Methods and apparatus for laser ablation for large scale tissue enrichment

Abstract

Methods and apparatus for enriching for biological material from a region of interest within a tissue sample mounted on a slide using a laser apparatus are provided. A reference position of the tissue sample is defined relative to coordinates of the laser apparatus and regions of interest within the tissue sample are identified. The regions of interest are mapped to the coordinates of the laser apparatus to generate a dissection map and tissue is selectively ejected or modified using the laser apparatus to apply laser energy along a plurality of laser paths defined within the dissection map. A dissection map for one slide can be extrapolated to a dissection map for a plurality of slides bearing contiguous sections of the tissue sample using the reference position of the tissue sample on each slide.

Description

METHODS AND APPARATUS FOR LASER ABLATION FOR LARGE SCALE TISSUE ENRICHMENTCROSS-REFRENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, United States provisional patent application No. 63 / 621719 filed 17 January 2024. The foregoing application is incorporated by reference herein in its entirety for all purposes.TECHNICAL FIELD

[0002] Some embodiments relate to methods and apparatus for isolating biological tissue from microscope slides using laser energy. Some embodiments relate to methods and apparatus for enriching for selected portions of a biological tissue sample on a microscope slide using laser energy.BACKGROUND

[0003] A combination of demographic factors[1], and increasing indications for genomic analyses is causing a rapid rise in tissue sample submissions to cancer genomics labs. For example, BC Cancer, the entity responsible for cancer control in British Columbia (5.5 million people), saw solid tumour sample submissions rise 40% from 3,567 in 2018-20 to a projected 6,370 in 2023. Enriching for tumour from these formalin fixed paraffin embedded (FFPE) tissue samples remains a laboratory bottleneck. Enrichment is generally done by knife dissection of slidemounted tissue sections or needle-coring of tissue blocks, both using a stained section immediately adjacent to the material to guide dissection.

[0004] For a fixed number of sequencing reads, the larger the genome region to be interrogated, the lower the read coverage and thus the sensitivity to genome variants. It is therefore desirable to have an efficient method of tumour enrichment for any sequencing scale. The cost, however, must be balanced against the effort and precision required in identifying regions to dissect and, to a point, increasing sequencing depth. The two challenges presented are (1) efficient methods for identifying and mapping tissue regions of interest at adequate precision and (2) dissecting a sufficient quantity of tissue rapidly enough to be cost-effective and of utility in diagnoses.

[0005] The inventors recently demonstrated a method for whole-slide laser capture microdissection (LCM) using traditional LCM cutting techniques, mapping candidate tumour regions across a stained slide and extracting sufficient quantities of nucleic acids for whole genome and transcriptome sequencing121. This approach was shown to raise tumour content by up to 67% over un-dissected tissue without loss of sequence quality. A conventional LCM instrument using an externally generated cutting map scribed around and collected each target region. This took 30-60 minutes for a ~100 mm2tissue section, depending on the complexity of the map.

[0006] Current laser capture microdissection (commonly called LCM or LMD) instruments use slides with membranes that have low adhesion to the glass substrate. Tissue may be extracted by cutting around the region of interest and having it either fall into a collection chamber as the Leica LMD system does, or as the Zeiss system does, eject the material upward using a laser pulse to the centroid of the cut section such that the material leaves the surface and sticks to an adhesive tube cap. Both Leica and Zeiss laser microdissection machines are optimized for dissecting small areas, down to single cells, and use manual targeting of regions of interest. The inventors previously investigated using particularly the Leica apparatus to dissect larger areas by creating a cutting map by a machine learning algorithm on a high resolution scan of a whole stained tissue section. The tissue sections were mounted on PEN (polyethylene naphthalate) low adhesion membrane slides to facilitate cutting patches of tissue. The cutting map was then exported to the laser capture microdissection instrument121. The process worked to increase tumour content, but a typical dissection of around 50% of a lOxlOmm tissue section would take 45 minutes to an hour. This is an obvious practical limitation, but this work demonstrated that the process of laser dissection by using a laser to cut out sections of the tissue sample does not reduce the recovery efficiency of DNA and RNA from tissue compared to scraping it off slides with a knife.

[0007] A more recently developed form of microdissection is by laser-mediated ejection of tissue facilitated by laser-induced forward transfer (LIFT). In LIFT, rather than cutting around the tissue and capturing patches as they fall, tissue is ejectedfrom a surface by energizing a surface coating directly under the tissue using laser energy[3]. LIFT was first described by Adrian et al. for transferring metal in 1986[3]and Bradley Ringeisen and colleagues of the US Office of Naval Research, reported using LIFT for live cell transfer in about 2005[4]. This demonstration of preserving cell viability after transfer, spawned a considerable literature on laser mediated bioprinting. Ringeisen et al have a now-expired 2005 US patent 6,905,738 using this method, and developed polymer slide coatings to facilitate it. The technique has subsequently been used for spatial analysis of fixed cells, notably by Seoul National University's Sunghoon Kwon[5'6]. Here tissue from different areas of a slide is ejected into different wells of a plate where genomic analysis may be performed. Kwon's technique uses a fixed laser and moves the slide and an independent receiving plate, so different regions of interest can be dissected. The focus of using LIFT to transfer tissue from a slide has so far been on dissection of single cells or very small areas of tissue and to that end these authors describe applying techniques appropriate for single cell quantities of nucleic acids, such as whole genome amplification, to these samples.

[0008] It may also be noted that laser induced forward transfer is different from matrix assisted laser desorption ionization (MALDI) coupled with electrospray ionization and mass spectroscopy. MALDI also releases biomolecules from a surface via laser energy from the same side of the substrate, but the sample has previously been dissolved into a matrix so spatial information about the origin of the molecules has been lost. The mass of material released is also considerably smaller so while the technique has been used successfully for genotyping, identifying specific genetic markers, it has not been used for larger scale sequencing.

[0009] There remains, therefore, a need for sample processing methods and apparatus that facilitate the enrichment of a tissue sample for specific cells of interest that is fast enough to facilitate high throughput analysis by nucleic acid sequencing or proteomic analysis.SUMMARY

[0010] One aspect provides a method of using a laser apparatus to enrich for biological material from a region of interest within a tissue sample mounted on aslide. A reference position of the tissue sample is defined relative to coordinates of the laser apparatus. The tissue sample is visually assessed to identify regions of interest. The regions of interest are mapped to the coordinates of the laser apparatus to generate a dissection map. Tissue is selectively ejected or modified within the tissue sample using the laser apparatus to apply laser energy along a plurality of laser paths defined within the dissection map.

[0011] In one aspect, at least three defined reference positions are marked on the slide or on a coverslip of the slide using the laser apparatus. A high resolution image of the slide including the reference positions is obtained. The tissue sample is visually assessed to identify regions of interest. The regions of interest of the tissue sample are mapped to the coordinates of the laser apparatus based on a position of the at least three defined reference positions to generate a dissection map. Tissue within the tissue sample is selectively ejected or modified using the laser apparatus to apply laser energy along a plurality of laser paths defined within the dissection map.

[0012] In one aspect, a laser apparatus is used to enrich for a region of interest within a plurality of contiguous sections of a tissue sample mounted on a plurality of slides, one of the plurality of the slides being a parent slide and a remainder of the plurality of the slides being child slides. A high resolution image of the parent slide is obtained. A shape of a reference tissue region is defined on the parent slide. The reference tissue region can be an outline of at least a portion of the tissue sample. The tissue sample on the parent slide is visually assessed to identify regions of interest in the tissue sample. The reference tissue region of the parent slide is then aligned with a reference tissue region of one of the child slides in the laser apparatus to define a reference position of the child slide relative to the coordinates of the laser apparatus. The regions of interest from the parent slide are mapped to the child slide to generate a dissection map for the child slide.Tissue is selectively ejected or modified within the tissue sample on the child slide using the laser apparatus to apply laser energy along a plurality of laser paths defined within the dissection map for the child slide. The steps can be repeated for each one of a plurality of child slides, and for the parent slide as well if desired.The parent slide can be stained to facilitate identification of the regions of interest and the child slides can be unstained slides.

[0013] In some aspects, substantially the entirety of the dissection map is covered by the plurality of laser paths. In some aspects, the plurality of laser paths are linear or substantially linear. In some aspects, the dissection map is a positive selection dissection map and the plurality of laser paths defined within the dissection map are substantially contiguous within the regions of interest. In some aspects, the dissection map is a negative selection dissection map, and the plurality of laser paths defined within the dissection map are substantially contiguous outside the regions of interest.

[0014] In some aspects, selectively ejecting the tissue within the tissue sample is done using the laser apparatus to excite a coating on the slide, the coating having an excitation wavelength corresponding to an emission wavelength of the laser of the laser apparatus. In some aspects, the slide is an uncoated slide and selectively ejecting tissue within the tissue sample is done using the laser apparatus to supply CO2 laser energy along the plurality of laser paths.

[0015] In some aspects, the dissection map is a positive selection dissection map, and selectively ejecting or modifying tissue within the tissue sample using the laser apparatus is done by ejecting the regions of interest from the slide by rastering laser energy along the plurality of laser paths positioned within the dissection map.

[0016] In some aspects, the dissection map is a negative selection dissection map, and selectively ejecting or modifying tissue within the tissue sample using the laser apparatus comprises ejecting tissue outside the regions of interest by applying scanning laser energy along the plurality of laser paths positioned within the negative selection dissection map. In some aspects, the dissection map is a negative selection dissection map and selectively ejecting or modifying tissue within the tissue sample using the laser apparatus is done by using laser energy to degrade biological material in regions of the tissue sample outside the regions of interest by applying scanning laser energy along the plurality of laser paths positioned within the negative selection dissection map. In some such aspects, after the tissue has been selectively modified, the tissue sample is recovered fromthe slide and enriched for the desired biological material. In some such aspects, enrichment is done by size selection to recover undegraded biological material. In some aspects, a sensitizing reagent is added to the tissue sample prior to the step of selectively ejecting or modifying tissue within the tissue sample using the laser apparatus to promote degradation of the biological material.

[0017] In some aspects, the ejected tissue is collected in a medium that is compatible with downstream extraction protocols. In some such aspects, the medium is mineral oil or hexadecane.

[0018] In some aspects, apparatus for conducting the foregoing methods is provided. In some aspects, the apparatus has a slide holder for receiving at least one slide bearing the tissue sample and a laser mounted to deliver laser radiation to the at least one slide along a plurality of adjacent laser paths defined by a dissection map provided for the tissue sample. In some aspects, the apparatus has a viewer that can be used to correlate a reference tissue region of a parent slide with a corresponding reference tissue region of a child slide to map regions of interest from the parent slide to the child slide to generate a dissection map for the child slide.

[0019] In some aspects, the biological material is deoxyribonucleic acid, ribonucleic acid, or protein. In some aspects, the laser apparatus is a galvo laser apparatus.

[0020] Further aspects will become apparent with reference to the drawings and the following detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows a schematic representation of a tissue sample and the positive selection and negative selection dissection maps that can be prepared based on a region of biological interest defined within the tissue sample.

[0022] FIG. 2 shows a schematic exploded view of an example embodiment of an apparatus for carrying out certain methods.

[0023] FIG. 3 shows schematically a side view of an example embodiment of a coated slide for use in some embodiments.

[0024] FIG. 4 shows an example embodiment of a method for enriching a tissue sample for regions of interest using laser induced forward transfer applied by scanning a laser across a slide within a dissection map.

[0025] FIG. 5 shows an example embodiment of a method for carrying out laser capture microdissection using a scanning laser for high resolution large scale tissue microdissection.

[0026] FIG. 6 shows an example embodiment of a method of extrapolating a region of interest in a cutting map from a parent slide to a plurality of child slides.

[0027] FIG. 7 including panels FIG. 7A, 7B, 7C, 7D and 7E shows a specific example embodiment of a method of extrapolating a region of interest in a cutting map from a parent slide to a child slide using a reference tissue region.

[0028] FIG. 8 including panels FIG. 8A and 8B shows that adjustments to the region of interest on the parent slide can be used to adjust the region of interest on a view of the child slide.

[0029] FIG. 9 shows an example embodiment of a method for carrying out laser ablation to enrich for desired biological material within a tissue sample using negative selection.

[0030] FIG. 10 shows a second example embodiment of a method for carrying out laser ablation to enrich for desired biological material within a tissue sample using negative selection.

[0031] FIG. 11 shows an exemplary sample collection workflow.

[0032] FIG. 12 shows yield in ng / mm3from 8 pm sections from laser microdissected and bulk knife dissected samples.

[0033] FIG. 13 shows mean quality score (Q bases). LMD performed similar to bulk tissue.

[0034] FIG. 14 shows a comparison of dissection time vs. dissection area.

[0035] FIG. 15 shows an example of using negative selection to eject tissue from an uncoated slide directly using CO2 laser energy.

[0036] FIG. 16 shows an exemplary sample collection strategy for collecting five different fractions from a tissue sample by laser microdissection.

[0037] FIG. 17 shows sample and sequence quality metrics.

[0038] FIG. 18 shows variant allele fraction density.DESCRIPTION

[0039] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

[0040] In one aspect, the present invention provides a method and apparatus for rapidly isolating tissue regions of tens to hundreds of square millimeters with arbitrarily complex boundaries from surfaces such as glass slides in a form suitable for further analysis including DNA sequencing. One specific embodiment of the present invention includes the following steps: 1) glass slides are coated with a material capable of absorbing laser energy at a specific frequency corresponding to the wavelength of light emitted by the laser, 2) cells in a thin tissue section are mounted on the coated slides and stained, 3) fiducial markers are added to the slides or a reference position for the tissue sample on the slides is otherwise defined, 4) slides are digitized and the boundaries of regions of interest on the image are manually, digitally or otherwise annotated and 5) after removal of any coverslip, the annotated regions are removed from the surface using the laser following scanning paths along a dissection map and collected.

[0041] In one embodiment, the slides are coated with indium tin oxide. In an alterative embodiment the slides may be coated with triazene. In further alternative embodiments, a laser tuned to a selected frequency in combination with a transparent coating absorbent at said laser frequency may be selected, such as PMMA (poly(methyl methacrylate) or acrylic) combined with a dye that is transparent in the visible frequencies but strongly absorbent at the laser frequency / wavelength. In still a further embodiment, a laser capable of ejecting tissue without the need for a slide coating, for example a CO2 laser, can be used with uncoated slides.

[0042] In one embodiment, the laser is a 20 W pulsed fiber laser. In an alternative embodiment, low power lasers tuned to near IR. can be used. In a further alternative embodiment, a 1W 405nm diode laser is used.

[0043] In one embodiment, fiducial marks are applied by laser. Other methods of adding fiducial marks to slides or otherwise defining reference points known in the art may be selected. In alternative embodiments, the visible outline of the sample is used instead of fiducial marks as a reference to locate the portions of the sample selected for dissection.

[0044] In a still further aspect, negative ablation of undesired regions is performed using a laser to degrade the nucleic acids or other biological material of undesired regions mounted on uncoated slides. Degradation may be a combination of cutting the DNA into small fragments, crosslinking to adjacent protein material or modifying the nucleic acids themselves. The entire tissue section is then removed from the slide and the degraded nucleic acids are removed by size selection during the nucleic acid extraction process. This process may be aided by application of a reagent that is highly reactive to the laser such as to locally heat or otherwise chemically modify the DNA.

[0045] In one embodiment, the laser is scanned over the regions of interest in a raster scan with a laser-galvo system or mirror galvanometers. Other scanning or rastering methods known in the art may be selected.

[0046] Distinct from conventional laser capture microdissection techniques which rely on cutting around regions of interest to enable selected portions of the tissue sample to be removed from the slide, in embodiments of the present invention the laser beam is rastered or scanned across each area of interest, at a sufficient density of adjacent lines to energize all of the coating material within the desired target region, thereby ejecting substantially all of the tissue of interest within the desired target region.

[0047] Specifically, as shown in FIG. 1, based on an image of the tissue sample to be processed 10 in which one or more regions of interest 12 have been defined, there will be a corresponding one or more undesired regions 14. For example, region of interest 12 may be a region identified by a pathologist and / or a machine learning model as being likely to contain tumour cells, while undesired region 14 may be a region identified by a pathologist and / or a machine learning model as likely containing normal cells. For purposes of genomic or proteomic analysis, it is desirable to enrich for or obtain cells from region of interest 12. Accordingly, basedon the regions of tissue sample 10 as defined with reference to the coordinate system of a laser cutting apparatus, a dissection map can be prepared to enable the laser cutting apparatus to selectively dissect the region of interest 12 or ablate the undesired regions 14 to enrich the tissue sample for tissue from region of interest 12.

[0048] In some embodiments in which it is desired to selectively dissect tissue from region of interest 12, a positive selection dissection map 20 is prepared. In positive dissection selection map 20, a plurality of laser paths 40 along which the laser is scanned are provided that correspond spatially to region of interest 12. To dissect the tissue within region of interest 12, rather than cutting around the perimeter 50 of the region of interest 12 as would conventionally be done for laser capture microdissection, the laser is actuated to travel along each one of laser paths 40 defined within positive selection dissection map 20, to eject the tissue within region of interest 12 via laser induced forward transfer.

[0049] In some embodiments in which it is desired to selectively dissect tissue outside of region of interest 12 or to ablate or destroy biological material outside of region of interest 12 (i.e. within undesired region 14), a negative selection dissection map 30 that is essentially the inverse of positive selection dissection map 20 is generated. In negative selection dissection map 30, the laser is scanned along the plurality of laser paths 40 that correspond spatially to undesired region 14. In this way, tissue corresponding to undesired region 14 can either be removed from the slide by laser induced forward transfer, or alternatively the laser can be used to damage biological material such as nucleic acids or proteins within undesired region 14, so that only nucleic acids or proteins within region of interest 12 are left intact for further analysis.

[0050] The laser paths 40 are defined so that substantially an entirety of the dissection map is covered by the plurality of laser paths 40, for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% of the surface area within the dissection map is covered by the plurality of laser paths 40. In some embodiments, each one of the laser paths 40 defined within the dissection map is linear or substantially linear, as illustrated in FIG. 1. In alternative embodiments, laser paths 40 could be provided with any desired shapes, e.g. laserpaths 40 could be defined as a series of concentric circles or the like. In some embodiments, each one of the laser paths 40 defined within the dissection map is contiguous with at least one adjacent laser path 40. In some embodiments, each one of the laser paths 40 extends from a first point on perimeter 50 to a second point on perimeter 50 of the region of interest, although optionally as illustrated each one of the laser paths 40 may be discontinuous, for example by reason of the presence of an undesired region 14 within a region of interest 12 as defined by an inner perimeter 52. In this way, laser energy is applied along the laser paths 40 to substantially fill the area defined by positive selection map 20 or negative selection dissection map 30, rather than cutting around perimeter 50 as would be done when conducting traditional laser capture microdissection.

[0051] Some embodiments are based on the concept of Laser Induced Forward Transfer where laser energy is strongly absorbed by a thin film that sits between a glass slide and cells or a section of tissue. Upon absorbing the laser energy, the film vaporizes and rapidly expands as a gas, ejecting the material mounted on it. The resolution can be on the order of the laser spot size (10-25 pm), so single cells, ten microns in diameter can be ejected this way. The inventors have combined this technique with scanning optics using a laser unit such as a galvo laser unit to enable very fast microdissection of entire tissue sections. This is done in one example embodiment by coupling a laser to spinning mirror galvos, and importantly, such devices can be purchased commercially as laser engraving machines. These "laser-galvo" systems as they are commonly known, with addition of a slide positioning stage, can readily become a dissection tool as shown in FIG.2. The galvo mirror system enables very rapid scanning or rastering back and forth of the laser over regions of interest, ejecting tissue from the slide wherever the laser is scanned. Such embodiments are useful for example in the field of clinical genomics or proteomics.

[0052] The inventors have now recognized that the forward transfer mechanism may be applied to tumour microdissection such that a region of tens to several hundred square millimeters with arbitrarily complex boundaries might be ejected from a slide mounted tissue section in the order of one minute rather than one hour for conventional laser microdissection techniques that rely on cutting aroundregions of interest. This system of using laser energized, thin film mediated active ejection of tissue, in combination with using rastering (scanning) laser paths, has not been previously conducted. This is potentially important because tissue dissection for genomic analysis has become increasingly important for clinical laboratories as genomic profiling of patient tumours has expanded. Additionally, the inventors have found that use of an appropriate laser such as a CO2laser emitting at around 10.6 pm can be used to eject tissue even from uncoated slides.

[0053] With reference to FIG. 2, an example embodiment of an apparatus that can be used to carry out the methods described herein is illustrated. In the illustrated embodiment, the apparatus is a commercial laser engraver coupled to a stage or slide holder where slides can be reproducibly placed and then returned.

[0054] With more detailed reference to FIG. 2, an example embodiment of an apparatus 100 that can be used to carry out the methods described herein is illustrated in more detail in an exploded view. Apparatus 100 has a laser unit 120 mounted to direct light from a laser towards a slide holder 130. As can also be seen in FIG. 3, in some embodiments, a slide 140 for use with apparatus 100 has a coating 142 applied thereon, and can have a coverslip 144 affixed thereto to secure a tissue sample 146 in place.

[0055] In some embodiments, laser unit 120 is provided as a conventional laser engraving galvo laser unit having a laser 122 and mirrors 124 for directing light emitted by laser 122 as is known in the art. Software is available as is known in the art to control the application of laser energy by the laser unit 120 to select spatial locations on the slide 140. In alternative embodiments, a laser gantry is used with mirrors on XY gantries as the laser unit 120, which may provide a larger range of motion. Any desired laser system and mechanism of actuating such laser system may be used for laser unit 120. Galvo laser units such as that illustrated in FIG. 2 may provide faster scanning of the laser 122 and may be desirable for high throughput applications in particular.

[0056] Apparatus 100 also has a viewing unit 126, for example a suitable camera, for viewing a tissue sample positioned on slide holder 130. Viewing unit 126 used with laser apparatus 100 generally has a comparatively low resolution,because the viewing unit has to focus through the very long working distance field lens of the laser engraver.

[0057] In some embodiments, the laser used in laser unit 120 has an Nd-YAG (neodymium-doped yttrium aluminum garnet) laser that emits a wavelength of 1064 nm. This wavelength can energize indium tin oxide (ITO) and triazene-based slide coatings. In alternative embodiments, other lasers with different wavelengths could be used in laser unit 120 provided the slide coating 142 used has the appropriate absorption spectrum such that the coating is sufficiently energized by the laser to eject the tissue as described herein, e.g. a 1W 405 nm UV diode laser, a laser emitting below 350 nm, or the like. In another embodiment, the laser used in laser unit 120 is a carbon dioxide (CO2) laser which can directly modify the tissue or eject the tissue without the need for using a coated slide.

[0058] A recent development has been the realization by the inventors that a near infrared (I R) laser used to excite triazene or indium tin oxide (ITO), is likely not the most efficient combination of laser frequency and coating for carrying out LIFT. ITO at least, is not very absorptive in the near IR, so a lot of excess energy is currently being applied. The inventors have realized that there may be more efficient pairings, notably poly(methyl methacrylate) (PMMA or acrylic) combined with a dye that is transparent in the visible but strongly absorbent at the laser frequency. It's also likely there will be enough energy from a 1W 405nm diode laser to conduct this technique, and there are dyes can be found to match. In some alternative embodiments, a laser emitting below 350 nm may be used to directly energize the PMMA. Thus, other embodiments with tuned coatings / dyes and lasers to carry out LIFT may be used. In still other embodiments, a CO2 laser emitting at around 10.6 pm may be used without a coated slide to eject portions of the tissue sample.

[0059] One example of a dye that can be used together with PMMA is VIS404A, which is available from QCR Inc., which makes a wide range of dyes having different characterized absorption spectra. The person skilled in the art could select from among many such available dyes to select a dye having a desired absorption profile for a particular application. For example, VIS404A has an absorption peak at 404nm so if dissolved in PMMA, it should efficiently absorb energy from a 405nm UV diode laser, which is an example of a comparatively inexpensive laser. PMMA is advantageous as it is low cost and with sufficient heating, will vapourize rather than melt, proving a driving mechanism for LIFT. In other embodiments, a laser emitting below 350 nm may be used to directly energize the PMMA to facilitate LIFT.

[0060] In some embodiments, stage 130 is provided as a means to support and collect ejected tissue from one or more slides. To reduce parallax error and to allow the use of a small (eg ~15-25 mm square) collection vessel, in some embodiments each slide to be dissected is positioned or can be positioned under the centre of laser unit 120. In some embodiments, slide holder 130 can be a carousel-type slide holder that can hold a plurality of slides 140, so that multiple different slides may be acted on by laser unit 120. For example, in some embodiments, up to six slides prepared from adjacent sections of a single tissue sample may be dissected on a single slide holder 130, to allow the acquisition of sufficient enriched tumor material to facilitate downstream analysis such as nucleic acid sequencing or proteomic analysis.

[0061] In some embodiments, coating 142 is indium tin oxide (ITO), for example as developed by Ringeisen et al., or a triazene-based polymer, for example as were formerly commercially available from Expression Pathology / Leica. In other embodiments, coating 142 can be a laser-sensitive dye that can be excited at a specific laser wavelength to cause Laser Induced Forward Transfer of a portion of the tissue sample 146. In some embodiments, coating 142 can be poly methyl methacrylate (PMMA) combined with a laser-sensitive dye that can be excited at a specific laser wavelength to cause Laser Induced Forward Transfer of a portion of the tissue sample 146, or coating 142 can be PMMA alone if the laser used has a wavelength suitable to excite the PMMA directly (e.g. below 350 nm). In some embodiments, coating 142 is omitted if the laser being used is able to eject tissue from the slide without a coating (e.g. a CO2 laser emitting at around 10.6 pm) or if carrying out negative selection by the destruction of biological material in undesired regions of the tissue sample as described below.

[0062] In some embodiments, tissue sample 146 is a formalin-fixed paraffin- embedded (FFPE) tissue, for example as are conventionally prepared for histopathology analysis.

[0063] In some embodiments, a receiving tray 160 is provided to receive the tissue that is ejected by LIFT.

[0064] A mechanism is used to allow the reproducible positioning of a dissection map describing the laser path of apparatus 100 relative to the sample when carrying out the methods described herein. The dissection map 154 corresponds to a region of interest of tissue sample 146 which cannot be directly identified on the apparatus 100, so must be prepared from the slides 140 on an external apparatus, for example as described in Coope et al.[2]In some embodiments, the slides 140 are marked with fiducial markings 150, for example by the laser unit 120, to facilitate accurate positioning of the slides 140 for tissue dissection after the dissection map 154 has been prepared. In various embodiments, dissection map 154 can be either a positive selection dissection map 20 or a negative selection dissection map 30.

[0065] In some embodiments, a dissection map 154 is prepared as described in more detail with reference to the methods discussed below. The dissection map represents a target region of interest (for example a region of the tissue sample believed to contain tumour cells) and is defined with respect to the coordinate system of the apparatus 100, to allow apparatus 100 to carry out selective microdissection or laser modification of the regions of interest within the dissection map 154 by rastering laser energy from laser 122 within the area defined by the dissection map 154.

[0066] With reference to FIG. 4, an example embodiment of a method 200 for enriching a tissue sample for regions of interest using scanning lasers is illustrated. While specific embodiments can vary, the common elements of the method are to obtain a tissue sample of interest to dissect, obtain an equivalent slide on which the region of interest to dissect can be visualized and define a set of reference points that can be identified in both the slide visualization and on the laser dissection apparatus.

[0067] In some embodiments, at step 202 a tissue sample of interest, for example a tissue sample obtained from a putative tumor, is mounted on a slide, stained and covered. In some embodiments, the tissue samples are stained with hematoxylin and eosin and then covered with a coverslip as is known in the art. Insome embodiments, the tissue samples are sections of formalin-fixed paraffin- embedded (FFPE) tissue samples, for example as obtained from a biopsy of a putative tumor. In some embodiments, step 202 is carried out by a pathology lab, and the slide-mounted tissue samples are provided for use in the remaining steps of method 200.

[0068] At step 204, reference positions are defined to relate the coordinates of the laser dissection apparatus (e.g. apparatus 100) and the imaging apparatus used to define the target regions of interest. The reference positions are a set of three or more points which are known in the coordinate system of the dissection apparatus (e.g. apparatus 100) and also visible in the image analysis system used to define the regions of interest for dissection. In some embodiments, at step 204, the slides are marked before visualization with fiducial markings to facilitate accurate positioning of the dissection map relative to the laser apparatus, where the slide has been reproducibly positioned for dissection in the laser apparatus after imaging. In some embodiments, the fiducial markings are spots in the coating and / or coverslip applied by a laser, such as the laser unit 120 of apparatus 100. Because the fiducial markings are applied by the laser of the cutting apparatus, the fiducial markings are located in a known position with respect to the coordinate system used by the apparatus 100. Thus, when the slide is imaged to identify the target regions of interest, the target regions of interest can be mapped to the coordinate system of the apparatus 100 because the position of the fiducial markings is known in both systems. In some embodiments, slide holder 130 is configured to allow the slide to be reliably positioned at the same spatial location relative to the coordinate system of apparatus 100. In some embodiments, a set of reference points, e.g. three reference points although more can be used in various embodiments, are defined on an image file produced from the slide, e.g. as described in Coope et al., 2021[2].

[0069] In some embodiments at step 204 to define reference positions relative to the coordinate system of the laser cutting apparatus that are also visible in the imaging apparatus used to define the target regions of interest, a reference tissue region is defined based on an image of the slide. For example, the tissue sample on the slide will have a characteristic outline or contain certain characteristic visualshapes which are visible both on the cutting apparatus (e.g. apparatus 100) and in the visualization system used to define the target regions of interest. This characteristic shape of the tissue sample serves to define a reference position of the tissue sample at step 204.

[0070] At step 210, the slide is visually assessed to identify both the reference positions and the target regions of interest, for example to identify regions that likely contain tumour tissue, regions that are likely adjacent to tumour tissue, and regions that are likely representative of normal tissue. For example, a pathologist may manually annotate putative tumour and normal regions of the tissue sample either physically on the slide or on scanned images of the slide, or a machine learning model that has been trained to differentiate between different cell types (e.g. cancer cells vs. non-cancer cells vs cells likely adjacent to cancer cells) can be used to annotate putative tumour and normal regions of the tissue sample. In some embodiments, the pathologist may verify or further guide the determination of target regions of the tissue sample by the machine learning model. The identified regions of interest may correspond to the putative tumour cells.

[0071] At step 216, the points defining the region of interest in the visualization coordinates are converted to coordinates for the laser dissection apparatus using the reference points defined for each coordinate system. At step 218 a dissection map is generated from the regions of interest. This generates a series of scan lines at a specified density within each region of interest so that when the laser is scanned over the tissue, these areas are ejected evenly from the slide. In some embodiments this is a positive selection for recovery of the tissue in the regions of interest. In other embodiments a negative selection may be use where the laser scans the undesired regions either to eject or modify unwanted tissue.

[0072] In some embodiments in which a reference tissue region is defined based on an image of the slide or where reference points are defined on an image file produced from the slide, steps 204 and 210 may be carried out concurrently.

[0073] At step 220, a laser, which in some embodiments is coupled to a galvo mirror system, such laser unit 120 as illustrated, is used to enrich the tissue sample for regions of interest, either by directing the laser along the dissection map to eject tissue from the regions of interest if conducting positive selection, or bydirecting the laser along areas outside the dissection map to modify or eject tissue outside the regions of interest if conducting negative selection. For example, as described in more detail below, positive selection laser-induced forward transfer can be conducted along a plurality of laser scan lines to carry out laser induced forward transfer of tumour and / or normal regions of the tissue sample, or the laser can be used to eject or to damage material such as nucleic acid or protein in undesired material, thereby enriching for nucleic acids or proteins in the regions of interest of the tissue sample via a negative selection technique.

[0074] With reference to FIG. 5, an example embodiment of a method 300 for carrying out high resolution laser capture microdissection using scanning lasers is illustrated. This method can be used where near single cell resolution of the dissection map is desired and where the addition of fiducial markings, identification of the regions of interest, and tissue dissection are performed on a single slide. Steps 302, 316, 318 and 320 are equivalent to steps 202, 216, 218 and 220 described above, and are not further described again in detail.

[0075] At step 306, fiducial markings at defined coordinates are added to the slide using the laser dissection apparatus (e.g. apparatus 100). A high resolution image of the stained tissue sample and fiducial markings on the slide is obtained at 308. At step 310, the high resolution image is used to identify regions of the tissue sample that are of interest (e.g. putative tumour cells). At step 312, the fiducial reference positions are identified on the same high resolution image to facilitate translation of the regions of interest to the laser coordinates, in step 316, creating the dissection map at step 318 which is used to eject or modify tissue using the laser at 320 by scanning the laser across a plurality of laser paths defined within the dissection map. The dissection map generated at step 318 can be either a positive selection dissection map or a negative selection dissection map.

[0076] In some cases when carrying out laser microdissection for tissue enrichment for genomic analysis, it is conventional that a plurality of slides (e.g. six slides) will be prepared from separate slices of a single tissue sample in order to obtain a sufficient amount of nucleic acid for further analysis. It is also conventional that only one of the six slides may be stained and visually evaluated (whether manually or via a machine learning model or a combination thereof) todifferentiate between likely tumour tissue and likely normal tissue. This stained slide may be referred to as a parent slide. Because all of the slices of the tissue sample are taken in close proximity to one another, the overall shape of the tissue sample (i.e. the reference tissue region) and the regions of interest for dissection will be generally similar, and so the regions for dissection can be extrapolated for each one of the unstained slides, referred to as child slides, based on the reference tissue region defined by the parent slide.

[0077] Conventionally for genomics analysis, six slides are prepared from six adjacent sections of a single formalin-fixed paraffin-embedded (FFPE) tissue sample, but in other embodiments different number of slides could be used, e.g. 3, 4, 5, 6, 7, 8, 9 or 10 depending on the amount of the region of interest present on each one of the slides.

[0078] FIG. 6 shows a method 400 of extrapolating a region of interest in an image of a parent slide to a dissection map on a child slide to facilitate laser microdissection of the child slide on a laser apparatus such as apparatus 100. Steps 402, 408, 410, 416, 418 and 420 are similar or equivalent to steps 302, 308, 310, 316, 318 and 320 and are not further described in detail again. Step 402 is carried out only for the parent slide.

[0079] At 403, unstained child slides are prepared from tissue sections cut contiguously with stained parent slide section. No fiducial marks are added to the parent or child slides. At step 408, a high resolution image of the parent slide is obtained as described above for step 308, but high resolution images of the child slides are not required. In step 410, the regions of interest for the parent slide can be defined at low or high resolution. Rather than using fiducial marks as described for method 300, at step 412 the reference region for the parent slide is defined based on the visual geography of the tissue sample thereon, for example as the outer boundary of the tissue section, for which equivalent reference shapes will be visible on the child slide sections as imaged on the laser dissection apparatus (e.g. apparatus 100).

[0080] At 414, the reference tissue region obtained at 412 from the parent slide is visually aligned to the corresponding reference tissue region of the child slide's tissue sample (e.g. the outline of the tissue sample), visible on the viewer of thelaser dissection apparatus, producing a reference region for the child slide in the laser coordinates of the laser dissection apparatus. In step 416, the regions of interest defined for the parent slide are mapped to laser coordinates for the child slide based on the reference regions for the two slides. These mapped regions of interest are then carried forward to generate the dissection map for the laser in 418 to facilitate ejection or modification of appropriate regions of the child tissue sample at 420 by passing the laser along a plurality of laser scan lines defined within the dissection map. At 422, steps 414, 416, 418 and 420 can be repeated for each one of a plurality of child slides, and optionally for the parent slide as well.

[0081] In some embodiments, the parent slide is also dissected using the laser dissection apparatus in addition to one or more of the child slides (after any cover slip has been removed). In some embodiments, steps 414, 416 and 418 are also carried out even when dissecting tissue from the parent slide, as for example the tissue sample on the parent slide may have shifted, stretched or moved during removal of the coverslip. In some embodiments, both the parent slide and a plurality of child slides, for example, five child slides, which is common in manual dissections in clinical genomic analysis, are dissected in method 400.

[0082] In some embodiments custom software can be used to create a linked replica of the reference tissue region from the parent slide for each child slide at 416. The replica can be sized and warped to match the tissue section visible through the viewer of the galvo laser dissection apparatus. Regions of interest dependent on the reference region will be automatically adjusted for the child slide based on the parent slide.

[0083] In some embodiments, the laser dissection apparatus is equipped with a slide holder that can hold and move up to the total number of parent and child slides selected for dissection or modification (e.g. six slides), so that all of the plurality of slides can be loaded at one time and the slide holder can manipulate the slides to present them one at a time for processing by the laser apparatus.

[0084] FIG. 7 illustrates a specific example embodiment of extrapolating a region of interest from a stained parent slide to an unstained child slide using an apparatus such as apparatus 100 having a galvo laser unit. The image in FIG. 7A is a conventional microscope image of a tissue sample that has been stained toenable review by a pathologist and / or machine learning model to identify regions of interest. In this example embodiment, the upper lobe of the tissue sample has a generally triangular outline, with some regions (analogous to inlets and bays on a geographic map) in which no tissue is present. A reference region corresponding to this lobe has been annotated (in green). The image in FIG. 7B shows the equivalent region on an unstained tissue sample on a child slide which has not been deparaffinised through the camera (i.e. viewer) of a laser apparatus such as apparatus 100 with the reference region translated to the equivalent tissue region (in green). The generally triangular outline of the upper lobe of the tissue sample is faintly visible within the viewer of the galvo laser unit. Even in the case of viewing a stained slide through the viewer of the laser galvo apparatus, the resolution is typically such that little detail beyond the outline of the tissue sample will be visible.

[0085] The image in FIG. 7C shows a region of interest that has been superimposed on the generally triangular upper lobe of the parent tissue sample (e.g. a region that has been identified as being likely to contain tumour cells) to generate a dissection map. A linked replica of the reference tissue region of the parent slide can then be added to the laser coordinates in the galvo laser apparatus such as apparatus 100. The image in FIG. 7D shows the view of the child tissue sample through the camera (i.e. viewer) of the galvo laser unit, with the replica of the reference tissue region of the parent slide superimposed thereon including the subregion of interest, but at a different scale.

[0086] As can be seen in the image in FIG. 7E in which the outline of the reference tissue region in blue has been scaled to fit the outline of the reference tissue region of the child slide visible within the viewer, the replica of the reference tissue region of the parent slide can be scaled, translated, rotated or warped as required to match the tissue section visible in the camera view of the galvo laser apparatus, and the desired region of interest will be scaled accordingly to produce a dissection map for the child slide which can be dissected using the laser apparatus.

[0087] With reference to FIG. 8, with appropriate software, if adjustments are made to the shape and configuration of the region of interest on the parent slide (shown in high resolution view in FIG. 8B in green), then correspondingadjustments are also made to the region of interest on the child slide (shown in FIG. 8A in green through the viewer of the laser apparatus).

[0088] With reference to FIG. 9, an example embodiment of a method 500 for carrying out rapid laser ablation to enrich for desired nucleic acids or proteins within a tissue sample using negative selection is illustrated. Method 500 is shown here as a variant of method 400, but could be also be similarly performed as a variant of method 300. Steps 502, 503, 508, 510, 512, 514, and 516 correspond to or are generally similar steps 402, 403, 408, 410, 412, 414, and 416 and are not further described in detail again.

[0089] In method 500, rather than ejecting the desired material from the slide, enrichment for regions of interest is carried out by using the application of scanning laser energy along a negative selection dissection map to degrade nucleic acids or other biological materials such as proteins in regions that are not of interest. In some embodiments, at step 505 a sensitizing agent such as NIR.1072C from QCR Inc., which is used in conjunction with a laser emitting at 1064, is added to cause the tissue sample to absorb more laser energy leading to enhanced degradation of the nucleic acids and / or proteins at step 520. In another example embodiment, a laser with a wavelength that can be directly absorbed by the tissue sample such as the 10.6 pm wavelength of a CO2 laser, or which can directly degrade nucleic acids such as in the UV range of 260-280nm, can be used to carry out degradation of nucleic acid and / or protein in the regions that are not of interest at step 520.

[0090] At step 518, an inverse laser dissection map (i.e. a negative selection dissection map 30) is created that covers all areas of the tissue sample that lie outside the regions of interest. In step 520 the laser is scanned along the plurality of laser paths defined within the dissection map and degrades or otherwise modifies nucleic acids and / or proteins outside the regions of interest. Then, at step 530, the tissue, including nucleic acids and / or proteins, is recovered from the slide in bulk and at 532 the desired biological material being the unmodified nucleic acids and / or proteins within the regions of interest in the tissue sample are separated from the degraded nucleic acids and / or proteins, for example by using size selection to remove the degraded (and therefore smaller) degraded nucleic acids and / or proteins.

[0091] At step 522, steps 514, 516, 518, 520, 530 and 532 can be repeated for each one of a plurality of child slides, and optionally for the parent slide as well.

[0092] In some embodiments, the nucleic acids that are recovered in method 500 are deoxyribonucleic acids (DNA). In some embodiments, the nucleic acids that are recovered in method 500 are ribonucleic acids (RIMA). In some embodiments, the nucleic acids that are recovered in method 500 are both DNA and RNA. In some embodiments, proteins are recovered in method 500.

[0093] While method 500 has been described above with reference to dissecting a plurality of slides, if desired the same method could be applied to only the single parent slide without processing any child slides.

[0094] In some embodiments, if desired, rather than being used in conjunction with a low resolution method such as method 400, method 500 could be used in a high resolution method such as method 300 by appropriately modifying the steps of method 300 to carry out negative selection, illustrated as method 600 in FIG.10. In method 600, steps 602, 606, 608, 610, 612 and 616 are generally the same as steps 302, 306, 308, 310, 312 and 316 and are not further described again. At step 618, rather than generating a positive selection dissection map, a negative selection dissection map is generated. In step 620 the laser degrades or otherwise modifies nucleic acids and / or proteins outside the regions of interest. Then, at step 630, the tissue, including nucleic acids and / or proteins, is recovered from the slide in bulk and at 632 the desired biological material, e.g. unmodified nucleic acids and / or proteins within the regions of interest in the tissue sample are separated from the degraded biological material, e.g. nucleic acids and / or proteins, for example by using size selection to remove the degraded (and therefore smaller) degraded nucleic acids and / or proteins.

[0095] In some embodiments as illustrated in FIG. 11, laser microdissection of a slide is carried out and the ejected material is collected in a medium which is compatible with downstream extraction protocols such as mineral oil, hexadecane or the like. Without being bound by theory, such a collection method allows for small tissue particles to be efficiently collected since mineral oil is chemically similar to the paraffin that the tissue sample is embedded in. For example, in oneembodiment, the receiving tray 160 of apparatus 100 is filled with mineral oil, so that the collected tissue will be received in mineral oil.

[0096] After the ablated tissue has been collected into mineral oil, the mineral oil from receiving tray 160 can be poured into a sample tube, and the receiving tray 160 can be rinsed with mineral oil to help collect as much of the ablated tissue as possible. The receiving tray 160 can optionally be centrifuged, for example through a funnel, to collect residual oil in a tube, and the level of mineral oil in the sample tube can optionally be topped up or some mineral oil can be removed to achieve a standardized sample volume.

[0097] While the exemplary embodiments described above have been described principally with reference to the analysis of nucleic acids within a tissue sample, the same methods may be conducted to enrich for portions of a tissue sample for proteomic analysis.Examples

[0098] Certain embodiments are further described with reference to the following examples, which are intended to be illustrative and not limiting in nature.Example 1.0 - Rapid Laser Ablation for Large Scale Tissue Microdissection

[0099] The inventors used a SPI ND-YAG laser

[0014] connected to a Raylase Superscan II- E galvo scanner as the laser-galvo apparatus. In this apparatus, this scanner is mounted above a fixed stage where slides can be accurately repositioned, with a space under the slide for a receiving tray. The inventors also tested a Trumpf engraver and it was faster but otherwise similar.

[0100] The Nd-YAG laser emits at 1064nm. The inventors have used this laser successfully with two types of slide coatings. The first were Director Slides, originally made by a company called Expression Pathology and distributed by Leica. These were coated with a triazene polymer and were purportedly specifically offered to get the Leica LMD to be more efficient by using the forward energy transfer technique using conventional laser cutting of patch boundaries.

[0101] In addition to the Director slides, the inventors successfully used slides coated with Indium Tin Oxide (ITO), the well known transparent conductive metaloxide that is mentioned by Ringeisen et al. ITO also appears to be the coating of choice for recent work in this area, such as by the Kwon lab in Korea. Both types of slides were shown to work equally well with the tested laser system. It does appear from the literature that considerable effort was spent to develop triazene polymer coatings and that it might have been considered an excellent coating though apparently not better than ITO as currently only ITO slides are available commercially15'6'11'131.Example 1.2 - Low Resolution Applications of Large Scale Laser Ablation for Tissue Microdissection

[0102] In some regions, pathology labs send for genomic analysis, typically, five contiguously sectioned tissue slides plus one stained slide which has been manually annotated by pathologist to identify the region of interest. The genomics lab then manually knife-scrapes regions on the five slides corresponding to the annotation. To the knowledge of the inventors, there are multiple hospitals handling on the order of 500 slides / day in this manner.

[0103] The aforementioned workflow is suitable for genomic assays that do not require cell by cell separation of tumour and normal tissue. This means that the annotation from the stained slide, whether made digitally or manually, can be propagated from the stained slides to unstained slides with tissue which is contiguous with the stained slides and this will be sufficiently accurate for successful tumour enrichment. The slides upon which these tissue sections are mounted therefore desirably have an energy absorbing coating for LIFT, or negative selection methods can be usedExample 1.3 - Proof-of Concept by Exome Sequencing on Laser Ablation Microdissected Tissue

[0104] The inventors have performed a proof of concept experiment performing exome sequencing on tissue from 9 colorectal cancer cases

[0014] obtained by LIFT. These were from cases that had previously undergone whole genome and transcriptome sequencing of (fresh frozen biopsies) of the metastatic tumours, and whole genome sequencing on a normal (blood) sample[7]. FFPE blocks from the primary tumours for these cases that had been made years earlier when thecancers first presented, were then obtained and these were dissected by laser ablation as described herein and then underwent exome sequencing. These different sequencing operations on the same tissue allowed better analysis of sequence quality of the laser ablated tissue[2'14].

[0105] Sections were mounted on ITO coated slides, H&E stained, scanned on a Lieca Aperio scanner, and then the coverslips were removed before laser ablation microdissection. Tumour boundaries were drawn manually in Qupath[8]and exported via in-house scripts (see

[0014] ) to create tumour and normal enriched samples. Samples were also scraped off by knife as a bulk control and then as tumour and bulk knife controls.

[0106] Some performance metrics are highlighted in FIGs. 12 and 13. Yield of nucleic acids is notably similar to scraped material, and the mean quality scores from sequencing are also comparable. There are further metrics from this experiment characterizing the general performance of the exome sequencing but the important conclusion is that use of the LIFT process does not reduce yield or sequence quality vs bulk scraping the other key conclusion is it is fast, around 0.1 seconds / mm2. FIG. 14 shows that the tested system dissects tissue at ~0.1 seconds / mm2which is 30-50x faster than conventional laser microdissection.Example 1.4 - Exemplary Sample Collection Workflow

[0107] An example sample collection workflow is shown in FIG. 11. This example demonstrates that the ablated material can be collected in mineral oil, which is chemically similar to the paraffin the tissue is in, and this allows all the small tissue particles to be efficiently collected. In some aspects, different sections of tissue are ablated into different containers as well.Example 2.0 - Plain (Uncoated) Slide Sample Enrichment using Negative Selection by Laser Ablation (NSLA)

[0108] Summary. This example discloses the idea of using a scanning laser to degrade the nucleic acids on a mounted FFPE tissue section which are in the regions that are not of interest for study. This then leaves untouched the nucleic acids in the tissue of interest for study. After this process, the entire section is then scraped into a tube for extraction, but the degraded DNA and RNA will be sufficientlyfragmented so as to be removed by size selection during the extraction process, or the material has been ejected from the surface, potentially undergoing degradation in the process. This will enable tumour enrichment on tissues mounted on plain slides (i.e. uncoated slides), which enhances the utility of the scanning laser ablation technology by removing the requirement to use specially coated slides.

[0109] The inventors have demonstrated this using a 100 W CO2 laser cutting machine, normally used for cutting plastics and wood. Without the use of an image registration, the laser was scanned over rectangular regions of sample FFPE mouse liver tissue sections on plan slide that had not been deparaffinized or stained. Tissue was ablated with a 100 W CO2 laser operated at 10% power and a 600 mm / second scan speed. After laser exposure the slides were stained and a coverslip mounted. The results are shown in FIG. 15 where the rectangular regions that were ablated from the tissue are clearly visible (indicated with arrows for two different portions of ablated tissue). This demonstrates that if set up with an imaging system for reference region alignment, this system will work well for high speed negative selection by laser ablation.

[0110] There is a need to use the laser scanning ablation microdissection technology described above to dissect tissue from plain laboratory slides, i.e. slides that are not coated with any specific material such as ITO. In certain use cases, genomics labs may receive tissue only on plain laboratory slides, and are not able to obtain tissue specifically mounted for dissection.

[0111] The inventors' laser ablation microdissection technique can dissect complex maps on typical large tumour sections at the rate of about 0.1 sections / mm2, 60-100x faster than conventional laser dissection.Example 3.0 - Rapid Microdissection of Tissue Sections Via Laser Ablation

[0112] Summary. This example demonstrates a method for tissue microdissection using scanning laser ablation that is approximately two orders of magnitude faster than conventional laser capture microdissection. The inventors' novel approach uses scanning laser optics and a slide coating under the tissue that can be excited by the laser to selectively eject regions of tissue for further processing. Tissue was dissected at 0.117 sec / mm2without reduction in yield,sequencing insert size or base quality compared to un-dissected tissue. From eight cases, 58-416mm2of tissue was obtained from 1-4 slides in 7 to 48 seconds total dissection time per case. These samples underwent exome sequencing and the inventors found the variant allelic fraction increased in regions enriched for tumour as expected. This suggests that the above-described ablation technique may be useful as a tool in both clinical and research labs.

[0113] The two challenges to be addressed are (1) efficient methods for identifying and mapping tissue regions of interest at adequate precision and (2) dissecting tissue rapidly enough to be cost-effective and of utility in diagnoses. This example focuses on a new approach to achieve (2), demonstrating rapid dissection for arbitrarily complex tumour samples.

[0114] The inventors hypothesized that a laser connected to scanning optics, such as are used for laser-engraving metal, could employ LIFT to rapidly dissect tissue of interest over arbitrarily complex dissection maps. The inventors set out to demonstrate this approach using sequencing to measure success as they did in their previous study[2]. Specifically, the inventors used exome sequencing of FFPE tissue, from cases that previously had whole genome sequencing, to characterize yield, speed, and the ability to call somatic variants, a key metric of sequence quality and utility.

[0115] Methods. An engraver consisting of a Raylase™ SS-IIE-15 galvo with an EPI™ model EP-S 20W pulsed fiber laser, using Raylase's weldMARK3™ software, was mounted on an 80 / 20™ extrusion frame above a slide stage consisting of a plate with three dowel pins for accurate slide repositioning. A ledge under the slide holds a tissue capture tray.

[0116] 10 pm thick FFPE sections of colorectal cancer biopsies (UBC ethics certificates H14-00681 and H17-01897) were mounted on slides coated with Indium Tin Oxide (ITO) [Delta Technologies CB-50IN-S111 5-15 ohms per square]. A total of ten cases were provided, and five slides were made from each. Nine of these cases had previously been analyzed using whole genome and transcriptome analysis (WGTA) of the tumour tissue from separate, fresh frozen biopsies and sequencing of blood-derived normal genomes[7].

[0117] The mounted sections were Hematoxylin and Eosin stained, and coverslips were added. The slides were then loaded on the engraver, and a rectangle of fiducial dots was burned into the ITO coating and coverslip around the tissue on each slide. The slides were scanned on a Leica Aperio slide scanner at 40X. After scanning, the slides had their coverslips removed by incubation in xylene.

[0118] Scanned images were loaded into QuPath[8]. In consultation with an expert pathologist, putative tumour and normal regions were manually annotated. This map was then converted to weldMARK coordinates using the positions of the fiducial marks and exported as .svg vector files via an in-house QuPath plugin

[0014] .

[0119] As shown in FIG. 16, five fractions were collected from the tissue, three by laser ablation and two by knife scraping. Tumour and normal regions were manually annotated and the tumour-enriched and normal-enriched regions were collected by laser ablation. Tumour-depleted (adjacent normal) tissue was scraped off by knife dissection. Separate but contiguous complete sections were knife- scraped or completely ablated by laser. Ablation was performed at 25% laser power, pulsed at 100 kHz with the beam, ~25 pm wide, scanning in parallel lines that were 19 pm apart. Tissue was ejected into a 1 mm deep tray under the slide containing a 200 pl aliquot of mineral oil. For each case, tissue from adjacent slides was pooled by ablating these areas into the same oil aliquot. Using a pipette, this material was transferred into a 1.4 mL screw cap tube (ThermoFisher 3712), residual oil was centrifuged from the tray into the tube using a funnel (TaKaRa 640032), and the oil was topped up to 400 pl.

[0120] Post-laser tumour-depleted sections, as well as an un-dissected control section, were knife-scraped, with the aid of a few drops of mineral oil to prevent uncontrolled fragment dispersion, into a 1.4 mL screw cap tube and rinsed with more oil to 400 pl total volume.

[0121] DNA purification of these samples was by a SPRI bead-based method19,10]. Library construction, exome capture, sequencing, and variant calling were performed as in[2]. Somatic tumour variants were identified from the previously generated whole genome sequencing of the tumour and blood, and genome positions containing somatic variants were queried in the matching laser-selectedsequence reads. Ratios of tumour and normal allele-supporting reads were used to generate variant allele density estimates.

[0122] Results. Table 1 shows experimental variables encompassing section mounting, movement after coverslip removal and tumour content. All fractions where material was recovered were used for yield and dissection speed measurements. Nine of the ten samples produced usable fractions, one was not mounted well enough to dissect and one was dissected but did not have previously generated whole genome sequencing of the tumour and blood for variant calling. 49 tissue regions were dissected, ranging in area from 16 to 270 mm2, and the ablation rate was found to be 0.117 sec / mm2+ 4.1 sec. Pooling equivalent tissue from one to four slides of each case gave totals of 58 to 416 mm2of laser dissected tissue area and 7 to 48 seconds of dissection time per case. FIG. 17 shows sample quality metrics of yield, insert size and base-quality score - all of which indicate that derived sequence data are suitable for further analysis. Genomic positions of expected variants were queried in the sequence reads to estimate the fraction of cells that contain tumour signal. FIG. 18 shows the smoothed histogram of variant allele fractions (VAFs) in each fraction-sample pair. The "tumour-enriched" fractions showed an increase in tumour content when compared to the "normal-enriched" results.

[0123] Discussion. The inventors have demonstrated dissection using LIFT that is 60-100x faster than was observed with conventional LCM[2], such that dissection of 100-300 mm2over three slides took <1 minute. FIG. 17 shows that the yield of nucleic acid and general sample performance of laser-ablated samples was comparable to knife-dissected material. The nucleic acid yield of the normal- enriched fractions was lower, and this correlates with the inventors' previous observation that these colorectal tumour samples have more DNA per unit area in the tumour due to increased ploidy[2]. A key metric of sequence quality for FFPE is the ability to call variants, and FIG. 18 shows the expected increase in variant allele fraction (VAF) of tumour-enriched tissue and reduced VAF density in the tumour- reduced tissue as expected.

[0124] The coordinate transfer strategy from slide scanner to laser engraver via laser-induced fiducials marking worked well, and slides could be repositioned withan error below that of the laser beam width of 25 pm. The coverslip removal process caused movement in some sections, and, as noted in Table 1, some target areas were manually adjusted at the laser as a tissue region had moved relative to the rest of the section. The inventors used 10 pm sections here as these previously showed good recovery, but such thick sections exhibit instability when removing coverslips. Thinner sections may be more stable, and given the speed of this method, more slides could readily be dissected to obtain equivalent material.

[0125] Conclusion. The inventors have shown that using laser ablation microdissection using LIFT with ITO-coated slides can dissect useful quantities of tissue in under a minute, at about 25 pm resolution, with no loss of yield or sample quality vs knife dissected tissue. This suggests the technique could be used to enrich tumour samples for clinical cancer panels, for example, as well as more precise separation of tumour and normal regions to obtain both tumour and reference normal tissue sample. The former may be done by dissecting unstained but contiguous sections using a stained and annotated slide as reference. A limitation of the method is the need for coated slides, necessitating a connection between pathology and genomics labs to prepare the slides correctly, although subsequently the inventors have demonstrated the utility of a CO2 laser to eject tissue from uncoated slides as described above.Table 1. Recovery of different fractions from the ten samples. The key to the source for each fraction is shown in FIG. 16.Small, very littlenormalLib = Nucleic acids extracted and libraries constructedSeq = SequencedVar = Variant allele fraction analysis* Case 8 had not been previously sequenced so variants could not be called

[0126] FIG. 17 shows sample and sequence quality metrics. The yield was measured by Qubit after DNA purification and before library construction. Sections were 10 pm thick, so the yield is about 10 ng / mm2.

[0127] FIG. 18 shows variant allele fraction density. These plots are convolutions of the variant allele fraction histogram for each fraction with a Gaussian distribution.References

[0128] The following references are of interest with respect to the subject matter herein. Each of the following references is incorporated by reference herein in its entirety.1. Canadian Cancer Statistics: A 2022 special report on cancer prevalence. In : Canadian Cancer Statistics Advisory in collaboration with the Canadian Cancer Society, Statistics Canada and the Public Health Agency of Canada.; 2022.2. Coope RJ, Schlosser C, Corbett RD, Pleasance S, Tessier-Cloutier B, Pandoh P, Kirk H, Haile S, Zhao Y, Mungall AJ et ah Whole-slide laser microdissection for tumour enrichment. J Pathol 2021, 253(2): 225- 233.3. Adrian F, Bohandy J, Kim B, Jette A, Thompson P: A Study of The Mechanism of Metal-Deposition by the Laser-Induced Forward Transfer Process. Journal of vacuum science & technology B, Microelectronics processing and phenomena 1987, 5(5): 1490-1494.4. Barron JA, Krizman DB, Ringeisen BR: Laser printing of single cells: statistical analysis, cell viability, and stress. Annals of biomedical engineering 2005, 33(2): 121-130.Ringeisen BR, Kim H, Barron JA, Krizman DB, Chrisey DB, Jackman S, Auyeung RY, Spargo BJ: Laser printing of pluripotent embryonal carcinoma cells. Tissue engineering 2004, 10(3-4):483-491.5. Lee AC, Lee Y, Choi A, Lee HB, Shin K, Lee H, Kim JY, Ryu HS, Kim HS, Ryu SY et al Spatial epitranscriptomics reveals A-to-I editome specific to cancer stem cell microniches. Nature communications 2022, 13(1): 2540.6. Kim S, Lee AC, Lee HB, Kim J, Jung Y, Ryu HS, Lee Y, Bae S, Lee M, Lee K et ah. PHLI-seq: constructing and visualizing cancer genomic maps in 3D by phenotype-based high-throughput laser-aided isolation and sequencing. Genome biology 2018, 19(1): 158.7. Pleasance E, Bohm A, Williamson LM, Nelson JMT, Shen Y, Bonakdar M, Titmuss E, Csizmok V, Wee K, Hosseinzadeh S et ah. Whole-genome and transcriptome analysis enhances precision cancer treatment options. Ann Oncol 2022, 33(9):939-949.8. Bankhead P, Loughrey MB, Fernandez JA, Dombrowski Y, McArt DG, Dunne PD, McQuaid S, Gray RT, Murray LJ, Coleman HG et ah. QuPath: Open source software for digital pathology image analysis. Scientific reports 2017, 7(1): 16878.9. Haile S, Pandoh P, McDonald H, Corbett RD, Tsao P, Kirk H, MacLeod T, Jones M, Bilobram S, Brooks D et ah. Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis. PloS one 2017, 12(6):e0178706.10. Haile S, Corbett RD, Bilobram S, Bye MH, Kirk H, Pandoh P, Trinh E, MacLeod T, McDonald H, Bala M et ah. Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin- fixed paraffin-embedded samples. Nucleic acids research 2019, 47(2):el2.11. James Shaw Stewart TL, Matthias Nagel, Frank Nuesch and, Wokaun A: Laser-Induced Forward Transfer Using Triazene Polymer Dynamic Releaser Layer. In: International Symposium on High Power Laser Ablation 2010: 2010\ American Institute of Physics; 2010.12. A. Doraiswamy, R.J. Narayan, T. Lippert, L. Urech, A. Wokaun, M. Nagel, B. Hopp, M. Dinescu, R. Modi, R.C.Y. Auyeung et ah Excimer laser forward transfer of mammalian cells using a novel triazene absorbing layer, . Applied Surface Science, 2006, 252(13):4743-4747.13. Lippert T, Hauer M, Phipps CR, Wokaun A: Fundamentals and applications of polymers designed for laser ablation. Appl Phys A 2003, 77:259-264.14. Coope RJ, Pleasance S, Pandoh P, Schlosser C, Corbett RD, Marra MA. Rapid microdissection of tissue sections via laser ablation. J Clin Pathol. 2024, 77(6):430-434.

Claims

WHAT IS CLAIMED IS:

1. A method of using a laser apparatus to enrich for biological material from a region of interest within a tissue sample mounted on a slide, the method comprising: defining a reference position of the tissue sample relative to coordinates of the laser apparatus; visually assessing the tissue sample to identify regions of interest; mapping the regions of interest of the tissue sample to the coordinates of the laser apparatus to generate a dissection map; and selectively ejecting or modifying tissue within the tissue sample using the laser apparatus to apply laser energy along a plurality of laser paths defined within the dissection map.

2. A method of using a laser apparatus to enrich for a region of interest within a plurality of contiguous sections of a tissue sample mounted on a plurality of slides, one of the plurality of the slides being a parent slide and a remainder of the plurality of the slides being child slides, the method comprising:(i) conducting the method as defined in claim 1 on a parent slide bearing one of the plurality of sections of the tissue sample;(ii) aligning a reference tissue region of the parent slide with a reference tissue region of a first one of the child slides in the laser apparatus to define a reference position of the first one of the child slides relative to the coordinates of the laser apparatus;(iii) mapping the regions of interest from the parent slide to the first one of the child slides to generate a dissection map for the first one of the child slides;(iv) selectively ejecting or modifying tissue within the tissue sample on the first one of the child slides using the laser apparatus to apply laser energy along a plurality of laser paths defined within the dissection map for the first one of the child slides; and(v) repeating steps (i) through (iv) for each one of the child slides.

3. A method of using a laser apparatus to enrich for biological material from a region of interest within a tissue sample mounted on a slide, the method comprising: marking at least three defined reference positions on the slide or on a coverslip of the slide using the laser apparatus; obtaining a high resolution image of the slide including the reference positions; visually assessing the tissue sample to identify regions of interest; mapping the regions of interest of the tissue sample to the coordinates of the laser apparatus based on a position of the at least three defined reference positions to generate a dissection map; and selectively ejecting or modifying tissue within the tissue sample using the laser apparatus to apply laser energy along a plurality of laser paths defined within the dissection map.

4. A method of using a laser apparatus to enrich for a region of interest within a plurality of contiguous sections of a tissue sample mounted on a plurality of slides, one of the plurality of the slides being a parent slide and a remainder of the plurality of the slides being child slides, the method comprising:(i) obtaining a high resolution image of the parent slide;(ii) defining a shape of a reference tissue region on the parent slide, optionally wherein the reference tissue region comprises an outline of at least a portion of the tissue sample;(iii) visually assessing the tissue sample on the parent slide to identify regions of interest in the tissue sample;(iii) aligning the reference tissue region of the parent slide with a reference tissue region of a first one of the child slides in the laser apparatus to define a reference position of the first one of the child slides relative to the coordinates of the laser apparatus;(iv) mapping the regions of interest from the parent slide to the first one of the child slides to generate a dissection map for the first one of the child slides;(v) selectively ejecting or modifying tissue within the tissue sample on the first one of the child slides using the laser apparatus to apply laser energy along a plurality of laser paths defined within the dissection map for the first one of the child slides; and(vi) repeating steps (iii) through (v) for each one of the child slides and optionally for the parent slide.

5. The method as defined in any one of claims 2 or 4, wherein the plurality of slides comprises six slides.

6. The method as defined in any one of claims 2 or 4 to 5, wherein the child slides comprise unstained slides.

7. The method as defined in any one of claims 1 to 6, wherein substantially an entirety of the dissection map is covered by the plurality of laser paths.

8. The method as defined in any one of claims 1 to 7, wherein the plurality of laser paths are linear or substantially linear.

9. The method as defined in any one of claims 1 to 8, wherein the dissection map comprises a positive selection dissection map and the plurality of laser paths defined within the dissection map are substantially contiguous within the regions of interest.

10. The method as defined in claim 9, wherein at least one of the regions of interest has an outer perimeter, and wherein the plurality of laser paths extend across the at least one region of interest from a first edge of the outer perimeter to a second edge of the outer perimeter, optionally wherein at least some of the plurality of laser paths are discontinuous within the at least one region of interest.

11. The method as defined in any one of claims 1 to 8, wherein the dissection map comprises a negative selection dissection map and the plurality of laser paths defined within the dissection map are substantially contiguous outside the regions of interest.

12. The method as defined in any one of claims 1 to 11, wherein selectively ejecting tissue within the tissue sample comprises using the laser apparatus to excite a coating on the slide, the coating having an excitation wavelength corresponding to an emission wavelength of the laser of the laser apparatus.

13. The method as defined in claim 12, wherein the coating comprises indium tin oxide, triazene, poly(methyl methacrylate), or poly (methyl methacrylate containing a dye that is transparent in visible light and strongly absorbent at a wavelength corresponding to an emission wavelength of the laser of the laser apparatus.

14. The method as defined in any one of claims 1 to 11, wherein the slide is an uncoated slide, and wherein selectively ejecting tissue within the tissue sample comprises using the laser apparatus to supply CO2 laser energy along the plurality of laser paths.

15. The method as defined in any one of claims 1 to 14, wherein defining the reference position of the tissue sample comprises applying at least three fiducial markings to the slide or to a coverslip of the slide.

16. The method as defined in any one of claims 1 to 14, wherein defining the reference position of the tissue sample comprises defining a shape of a reference tissue region, optionally wherein the reference tissue region comprises an outline of at least a portion of the tissue sample.

17. The method as defined in any one of claims 1 to 16, wherein the tissue sample has been stained, and wherein visually assessing the tissue sample to identify regions of interest comprises one or more of: a pathologist manually annotating the slide to mark the regions of interest, a pathologist annotating a digital representation of the tissue sample to mark the regions of interest, or a machine learning model that has been trained to differentiate between normal cells and tumor cells or likely tumor cells annotating a digital representation of the tissue sample to mark the regions of interest.

18. The method as defined in any one of claims 1 to 10 or 12 to 17, wherein the dissection map comprises a positive selection dissection map, and wherein the selectively ejecting or modifying tissue within the tissue sample using the laser apparatus comprises ejecting the regions of interest from the slide by rastering laser energy along the plurality of laser paths positioned within the dissection map.

19. The method as defined in any one claims 1 to 8 or 11 to 17, wherein the dissection map comprises a negative selection dissection map, and wherein the selectively ejecting or modifying tissue within the tissue sample using the laser apparatus comprises ejecting tissue outside the regions of interest by applying scanning laser energy along the plurality of laser paths positioned within the negative selection dissection map.

20. The method as defined in any one of claims 1 to 8 or 11 to 17, wherein the dissection map comprises a negative selection dissection map, and wherein the selectively ejecting or modifying tissue within the tissue sample using the laser apparatus comprises using laser energy to degrade biological material in regions of the tissue sample outside the regions of interest by applying scanning laser energy along the plurality of laser paths positioned within the negative selection dissection map.

21. The method as defined in claim 20, further comprising, after selectively modifying tissue within the tissue sample, recovering the tissue sample from the slide and enriching for the desired biological material, optionally by conducting size selection to recover undegraded biological material.

22. The method as defined in either one of claims 20 or 21, comprising adding a sensitizing reagent to the tissue sample prior to the step of selectively ejecting or modifying tissue within the tissue sample using the laser apparatus.

23. The method as defined in any one of claims 1 to 22, wherein the laser apparatus comprises a galvo laser apparatus.

24. The method as defined in any one of claims 1 to 23, wherein the laser is a pulsed fiber laser, a 405 nm diode laser, a CO2 laser emitting at 10.6 pm, a neodymium-doped yttrium aluminum garnet laser emitting at a wavelength of 1064 nm, or a laser emitting below 350 nm.

25. The method as defined in any one of claims 1 to 19 or 23 to 24, wherein the ejected tissue is collected in a medium that is compatible with downstream extraction protocols, optionally wherein the medium is mineral oil or hexadecane.

26. Apparatus for conducting a method as defined in any one of claims 1 to 25.

7. Apparatus for enriching a tissue sample for a biological material of interest, the apparatus comprising: a slide holder for receiving at least one slide bearing the tissue sample; and a laser mounted to deliver laser radiation to the at least one slide along a plurality of adjacent laser paths defined by a dissection map provided for the tissue sample.

28. The apparatus as defined in claim 27, further comprising a viewer for correlating a reference tissue region of a parent slide with a corresponding reference tissue region of a child slide to map regions of interest from the parent slide to the child slide to generate a dissection map for the child slide.

29. The apparatus as defined in any one of claims 27 to 28, wherein the apparatus comprises a source of energy and one or more galvo mirrors to direct the laser energy along the plurality of adjacent laser paths.

30. The apparatus as defined in any one of claims 27 to 29, wherein the at least one slide comprises a coating that can be energized by application of laser energy, optionally wherein the coating that can be energized by application of laser energy comprises indium tin oxide, a triazene polymer, poly(methyl methacrylate), or poly(methyl methacrylate) combined with a laser-excitable dye.

31. The apparatus as defined in any one of claims 27 to 29, wherein the at least one slide comprises an uncoated slide and wherein the laser comprises a CO2 laser.

32. The apparatus as defined in any one of claims 27 to 31, wherein the at least one slide comprises fiducial markings.

33. The apparatus as defined in any one of claims 27 to 32 or method as defined in any one of claims 1 to 25, wherein the biological material comprises deoxyribonucleic acid, ribonucleic acid or protein.

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