acetone is shown on Fig. 7A. The tenderloin tissue was injected with acetone to obtain several injection points. Injecting was performed using the linear multi-needle system connected to a syringe filled with acetone. The tenderloin tissue after injecting acetone is shown on Fig. 7B. As a result of injecting acetone, the fixed areas were observed as gray volumes on the surface of the tenderloin tissue. Fig. 7C shows an image of the tenderloin tissue after cutting said tissue along a virtual cutting line defined by the injections points, and opening said tissue along the cutting line using a set of tweezers. Circles in Fig. 7C indicate a portion or a whole injection point of the injection points, after the opening of the tenderloin tissues using the set of tweezers. Cutting was performed such as that two injection points were approximately cut in half. The fixed areas were observed spreading from the injection points towards the inner areas of the tissue.

[0147] Itemized list of embodiments

[0148] 1. A method for intraoperative assessment of a tissue sample obtained from a patient, comprising the steps of:

[0149] (a) fixing the tissue sample by subjecting it to a flow of a dehydrating liquid caused by a pressure difference between the tissue sample and the dehydrating liquid;

[0150] (b) subjecting the fixed tissue sample from step (a) to X-ray microimaging to generate multiple X-ray projections;

[0151] (c) obtaining a 3D image of the fixed tissue sample by X-ray microtomography using the X-ray projections obtained in step (b) .

[0152] 2. The method of embodiment 1, wherein the patient is undergoing surgery for removal of a solid tumor.

[0153] 3. The method of embodiment 2, wherein the tumor resection margin of the tissue sample in step (a) is assessed.

[0154] 4. The method of any one of the preceding embodiments, wherein step (a) is a single-step chemical fixation process.

[0155] 5. The method of any one of the preceding embodiments, wherein the tissue sample in step (a) is a non-sliced tissue sample.

[0156] 6. The method of embodiment 5, wherein the thickness of the tissue sample in step (a) is at least 3 mm.

[0157] 7. The method of embodiment 6, wherein the thickness of the tissue sample in step (a) is at least 10 mm.

[0158] 8. The method of embodiment 7, wherein the thickness of the tissue sample in step (a) is at least 12 mm, such as at least 15 mm.

[0159] 9. The method of any one of the preceding embodiments, wherein the fixed tissues sample from step (a) is not sliced before subjecting it to step (b).

[0160] 10. The method of any one of the preceding embodiments, wherein the fixed tissue sample from step (a) is sliced before subjecting it to step (b). 11. The method of any one of the preceding embodiments, wherein the fixing in step (a) is up to 30 minutes long.

[0161] 12. The method of any one of the preceding embodiments, wherein the dehydrating liquid used in step (a) comprises at least one protein-precipitating organic solvent.

[0162] 13. The method of any one of the preceding embodiments, wherein the dehydration liquid used in step (a) comprises acetone, methanol, or ethanol, or any combination thereof.

[0163] 14. The method of any one of the preceding embodiments, wherein the dehydration liquid used in step (a) comprises at least 70 vol% acetone.

[0164] 15. The method of embodiment 14, wherein the dehydration liquid used in step (a) comprises at least 80 vol% acetone.

[0165] 16. The method of embodiment 15, wherein the dehydration liquid used in step (a) is acetone.

[0166] 17. The method of any one of the preceding embodiments, wherein the flow in step (a) is achieved by injecting the dehydration liquid into the tissue sample to achieve a locally increased pressure of the dehydration liquid at the injection point.

[0167] 18. The method of embodiment 17, wherein injecting the dehydration liquid in the tissue sample is performed using needles.

[0168] 19. The method of any one of embodiments 17 and 18, wherein injecting the dehydration liquid in the tissues sample is performed using needles with gauge number of 25 or larger.

[0169] 20. The method of any one of embodiments 17-19, wherein injecting the dehydration liquid in the tissue sample is performed by a single injection or by multiple parallel injections. 21. The method of embodiment 20, wherein injecting the dehydration liquid in the tissue sample is performed by multiple parallel injections with a constant separation between injection points.

[0170] 22. The method of any one of embodiments 20 and 21, wherein injecting the dehydration liquid in the tissue sample is performed by multiple parallel injections with needles of different lengths.

[0171] 23. The method of any one of embodiments 17-22, wherein injecting the dehydration liquid in the tissue sample is performed using needles comprising a single side hole or multiple side holes on each needle.

[0172] 24. The method of any one of embodiments 17-23, wherein injecting the dehydration liquid in the tissue sample is performed using needles, wherein each needle is connected to a flow actuator and optionally a flow sensor.

[0173] 25. The method of any one of the preceding embodiments, further comprising a step:

[0174] (d) analyzing the 3D image to assess whether the tissue sample contains cancerous tissue or not.

[0175] 26. The method of any one of the preceding embodiments, wherein the X-ray microimaging of step ( b) is a phase-contrast X-ray imaging.

[0176] 27. The method of embodiment 26, wherein the phase-contrast X-ray imaging is propagation-based phase-contrast X-ray imaging (PBI ).

[0177] 28. The method of any one of the preceding embodiments, wherein the X-ray microtomography of step (c) is a phase-contrast computed tomography (PC-CT).

Claims

CLAIMS1. A method for intraoperative assessment of a tissue sample obtained from a patient, comprising the steps of:(a) fixing the tissue sample by subjecting it to a flow of a dehydrating liquid caused by a pressure difference between the tissue sample and the dehydrating liquid;(b) subjecting the fixed tissue sample from step (a) to X-ray microimaging to generate multiple X-ray projections;(c) obtaining a 3D image of the fixed tissue sample by X-ray microtomography using the X-ray projections obtained in step (b) .

2. The method of claim 1, wherein the patient is undergoing surgery for removal of a solid tumor.

3. The method of claim 2, wherein the tumor resection margin of the tissue sample in step (a) is assessed.

4. The method of any one of the preceding claims, wherein step (a) is a single- step chemical fixation process.

5. The method of any one of the preceding claims, wherein the tissue sample in step (a) is a non-sliced tissue sample, and wherein the thickness of the tissue sample in step (a) is at least 3 mm.

6. The method of any one of the preceding claims, wherein the dehydrating liquid used in step (a) comprises at least one protein-precipitating organic solvent.

7. The method of any one of the preceding claims, wherein the dehydration liquid used in step (a) comprises acetone, methanol, or ethanol, or any combination thereof.

8. The method of any one of the preceding claims, wherein the flow in step (a) is achieved by injecting the dehydration liquid into the tissue sample to achieve a locally increased pressure of the dehydration liquid at the injection point.

9. The method of claim 8, wherein injecting the dehydration liquid in the tissue sample is performed using needles, preferably using needles with gauge number of 25 or larger.

10. The method of any one of claims 8 and 9, wherein injecting the dehydration liquid in the tissue sample is performed by a single injection or by multiple parallel injections.

11. The method of claim 10, wherein injecting the dehydration liquid in the tissue sample is performed by multiple parallel injections with a constant separation between injection points.

12. The method of any one of claims 10 and 11, wherein injecting the dehydration liquid in the tissue sample is performed by multiple parallel injections with needles of different lengths.

13. The method of any one of claims 8-12, wherein injecting the dehydration liquid in the tissue sample is performed using needles comprising a single side hole or multiple side holes on each needle.

14. The method of any one of claim 8-13, wherein injecting the dehydration liquid in the tissue sample is performed using needles, wherein each needle is connected to a flow actuator and optionally a flow sensor.

15. The method of any one of the preceding claims, further comprising a step:(d) analyzing the 3D image to assess whether the tissue sample contains cancerous tissue or not.

16. The method of any one of the preceding claims, wherein the X-ray microimaging of step (b) is a phase-contrast X-ray imaging, such as propagationbased phase-contrast X-ray imaging ( PBI).

17. The method of any one of the preceding claims, wherein the X-ray microtomography of step (c) is a phase-contrast computed tomography (PC-CT).

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