Device and method for dissecting and analyzing individual cell samples

A method for dissecting and collecting one or more cells from a tissue sample fixed to an inner surface of a microfluidic device is described. The tissue sample is in fluid communication with a channel having an inlet end and an outlet end defined by the microfluidic device. The method comprises flowing a first fluid through the channel with a fluid flow from the inlet end to the outlet end; powering a laser to direct laser energy into the channel to impinge upon the first fluid proximate a first region of the tissue sample and cause fluid cavitation to thereby ablate a first set of one or more cells from the tissue sample; and collecting the first set of one or more cells within a first sample container coupled to the outlet end.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage filing under 35 U.S.C. § 371 of International Application No. PCT/US2017/024532, filed Mar. 28, 2017, which claims the benefit of U.S. Provisional Patent Application No. 62/390,431, filed Mar. 29, 2016, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This invention generally relates to methods for isolating, dissecting, collecting, sorting and analyzing individual cells or groups of cells from fresh, frozen, or fixed biological laboratory samples; and more particularly to isolating, dissecting, collecting, sorting and analyzing individual cells or groups of cells from biological laboratory samples formalin-fixed in paraffin; still more particularly to the conservation and visualization of spatial and morphological information of the dissected and collected individual cells or groups of cells.

BACKGROUND OF THE INVENTION

Current methods to isolate individual cells from formalin-fixed paraffin-embedded (FFPE) tissue samples lack out-of-plane (z-axis) resolution. These techniques typically utilize laser capture microdissection (LCM) to detach cells glued to a plastic film or a glass plate (x-y plane) either by heat through use of a pulsed infrared laser (see e.g., the PixCell II infrared LCM system commercialized by Arcturus Engineering of Mountain View, Calif., US), or by force against gravity using ultraviolet laser capture microdissection (see e.g., the Palm Zeiss ultraviolet LCM system commercialized by P.A.L.M. Microlaser Technologies AG of Bernried, Germany). Each of these systems are dry systems where the laser energy impinges upon the tissue sample to ablate cells and cell material. The recovery rate of ablated cells is very low and the direct impingement of the laser on the tissue may potentially damage the cells and chemical/structural constituents thereof, thereby negatively impacting study analysis and results. Also, these LCM techniques require the use of an Eppendorf micro-centrifuge-tube which greatly restricts throughput. Moreover these techniques are restricted to two-dimensional (2-D) analysis and cannot be used with samples having layers of cells in the z axis. In other words, traditional LCM approaches are restricted to the x-y plane and lack out-of-plane resolution in the z axis.

One attempt to address the above shortcomings of traditional LCM techniques is to couple a microfluidic device with the laser source. The microfluidic device holds the tissue sample within a chamber and a fluid flows through the chamber where the laser directly impinges upon the tissue sample. One or more cells may be ablated from the tissue sample upon laser impingement. The ablated cells are then received in and carried by the fluid flow to a sample collecting element. Cells may be serially ablated and collected in respective collecting elements. Each respective collecting element may be used to maintain spatial information regarding the collected cells. In this manner, the tissue sample may be interrogated across the x-y plane in a first instance. The laser can then be used to ablate the next successive layer in similar fashion. All of the above cell samples may then be analyzed and any data collected may be correlated to locate the spatial location of the respective cell sample within the entire original tissue sample. However, while achieving resolution in the z-axis with improved sampling efficiencies, direct laser impingement upon the tissue sample may cause damage to the ablated cells or the underlying cell layer of the tissue sample thus negatively impacting any information collected or reported study results.

Accordingly, what is needed in the art is a device and method for isolating, dissecting, collecting, sorting and analyzing individual cells or groups of cells without direct laser impingement upon the tissue sample, as well as the conservation of spatial and morphological information of individual cells or cell groups to enable visualization, recordation and study through multimodal molecular analysis of quantum dissected qubits of tissue voxels.

SUMMARY OF THE INVENTION

Briefly described, a method for dissecting and collecting one or more cells from a tissue sample fixed to an inner surface of a microfluidic device where the tissue sample is in fluid communication with a channel having an inlet end and an outlet end defined by the microfluidic device comprises flowing a first fluid through the channel with a fluid flow from the inlet end to the outlet end; powering a laser to direct laser energy into the channel to impinge upon the first fluid proximate a first region of the tissue sample and cause fluid cavitation to thereby ablate a first set of one or more cells from the tissue sample; and collecting the first set of one or more cells within a first sample container coupled to the outlet end. Ablation of the tissue sample may be optically monitored using a microscope.

In a further aspect of the present invention, the method may further include flowing a second fluid within the channel wherein the first fluid forms discretized fluid slugs comprised of the first fluid. To that end, the laser may be powered when a respective fluid slug of the first fluid communicates with the first region of the tissue sample and may be unpowered when a respective wash droplet communicates with the first region of the tissue sample. In one aspect of the invention, the first fluid is an oil and the second fluid is a gas, while in another aspect, the first fluid is an oil and the second fluid is a liquid immiscible with the oil.

In another aspect of the present invention, the method may further include injecting a third fluid within the channel wherein the third fluid forms discretized wash droplets comprised of the third fluid. The wash droplets may be interposed between successive fluid slugs of the first fluid. The first fluid may be an oil, the second fluid may be a gas and the third fluid may be a liquid immiscible with the oil.

In still another aspect of the present invention, the channel may be a serpentine channel including alternating linear channel segments and curved channel segments with the linear channel segments arranged in parallel relation to one another. Each linear channel is configured to overlap a portion of the tissue sample. Alternatively, the microfluidic device may define a plurality of channels arranged in spaced parallel relation. Each channel may include a respective inlet end and outlet end and each channel may be configured to overlap a portion of the tissue sample. Fluid flow may be through one channel of the plurality of channels at a time.

In still a further aspect of the present invention, the microfluidic device may further comprise a planar bottom slide affixed to a microfluidic substrate thereby defining the channel therebetween. The channel has a length, width and depth wherein the resilient member is configured to be addressable to selectively reduce at least a portion of the width of the channel. In one aspect, the resilient membrane may be addressable by a plunger, where the plunger may be actuatable to direct the resilient membrane in touching engagement with the planar bottom slide. Alternatively, the resilient membrane may be covered by a top cover opposite the planar bottom slide. The resilient membrane may define a membrane channel where the membrane channel may be actuatable to direct the resilient membrane in touching engagement with the planar bottom slide.

In yet another aspect of the present invention, the channel may overlap with more than one tissue sample. The laser may then be powered to impinge the first fluid proximate only one tissue sample at a time.

In still another aspect of the present invention, the method may further include the additional step of infusing a solution containing nanoparticles selected to absorb the laser energy prior to powering the laser. The nanoparticles may penetrate a portion of the tissue sample to form a nanoparticle saturated tissue layer and the laser energy may be directed to the nanoparticle saturated tissue layer whereby the nanoparticles absorb the laser energy and ablate the first set of one or more cells from the tissue sample. Additionally or alternatively, a target cell within the tissue sample may be selectively labeled with a fluorescent dye or biomarker to produce a labeled cell. The laser energy may be directed to the first fluid proximate the labeled cell thereby causing fluid cavitation and ablation of the labeled cell from the tissue sample.

In yet a further aspect of the present invention, the method may further include the steps of powering the laser to direct laser energy into the channel to impinge upon the first fluid proximate a second region of the tissue sample and cause fluid cavitation to thereby ablate a second set of one or more cells from the tissue sample; and collecting the second set of one or more cells within a second sample container coupled to the outlet end. The first sample container and second sample container may be respective wells within a multi-well plate. A further step may be effectuating relative movement between the microfluidic device and the laser after collecting the first set of one or more cells within the first sample container and before powering the laser to direct laser energy into the channel to impinge upon the first fluid proximate a second region of the tissue sample.

Further steps may include uniquely identifying the first and second sample containers to conserve spatial and/or morphological information of the respective first and second sets of one or more cells relative to the fixed tissue sample; storing the spatial and/or morphological information of the respective first and second sets of one or more cells within in a database; performing molecular analysis on one or both of the first and second sets of one or more cells to create cell data; correlating the cell data with the respective spatial and/or morphological information of the respective first and/or second sets of one or more cells to create a compiled data file for each cell or group of cells within the first and second sets of one or more cells; retrieving at least one of the spatial and/or morphological information, the cell data or the compiled data from the database; and electronically reconstructing at least a portion of the tissue sample using at least one of the spatial and/or morphological information, the cell data and the compiled data. The database may be resident within a cloud and the spatial and/or morphological information, the cell data and the compiled data may be stored in the cloud and retrieved from the cloud through a network.

Additional objects, advantages and novel features of the present invention will be set forth in part in the description which follows, and will in part become apparent to those in the practice of the invention, when considered with the attached figures.

DETAILED DESCRIPTION

Attention is now turned toFIG. 1, which by assembly illustrates a cluster of cells12in an exemplary formalin-fixed paraffin-embedded (FFPE) tissue sample10. Cells12are in layers in tissue10with the layer extension measured in x and y directions and the layer thickness in direction z. Tissue10has cell layers A, B, C and D whereby individual cells12in any particular cell layer are numbered as cell layer/cell number, such as A1, A2, etc., and D1, D2, D3, etc. Laser capture microdissection (LCM) strips or captures the top layer of cells first (i.e., cell layer A). Ideally, the newly exposed second layer (i.e., cell layer B) would be captured next, and so on in direction z. However, only cell layer A can typically be removed as traditional LCM methods cause damage to cell layer B, thereby rendering the underlying cells unsuitable for capture and analysis. Thus, LCM is not sensitive to direction z, and as such, it is unsuitable to preserve 3-D information (x-y-z) of individual cells in a tissue sample. LCM, therefore, operates on 2-D layers (x-y), which is a significant drawback to tumor cell recognition and morphology studies. As discussed in greater detail below, aspects of the present invention seek to overcome this shortcoming, as well as provide additional benefits.

Turning now toFIGS. 2 and 3, an exemplary embodiment of a microfluidic device in accordance with an aspect of the invention is generally indicated by reference numeral20. Microfluidic device20may generally comprise a bottom planar slide, such as glass slide22, compressed, adhered, bonded or otherwise coupled to microfluidic substrate24. A recess25may be fabricated within microfluidic substrate24whereby a channel26may be formed upon coupling glass slide22to microfluidic substrate24. Channel26may be fabricated to have a width W (FIG. 2) between about 50 micron and about 500 micron, a depth D (FIG. 3) between about 50 micron and about 500 micron, and a length L (FIG. 2) between about 2 cm and about 10 cm. A tissue sample28may be fixed to inner surface30of glass slide22such that at least a portion of tissue sample28is in communication with channel26. Channel26may further include an inlet end32and outlet end34defined within microfluidic device20whereby a first fluid36may be introduced at inlet end32to flow in a fluid flow (generally indicated by arrows33) through channel26(and thereby communicate with tissue sample28) before exiting out of outlet end34. To that end, a sample container (see e.g.,FIG. 18wherein sample container is a respective well A1, etc. within a 96-well plate180) may be coupled to outlet end34so as to receive and store the exiting fluid for offline analysis.

As seen more clearly inFIG. 3, a laser38may direct laser energy40into channel26to impinge upon first fluid36proximate tissue sample28, such as within about 100 nm to about 1 micron of the surface of tissue sample28. In one aspect of the invention, laser38may be a pulsing two photon infrared laser having a wavelength between about 750 nm and about 1200 nm, and more particularly between about 800 nm and about 850 nm. In a further aspect of the invention, laser38may be a pulsing two photon ultraviolet (UV) laser having a wavelength between about 200 nm and about 400 nm, and more particularly between about 300 nm and 350 nm. Laser energy40may operate to cause cavitation of first fluid36such that one or more cells42may be ablated from tissue sample28. It should be noted that, while UV radiation may damage biological tissues, and more specifically DNA and RNA which absorb wavelengths within the UV spectrum, such damage is greatly reduced, and potentially eliminated, when employing a method in accordance with the present invention. That is, UV laser energy may be selectively targeted so as to impinge upon first fluid36and not tissue sample28. In this manner, cells42may be hydrodynamically ablated from tissue sample28via cavitation bubbles rather than direct impingement of laser energy40on tissue sample28. As a result, tissue sample28may be exposed to little or no UV radiation thereby decreasing the breadth and magnitude of any subsequent sample damage, if any. Accordingly, the present invention may recover better quality cell (e.g., DNA and/or RNA) samples, and resultant data, than traditional UV laser microdissection techniques.

Following ablation from tissue sample28, cells42may then be captured within first fluid36for transport to outlet end34, and resultant collection by the sample container coupled thereto. The laser energy does not directly impinge upon the tissue sample, and as a result, higher power laser pulses may be used without damaging cells or cell structures than are typically used in laser microdissection techniques. The collected cells42may then be further interrogated offline, such as through multimodal molecular analysis which will be discussed in greater detail below. While shown and described inFIG. 3as being directed through microfluidic substrate24in a top-down direction, laser38may also be directed through glass slide22in a bottom-up direction. To that end, both glass slide22and microfluidic substrate24may be transparent to laser38. Further, ablation of the tissue sample28may be optically monitored using a microscope (not shown) and may also be transparent to white light. In a further aspect of the present invention, there may be relative movement between microfluidic device20and laser38after collecting a first set of cells within a dedicated container and before powering laser38to impinge upon the fluid and ablate a second set of cells that may be collected in a second, dedicated sample container. In this manner, the microfluidic device may be rastered relative to laser38such that multiple regions of the sample may be collected within channel26with each individual region being individually sequestered so as to minimize or eliminate cross contamination.

As further shown inFIG. 3, in accordance with another aspect of the present invention, a second fluid44may flow within channel26whereby the first fluid creates discretized fluid slugs46of first fluid. In this aspect, first fluid36may be an oil while second fluid44is a gas or fluid immiscible within first fluid36, and more particularly, second fluid44may be a gas (such as, but not limited to N2). First fluid36may form a series of discrete fluid slugs46such that laser38may be powered when a respective fluid slug46of first fluid36communicates with tissue sample28. Laser38may then be unpowered when second fluid44(i.e., gas) communicates with tissue sample28. As further shown inFIG. 3, a third fluid48may also be injected within inlet end32. Third fluid48may be comprised of detergent that is immiscible with first fluid36and/or second fluid44. Third fluid48may operate to wash of otherwise sweep debris from channel26in between respective fluid slugs46. Third fluid48may be discarded at outlet end34. In this manner, ablated cells42may be sequestered exclusively within fluid slugs46where each respective fluid slug46may be collected within a respective, dedicated sample container which correlates to a specific laser pulse and cavitation event. As such, cross contamination of cells42within serial fluid slugs46may be reduced or eliminated.

To promote laser target location and/or laser energy absorption, tissue sample28may be conditioned with one or both of a nanoparticle solution tuned to absorb laser energy and a solution containing fluorescent dye and/or biomaterials configured to selectively bind with specific regions within the sample. As shown inFIG. 4, a sample28nmay be affixed to glass slide22as generally described above. A solution containing nanoparticles30nmay entrain sample28nat a specific depth so as to form a nanoparticle saturated layer32n. Nanoparticles30nare tuned to absorb laser energy40from laser38. The energy-absorbed portion of nanoparticle saturated layer32nmay then ablate one or more cells42nwhich then are transported and collected as described above. Laser energy is absorbed by the nanoparticles and not by the cells such that little to no cell damage results from the laser impingement. Additionally, or alternatively, a fluorescent dye or biomarker solution may be entrained to a sample, such as sample28n, whereby the dye or biomarker are selectively bound by specific structures/compounds within the bulk tissue sample. Laser energy40may then be specifically directed toward the bound regions for subsequent ablation of the marked cells.

As generally shown inFIG. 2, channel26may have a width W that may be too narrow to overlap an entire tissue sample28. As such, only that portion of tissue sample28overlapped by channel26may be potentially ablated and collected as described above. One alternative to providing greater coverage of tissue sample28may be through use of a microfluidic device20agenerally shown inFIG. 5. In accordance with an aspect of the present invention, microfluidic substrate24amay be fabricated to include a wider recess25a, such that, upon coupling microfluidic substrate24awith glass slide22, a channel26amay be formed having a wider width Wawhen compared to channel26of microfluidic device20. Without limitation thereto, width Wamay be selected to be between about 1 mm and about 25 mm. Channel26amay include a single inlet end32aand a single outlet end34a. As a result, more or all of sample28may be in fluid communication with channel26a.

FIGS. 6-12show alternative microfluidic devices20b-20fconfigured to provide greater tissue sample overlap while using microfluidic channel(s) having widths less than about 1 mm. Discussing each in turn, and with immediate reference toFIG. 6, microfluidic device20bmay comprise glass slide22coupled to microfluidic substrate24b. Microfluidic substrate24bmay be configured to include a serpentine recess2b, which, when coupled to glass slide22, forms a serpentine channel26bhaving an inlet end32band an outlet end34b. In one aspect of the invention, the serpentine channel includes alternating linear channel segments27band curved channel segments29bwherein linear channel segments27bare arranged in parallel relation to one another. Serpentine channel26bmay be configured to overlap all or a significant portion of tissue sample28. In this manner, fluid may flow from inlet end32bto outlet34bwhile the laser (not shown) may selectively cavitate the fluid and ablate selected cells as described above, but over a larger field while also providing improved fluid dynamics than when using a wide channel.

Turning now toFIG. 7, microfluidic device20cmay comprise glass slide22coupled to microfluidic substrate24c. Microfluidic substrate24cmay be configured to include a plurality of recesses25carranged in spaced parallel relation to one another. When coupled to glass slide22, microfluidic substrate24cthus forms a plurality of parallel spaced channels26c, each respective channel26chaving a respective inlet end32cand an outlet end34c. The collective channels26cmay be configured to overlap all or a significant portion of tissue sample28. Depending upon the location of the tissue sample selected to be sampled through laser cavitation and ablation as described above, fluid may flow from a respective inlet end32cto a respective outlet34cof the overlapping channel26cwithin the array of parallel channels. As such, a larger field of tissue sample28may be interrogated while also providing improved fluid dynamics than when using the wide channel and while also reducing fluid travel length, resultant back pressure issues, and increased sample collection times when using long channels.

FIGS. 8 and 9generally depict a microfluidic device20dincluding a glass slide22coupled to microfluidic substrate24dwhich may be comprised of a selectively actuatable membrane23ddefining an initial channel26dhaving an initial width Wid(FIG. 7). Actuatable membrane23dmay be mechanically addressable so as to reduce the channel width of channel26d. By way of example and without limitation solely thereto, actuatable membrane23dmay be actuated by one or more plungers50whereby a plunger50imparts a force upon a selected portion52of microfluidic substrate24dso as to direct selected portion52into touching engagement with slide22. In this manner, initial channel26dis segregated into two or more reduced channels26r1and26r2having a reduced width, such as width Wr1and Wr2as shown inFIG. 9.

As also shown inFIG. 9, two or more plungers50may be employed to define the reduced channels (e.g., Wr1, Wr2). In this manner, and as shown inFIG. 9, a microfluidic channel26d′ may be selectively created, where channel26d′ is selected to overlap with a portion of the tissue sample (not shown, see e.g.,FIGS. 2 and 6) which will be subjected to impingement by the laser and resultant cavitation and cell ablation as described above. In accordance with one aspect of the present invention, microfluidic substrate24dis a flexible yet resilient member whereby initial channel26dwill substantially reform following removal of downward pressure from plunger(s)50. Plunger(s)50may then be relocated over another portion of microfluidic substrate24dso as to form another microfluidic channel over another selected portion of the tissue sample. To assist in locating plunger(s)50, microfluidic substrate24dmay include a plurality of upwardly extending ribs54arranged in uniform spaced parallel relation so as to define a plurality of alternating grooves56. Grooves56may be proportioned to receive plunger(s)50so as to enable selective actuation of plunger(s)50to form a plurality of microfluidic channels having a substantially constant width, such as Wr2. As a result, all or a substantial portion of the tissue sample may be interrogated by sequentially creating serial channels26d′.

FIGS. 10 and 11generally depict a microfluidic device20eincluding a bottom glass slide22, a top cover21eand a microfluidic substrate24ecoupled therebetween. Microfluidic substrate24emay be comprised of a selectively actuatable membrane23edefining an initial channel26ehaving an initial width Wie(FIG. 10). Actuatable membrane23emay further define a plurality of conduits56eseparated by membrane sidewalls54e. Each conduit56emay be selectively, individually addressable so as to reduce the channel width of channel26e. By way of example and without limitation solely thereto, actuatable membrane23emay be pneumatically actuated by a high pressure air source (not shown) so as to impart a force upon a selected conduit56eto thereby expand a selected portion50eof actuatable membrane into touching engagement with slide22. In this manner, initial channel26emay be segregated into two or more reduced channels26r3and26r4having a reduced width, such as width Wr3and Wr4as shown inFIG. 11.

As also shown inFIG. 11, two or more conduits56e(e.g.,56e′/56e″) may be expanded to define the reduced channels (e.g., Wr3, Wr4). In this manner, and as shown inFIG. 11, a microfluidic channel26e′ may be selectively created, where channel26e′ is selected to overlap with a portion of the tissue sample (not shown, see e.g.,FIGS. 2 and 7) which will be subjected to impingement by the laser and resultant cavitation and cell ablation as described above. In accordance with one aspect of the present invention, microfluidic substrate24eis a flexible yet resilient member whereby initial channel26dwill substantially reform following removal of any applied high pressure air. A second conduit56eor conduit pair56e′/56e″ may then be pneumatically actuated so as to form another microfluidic channel over another selected portion of the tissue sample. As a result, all or a substantial portion of the tissue sample may be interrogated.

Turning now toFIG. 12, microfluidic device20fmay comprise glass slide22coupled to microfluidic substrate24f. Microfluidic substrate24fmay be configured to include a plurality of recesses25fhaving linear channel segments27farranged in spaced parallel relation to one another and angled end portions29fin communication with either common inlet end32for common outlet end34f. A top cover (not shown) includes a set54fof channel inserts56f(elevated for clarity) such that, when coupled to microfluidic substrate24f, the top cover and glass slide22form a plurality of spaced channels similar to channel26. The collective channels may be configured to overlap all or a significant portion of tissue sample28affixed to glass slide22. Depending upon the location of the tissue sample selected to be sampled through laser cavitation and ablation as described above, a selected channel26fmay enable fluid flow from inlet end32fto outlet34f. In one aspect of the invention, channel insert set54fmay be configured to socket each channel insert56fwithin its respective corresponding channel, whereby a selected channel insert56fmay then be actuated to lift from the selected channel thereby permitting fluid flow through only that selected channel. Alternatively, channel insert set54fmay be configured to lie above recesses25fso as to define a plurality of open channels. In operation, all but one channel insert56fmay then be actuated to block fluid flow within its respective channel. In this manner, only one channel will remain open such that any fluid flow would be directed into that open channel. As a result, all or a substantial portion of the tissue sample may be interrogated by sequentially selecting which respective channel is open at one time.

Heretofore, microfluidic devices20-20fhave been described for use with a single tissue sample28. However, it should be understood by those skilled in the art that the above devices may be suitable for use with multiple tissue samples affixed to a single slide. With attention toFIGS. 13 and 14, an exemplary microfluidic device20gfor use with an array28′ of multiple tissue samples28a-28(n) is shown. As can be seen inFIG. 13, glass slide22may be configured to receive an array28′ of tissue sample28a-28(n), such as in a grid-like pattern. A microfluidic substrate, such as but not limited to microfluidic substrate24c, may be couple to glass slide22so as to form channels26c, as described above (seeFIG. 7). Each individual sample28a-28(n) may be sequentially interrogated by selectively flowing fluid(s), such as a first fluid36, a fluid slug46of second fluid44and wash droplets of a third fluid48(seeFIG. 14), from a respective inlet end32cto the corresponding outlet end34cof a channel26cwhich overlaps the particular sample28being interrogated. A laser (i.e., laser38) may then impinge upon the fluid within channel26cas described above so as to cause fluid cavitation and cell ablation from the tissue sample. Each tissue sample (or portion thereof) may then be collected within a dedicated sample container at outlet end34cas described above. In this manner, multiple cell samples may be selectively extracted and collected with minimal to no cross contamination between samples.

As generally indicated inFIG. 15, a method70for dissecting and collecting one or more cells from a tissue sample affixed within a microfluidic device may comprise: a) flowing a first fluid through the channel with a fluid flow from the inlet end to the outlet end (step71); b) powering a laser to direct laser energy into the channel to impinge upon the first fluid proximate a first region of the tissue sample and cause fluid cavitation to thereby ablate a first set of one or more cells from the tissue sample (step72); and c) collecting the first set of one or more cells within a first sample container coupled to the outlet end (step73). The method may further include: d) powering the laser to direct laser energy into the channel to impinge upon the first fluid proximate a second region of the tissue sample and cause fluid cavitation to thereby ablate a second set of one or more cells from the tissue sample (step74); and e) collecting the second set of one or more cells within a second sample container coupled to the outlet end (step75). Still further, the method may comprise: f) uniquely identifying the first and second sample containers to conserve spatial and/or morphological information of the respective first and second sets of one or more cells relative to the fixed tissue sample (step76); and g) storing the spatial and/or morphological information of the respective first and second sets of one or more cells within in a database (step77). Additional steps may include: h) performing molecular analysis on one or both of the first and second sets of one or more cells to create cell data (step78); and i) correlating the cell data with the respective spatial and/or morphological information of the respective first and/or second sets of one or more cells to create a compiled data file for each cell or group of cells within the first and second sets of one or more cells (step79), as well as j) retrieving at least one of the spatial and/or morphological information, the cell data or the compiled data from the database (step80); and k) digitally reconstructing at least a portion of the tissue sample using at least one of the spatial and/or morphological information, the cell data and the compiled data (step81).

With the above method70outlined inFIG. 15, attention is now directed toFIG. 16which illustrates a multimodal mapping flow diagram illustrating microdissection for qubit study in accordance with one aspect of the present invention. Slide160includes a tissue sample (such as tissue sample28, which in this exemplary case may be a histological FFPE sample). Prior to dissection and collection, slide160may be imaged, such as through a microscope, where slide160may then be subdivided into areas by grid or custom shapes as generally indicated by subdivisions162a-162(n) in microdissection162. Such subdivision may be computer-implemented or computer-aided according to presets, manual selection or an algorithm as is known in the art. Each defined subdivision162a-162(n), or any specifically identified subdivision(s) may then be dissected/ablated and delivered for molecular analysis as described above. Each subdivision may be segregated and labeled whereby the analytical results may be correlated to the original location of the subdivision within the sample slide. As a result, each subdivision may be visualized as map164. For those analyses employing more than one biomarker (see above with regard toFIG. 4), multiple maps164athrough164(n) may be generated corresponding to different biomarkers. In this manner, maps may be displayed preserving the original spatial locations of the dissected samples and images. By way of example and not to be limited thereto, maps164a-164(n) may be heat maps, wherein the magnitude of shading within each grid correlates to magnitude of detection. That is, a darker grid location may indicate a higher or lower magnitude that a lighter shaded location. This may be generally referred to as a 2-dimensional analysis.

Turning now toFIG. 17, a general schematic of a 3-dimensional dissection method is generally indicated by reference numeral170. Dissection170employs similar techniques as described above with regard toFIG. 16, except wherein slide160was confined to x- and y-directions, microdissection170may be expanded in the z-direction. To that end, microdissection170may comprise layered imaging planes172a-172(n). Tissue sample28may then be imaged on parallel planes Z1-Z(n) to determine regions of interest. Such subdivision may be computer-implemented or computer-aided according to presets, manual selection or an algorithm as is known in the art. Regions may range in size from bulk cell to single cell to fraction of cells. Dissection with laser38, as described above, may discretely ablate targeted cells while recording the x, y, z coordinates of each ablation such that the ablated cells may be correlated to their original location (x, y and z) within the sample.

As shown inFIG. 18, a method of sorting and addressing cell samples (such as via fluid slugs46described above) for qubit study in accordance with the present invention may utilize a 96-well plate180. Well plate180may be include well coordinate rows182(from A through H) and columns184(from1through12) to define specific individual wells (i.e., sample containers), such as A1, B2, etc. Well plate180may communicate with a well correlation database184as is known in the art. Dissected samples are deposited into the wells (which may also include vessels or tubes or vials) and coordinates182/184are correlated to the sample dissection coordinates x-y-z such that database184is populated accordingly.

With reference toFIG. 19, a multimodal molecular analysis method190includes a molecular analysis output192and database194. All or selected samples from well plate180may be independently analyzed, including but not limited to analyses of molecular content and composition using exemplary techniques such as qRT-PCR, RNA Seq, DNA Seq, NGS, etc. Analytical results are evaluated on plot192or similar (hysteretic loop) output display and an analytical results database194is populated accordingly. Database194may then be correlated with data base186and its spatial coordinate database x-y-z. As a result, analytical data may be directly attributable to a specific location within the original tissue sample.

FIG. 20generally illustrates an exemplary 3-D qubit separation from voxel process as described above with reference toFIGS. 17-19. Voxel200may be subdivided into qubits202(V1V2and V3) which have designated x, y, and z coordinates. Voxel200(all or a portion of tissue sample28under study) may be subdivided and dissected by laser38and laser energy40as described above. The spatial coordinates (x, y and z) for the laser is known and tracked such that, as each individual qubit202(V1, V2, V3) is ablated and transported sequentially as described above, the location of each dissected subdivision within the original tissue sample is known and tracked by spatial database. Each qubit202may then be sorted into respective wells of plate182for molecular analysis190to obtain database192as described above. Visual presentation of analytical results may take the form of a heat map similar to that described above with regard toFIG. 16. However, as voxel200possess information in three dimensions, voxel visualization reconstructed from qubits, may generally include a voxel computer model210which may be displayed, by example, along cross-sectional plane212, as a planar layer214. Planar layer214may by further dissected into fragments216(qubits data). In this manner, model210may be sliced layer-by-layer in any desired sectional orientation, with each layer214may be further broken down to qubits214. As a result, model210may be a true computer data representation of physical voxel200wherein model210may be built from databases x-y-z (laser location information),163or186(tissue sample location within 96-well plate), and194(analytical data corresponding to location within 96-well plate).

As will be appreciated by those skilled in the art, the digitalized model210will be suitable to study long after the physical samples are gone, destroyed, discarded or degraded. Moreover, model210may be shared by scientists and practitioners as digital files may be electronically distributed across the globe wherein physical specimens were solely within the domain of the research/clinical laboratory possessing those samples. To that end, all information encoded within model210, along with all data within databases163,186and194, may be stored on a cloud-based data storage device. The cloud-based data storage device may be an access-controlled, shared-computing device accessible wirelessly. As a result, samples may be studied by a host of remote experts and practitioners where they may share their findings instantly, even long after the physical samples are destroyed by nature or on purpose in laboratories. This may globalize currently local biological tissue cell research, advancing microbiology related to tumor recognition and study.

The present invention is described above with reference to a preferred embodiment. However, those skilled in the art will recognize that changes and modifications may be made in the described embodiment without departing from the nature and scope of the present invention. For instance, employing bidirectional microchannel arrays spatially separated but connected by across microfluidic holes (sequencer) is considered obvious modification to employing unidirectional micro-channels and thus hereby considered to be within the scope of the invention.

Various further changes and modifications to the embodiment herein chosen for purposes of illustration will readily occur to those skilled in the art. To the extent that such modifications and variations do not depart from the spirit of the invention, they are intended to be included within the scope thereof. Accordingly, it is intended that the invention not be limited to the described embodiments, but will have full scope defined by the language of the following claims.