Use of ir spectroscopy in assessing the penetration of reagents into biological specimens

Abstract

Devices and associated methods and apparatus for measuring sample material (e.g., tissue samples) use involve a spectroscopic system or device configured with a sample measurement device (e.g., an attenuated total reflectance infrared (ATR-IR) sample measurement device) that includes a crystal defining a sample receiving surface / interface that is operatively connected to an IR source and an IR detector. The sample receiving surface / interface facilitates the assessment of whether a fixative (e.g., formaldehyde) (and optionally, the amount thereof) is present in a sample material, such as a tissue sample. Measurements are taken at one or more locations within a sample placement structure / region, which can be provided in the form of a probe or raised piercing structure containing one or more ATR-IR (or other) sample measurement devices. The probe can include a piercing structure or mechanism at a distal (open) end of the probe that is configured to form a channel into a selected location (depth) within the sample material in a minimally invasive manner so that the sample is not severely damaged during testing.

Description

TECHNICAL FIELD

[0001] Various embodiments of the present invention generally relate to tissue processing, and in particular to techniques, instrument usage, and methods for assessing whether a fixative (e.g., formalin) is present within a tissue sample using IR spectroscopy. BACKGROUND

[0002] Body tissue for disease process diagnosis is typically processed in a histology lab to produce thin tissue sections that can be mounted on glass slides, stained, and observed under a microscope by a pathologist for analysis. These pre-analytic processes typically include, in sequence, gross examination, fixation, dehydration, clearing (or transparency), paraffin infiltration, and embedding. This procedure is used to process tissue, including biopsies, larger specimens removed intraoperatively, or autopsy tissue.

[0003] Gross examination typically consists of describing the macroscopic specimen and placing all or selected portions of it into small plastic boxes that hold the tissue as it is processed into paraffin blocks. Initially, the boxes are placed into a fixative.

[0004] Following gross examination, the tissue is fixed. The purpose of fixation is to permanently preserve the tissue in as lifelike a state as possible by changing the structure of proteins so that autolytic degradation does not occur. Depending on the type of tissue present and the features to be represented, a variety of fixatives can be used. Major fixative groups, classified according to mechanism of action, include aldehydes, mercury, alcohols, oxidizing agents, and picrates. Formalin is an aqueous solution of formaldehyde (gas). Actual fixation is by the formaldehyde. Formalin fixation is best performed near neutral pH (e.g., in the range of 6-8). Hypoxia of the tissue tends to lower the pH, so the fixative should have buffering capacity to prevent excessive acidity. Common buffers include phosphate, bicarbonate, malate, cacodylate, and veronal. For example, commercially available formalin can be buffered with phosphate at a pH of 7. Penetration of the tissue is dependent on the diffusivity of the individual fixatives. One method to improve penetration of the fixative is to gross the tissue thinly (cut) (2 to 3 millimeters (mm)). Penetration to thin sections occurs more quickly than to thick sections. The volume of fixative is often important, with a ratio of fixative to tissue typically targeted at 10: 1 or higher. Agitation of the specimen in the fixative also typically enhances fixation.

[0005] Once the tissue is fixed, the tissue needs to be processed into a form that can be made into thin sections for microscopic examination. The usual method is to use paraffin. The tissue is embedded in paraffin, which provides a solid support matrix for the tissue that enables it to be sectioned at a thickness of about 2 to 20 microns. The process of placing the fixed tissue into paraffin for sectioning is called tissue processing, where the main steps in the process are dehydration, clearing, infiltration, and embedding.

[0006] Tissue that is fixed in an aqueous solution cannot be directly infiltrated with paraffin. First, the water in the tissue must be removed by dehydration. This can be done by a series of alcohols of different concentrations (e.g., 70% to 95% to 100%). Alternatively, a mixture of formalin and alcohol is used for dehydration. Other dehydrating agents can also be used, such as acetone or a mixture of different solvents.

[0007] After dehydration, the tissue is cleared. "Clearing" consists of removing the dehydrating agent and some lipids using a substance that is miscible with the embedding medium (e.g., paraffin). The most common clearing agent is xylene.

[0008] Once cleared, the tissue is infiltrated with an embedding agent such as paraffin. Finally, the tissue in the cassette or removed from the cassette is placed into molten paraffin, which is then cooled to form a solid block that embeds or encases the tissue so that it can be sectioned. Alternatively, the tissue can be processed in a cassette, embedded in paraffin with the cassette, and sectioned. Once the tissue is embedded in a solid paraffin block, the tissue can be cut into sections that can be placed on a slide. This is done with a microtome. Once the sections are cut, they are floated on a warm water bath, which helps to remove any wrinkles. The tissue section paraffin block is then picked up from the water bath and placed on a glass microscope slide.

[0009] Further to fixation, it would be helpful to be able to assess the penetration of the fixative (e.g., formaldehyde present in formalin) in a tissue sample in a manner that would be helpful in understanding the degree of fixation of the tissue to determine if the tissue is ready for processing.

[0010] It would be helpful to be able to assess whether a fixative substance (e.g., formaldehyde present in formalin) is present within a tissue sample of a thicker and / or larger size (area).

[0011] It would be helpful to be able to configure or provide an IR spectroscopy system / device with a tissue sample measurement device that is capable of and facilitates obtaining measurement data indicative of whether a fixative substance (e.g., formaldehyde in formalin) is present at a selected depth location within a tissue sample.

[0012] It would be helpful to be able to configure or provide an IR spectroscopy system / device as or with a tissue sample measurement instrument to simultaneously obtain measurement data indicative of whether a fixed substance (e.g., formaldehyde in formalin) is present at multiple different locations of a tissue sample (and at respective selected depths within the tissue sample). BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1A IR spectra plots (absorbance and wave number [cm"1]) of kidney tissue measured at different times during formalin / formaldehyde infiltration into the kidney tissue are shown;

[0014] Figure 1B IR spectra plots (absorbance and wave number [cm"1]) of lung tissue measured at different times during formalin / formaldehyde infiltration into the lung tissue are shown;

[0015] Figure 2 An example embodiment of a spectroscopy system for evaluating samples from tissue subjected to formalin infiltration is shown;

[0016] FIG. 3A shows an example embodiment of a sample placement structure (a surface on which a tissue sample is placed) having a single sample receiving surface / interface defined by a crystal of a sample measurement device;

[0017] FIG. 3B shows another example embodiment of a sample placement structure (a surface on which a tissue sample is placed) having four (4) different spatially separated sample receiving surfaces / interfaces, each defined by a crystal of a sample measurement device;

[0018] Figure 3C Another example embodiment of a sample placement structure (a surface on which a tissue sample is placed) having nine (9) different spatially separated sample receiving surfaces / interfaces, each defined by a crystal of a sample measurement device, is shown;

[0019] Figure 4 An example embodiment of a spectroscopy system for evaluating samples from tissue subjected to formalin infiltration is shown, provided in the form of an attenuated total reflectance infrared (ATR-IR) sample measurement device containing a crystal defining a sample receiving surface / interface;

[0020] Figure 5One exemplary embodiment of a tissue probe configured as an attenuated total reflection infrared (ATR-IR) sample measurement device containing a crystal defining a sample receiving surface / interface is shown, the tissue probe having been advanced (pushed) into a tissue sample such that tissue (surface or mass) at a selected depth location (denoted as "L") within the tissue sample is in direct contact with the sample receiving surface / interface;

[0021] Figure 6A Another exemplary embodiment of a tissue probe is shown, the tissue probe containing a piercing structure at a distal open end of the probe; the tissue probe containing a sample receiving surface / interface configured repositionable between a forward / extended position adjacent the distal end (of the probe) and a rearward / retracted position recessed within the probe housing away from the distal open end;

[0022] Figure 6B Another exemplary embodiment of a tissue probe is shown, the tissue probe containing a piercing structure at a distal open end of the probe; the tissue probe containing a sample receiving surface / interface configured repositionable between a forward / extended position adjacent the distal end (of the probe) and a rearward / retracted position recessed within the probe housing away from the distal open end;

[0023] Figure 7 is a flowchart showing an exemplary attenuated total reflection infrared (ATR-IR) sample (e.g., tissue sample) measurement method or steps associated therewith;

[0024] Figure 8 is a flowchart showing an exemplary method of instrument usage design; and

[0025] Figure 9 is a flowchart showing an exemplary attenuated total reflection infrared (ATR-IR) sample (e.g., tissue sample) measurement method or steps associated therewith that does not involve (or require) use of a probe to penetrate sample material. DETAILED DESCRIPTION

[0026] Various embodiments of the present invention relate to configuring and / or utilizing attenuated total reflection infrared (ATR-IR) sample measurement devices that include a crystal defining a sample receiving surface / interface to assess (and optionally, quantify) the presence of a fixative (e.g., formaldehyde present in formalin), for example, from a sample from a tissue subjected to a fixative (e.g., formalin) infiltration. In exemplary embodiments, the sample receiving surface / interface is provided in the form of, or included within, a probe or other structure suitable for advancing (pushing or otherwise repositioning) the sample receiving surface / interface to a selected location (or depth) within a sample material. In exemplary embodiments, the probe includes a piercing structure or mechanism at a distal (open) end of the probe that is configured to form a channel within the sample material to the selected location (depth) in a minimally invasive manner so that the sample material is not substantially damaged during testing. Exemplary embodiments and implementations relate to instrument usage that is configured or provided to facilitate such assessment at multiple different locations within a sample placement structure / area of a measurement instrument for simultaneous measurement at the different locations.

[0027] Referring to Figure 2 In the present exemplary embodiment, the spectroscopic system 200 is provided / configured for assessing a sample from a tissue subjected to formalin infiltration. The spectroscopic system 200 includes an IR spectrometer 202, which can be supported by a stand 220 for placement in a rough handling station (optional), and a base plate 230, which can include a computer / processor assembly supporting the stand 220 (optional). The spectroscopic system 200 includes a sample placement structure 300 (a surface on which a tissue sample is placed) and several indicators, which can be processor controlled lights or other human and / or machine perceptible phenomena, including in the present exemplary system: an analysis status indicator 210 (e.g., complete / in progress), a fixative presence insufficient indicator 212 (e.g., insufficient formaldehyde detected), and a fixative presence sufficient indicator 214 (e.g., formaldehyde detected).

[0028] Referring also to Figure 1A And 1B In operation, the spectroscopic system 200, which can be configured to utilize one or more of IR, FTIR, and ATR-IR spectroscopy techniques, facilitates testing of a tissue sample to determine whether a fixative chemical has sufficiently penetrated the tissue (to an internal location of the tissue or a specified location within the tissue). In exemplary embodiments and implementations, IR spectroscopy is utilized to provide information of the presence of formaldehyde indicated by C=0 stretching. The inventors tested several animal tissues— Figure 1A And 1B, first check their IR spectra before fixation in 10% neutral buffered formalin (NBF) solution (i.e., before the fixative begins to infiltrate) and periodically during fixation (i.e., during fixative infiltration), which is typically a solution of about 3.7% formaldehyde CH2O in 95-97% water H2O with a small amount of buffer salts included). It is observed that a new stretch appears with an absorbance peak between about 1000 cm -1 and 1100 cm -1 (represented as P A in Figure 1A and 1B ) that gets stronger every hour, indicating the presence of formalin in the tissue. The presence of methylene bonds in the spectrum can also be used to assess fixation. For example, the instrument used to test the tissue includes (or is provided with or configured to have) a sample placement surface having a sample receiving surface / interface defined by an ATR-IR diamond on which a tissue sample to be tested is placed - after washing, if needed to remove fixative solution.

[0029] In one testing method, a tissue sample undergoing fixation is bisected or cut to expose its deepest or most difficult to reach regions or to expose a surface to be tested. A clean knife is used to expose the region of interest without contaminating it. If needed, the exposed region can be washed with water and excess water is absorbed using a paper towel or water-absorbing material. The tissue region of interest is then placed on the ATR-IR crystal / diamond and analyzed. With general programming techniques and practices understood by those skilled in the art, the software (e.g., executable computer program) is configured / programmed in a manner that facilitates processing of the IR spectra (data) to identify the presence and intensity of particular IR stretches of interest and to provide a go / no-go indication by green / red light or pass / fail, etc. The presence of a stretch can mean the presence of formaldehyde or a new bond formed; thus, even if fixation is not complete, it has been observed (and in some cases can be assumed) that the tissue will behave as a sufficiently fixed tissue during the processing temperature and exposure.

[0030] FIG. 3A depicts one example of a sample placement structure / region, i.e., a sample placement structure 300-1 (e.g., a surface on which a tissue sample to be measured is placed), having a (single) sample measurement device 310 having a sample receiving surface / interface defined by a crystal such as, for example, an ATR-IR diamond. In some tissue sample measurement device configurations, a crystal material other than a diamond can be utilized, such as, for example, a germanium (Ge) ATR crystal. In exemplary embodiments and implementations, the sample receiving surface / interface is part of and defined by the crystal of the ATR-IR sample measurement device.

[0031] Figure 4 One exemplary embodiment of a spectroscopic system 400 for evaluating a sample material (e.g., from a sample of tissue subjected to formalin infiltration) is shown, the system 400 including a sample measurement device 310 (e.g., provided in the form of an ATR-IR sample measurement device) including a sample receiving surface / interface 402 defined by a crystal 404 and located within a peripheral boundary 406, the sample receiving surface / interface 402 operably connected to an IR source 410 and an IR detector 420 via associated optics 412, 422 (at opposite ends of the crystal 404), respectively. Other couplings of the crystal 404 to the IR source 410 and the IR detector 420 are also contemplated. In operation, a tissue sample (denoted as TS) is placed on the sample receiving surface / interface 402. Internal reflections of the crystal 404 produce evanescent waves (denoted as EW) that extend beyond the sample receiving surface / interface 402 of the crystal into the sample in contact with the crystal. The presence of formaldehyde or newly formed bonds in the fixative infiltrated tissue contributes to infrared absorption, and the attenuated energy from each evanescent wave is transmitted back to the IR beam. The sample receiving surface / interface 402 can be provided / defined by a single or multiple reflective ATR elements to suit the particular measurement device, application requirements, and sample material (e.g., tissue sample) properties. Thus, it should be understood that, Figure 4 The illustration and description herein of the sample measurement device 310 including multiple reflective ATR elements additionally provides explicit support for embodiments of the sample measurement device 310 including single reflective ATR elements. In the present exemplary embodiment, the spectroscopic system 400 includes a data collection, spectral analysis, and user interface assembly 430 operably coupled or interfaced with the IR detector 420. The spectroscopic system 400 can be configured to conduct qualitative and quantitative analysis. Quantitative: the software can be configured / programmed to facilitate providing an indication of the level / amount / extent of formalin present, which can also be further used to determine fixation. One method to accomplish this is to determine a series of different concentrations of fixative present in tissue and absorbance, and then use that data to estimate the concentration of fixative in a test sample. The data collection, spectral analysis, and user interface assembly 430 can include a user interface (e.g., one or more display screens, touchscreens, or remote displays / monitors) and a computer, server, etc. configured with software programmed to facilitate the required data collection, detector output signal / IR spectral processing, application programming (and other) interfaces, and provide / generate visual displays, indicators, and other outputs (e.g., described herein). By way of example, a Fourier Transform Infrared (FTIR) spectroscopic method is utilized to convert the output of the IR detector 420 into an interpretable spectral pattern (such as, for example, a plot of absorbance versus wavenumber) that can be used to determine the presence and / or amount of formalin in the sample. The data collection, spectral analysis, and user interface assembly 430 can be configured to facilitate the required data collection, detector output signal / IR spectral processing, application programming (and other) interfaces, and provide / generate visual displays, indicators, and other outputs (e.g., described herein). By way of example, a Fourier Transform Infrared (FTIR) spectroscopic method is utilized to convert the output of the IR detector 420 into an interpretable spectral pattern (such as, for example, a plot of absorbance versus wavenumber) that can be used to determine the presence and / or amount of formalin in the sample. Figure 1A and 1BThe data collection, spectral analysis, and user interface components 430 can include data storage devices (e.g., on the instrument) for record keeping and providing access to control libraries and other data resources. Alternatively or additionally, the data collection, spectral analysis, and user interface components 430 can be configured to facilitate transmission and remote storage of collected data and access to network and external data and software resources and control inputs.

[0032] The sample measurement device 310, including its sample receiving surface / interface 402, can be provided in the form of a probe (e.g., a tissue probe) or other tool, implement, or structure suitable for advancing (pushing or otherwise repositioning) the sample receiving surface / interface into a selected location (or depth) within a sample material (e.g., a tissue sample).

[0033] Figure 5 One exemplary embodiment of a sample probe 500 (e.g., a tissue probe) configured with a sample measurement device 310, such as an attenuated total reflection infrared (ATR-IR) sample measurement device, is shown, including a crystal defining a sample receiving surface / interface, such as previously described with respect to FIG. 2, for example. Figure 4A sample probe 500 is depicted. The sample probe 500 includes a distal open end 504 near which the sample measurement device 310 is located, with its sample receiving surface / interface facing distally (i.e., positioned and oriented relative to the distal open end 504 such that sample material advanced through the distal open end is deposited (pressed against) on the sample receiving surface / interface. In this illustration, the probe 500 is shown advanced (pushed) into sample material 50 (e.g., a tissue sample), such that material (e.g., a tissue surface or mass) at a selected depth location (denoted as "L") is in direct contact with the sample receiving surface / interface. The sample probe 500 includes a surface / edge 506, which in this example embodiment tapers inwardly moving distally along the probe toward the open end 504. The surface / edge 506 can be provided in various forms, generally falling into the categories of non-sharp and sharp (or piercing) for purposes of the present description. In one embodiment in which the surface / edge 506 is not sharp, the probe 500 can be used as a measurement rod or measurement device suitable for (non-destructively) advancing into an already formed opening, passageway, etc. In one embodiment in which the surface / edge 506 is sharp (e.g., is a cutting edge), the probe 500 allows the user to cut (excise or drill) a path through the sample material to a selected depth location therein. Thus, it should be appreciated that the probe 500 can be variously configured (e.g., with a changeable / replaceable housing) to measure sample material at a top surface or exposed surface (e.g., formed by roughing) of the sample material as well as at a selected depth location within the sample material when the probe is configured as a sharp or cutting device. It should also be appreciated that the surface / edge described herein can be provided as variously shaped sharps (e.g., cutting edges), including but not limited to straight, curved, and circular cutting edges.

[0034] In example embodiments, a sample probe (e.g., a tissue probe) includes a piercing structure and / or cutter at a distal (open) end of the probe configured to form a passageway within a sample material (e.g., tissue) to a selected location (depth) in a minimally invasive manner such that the sample material is not severely damaged during testing.

[0035] Figure 6A Another example embodiment of a sample probe 600 (e.g., a tissue probe) configured with a sample measurement device 310 is shown, such as an attenuated total reflectance infrared (ATR-IR) sample measurement device, including a crystal defining a sample receiving surface / interface, such as previously described with respect to FIG. 2, for example. Figure 4The sample probe 500 includes a distal open end 504, near which is located the sample measurement device 310, with its sample-receiving surface / interface facing distally (i.e., positioned and oriented relative to the distal open end 504 so as to place (press against) sample material advanced through the distal open end on the sample-receiving surface / interface. In this exemplary embodiment, the probe 600 includes a surface / edge 506 configured (shaped) to provide a piercing structure at the distal open end of the probe; and the sample measurement device 310 is configured to be repositionable (as represented by the double-headed arrow 602) between a forward / extended position adjacent the distal end of the probe and a retracted position 610 (shown in dashed lines) within the probe housing distal from the distal open end 504 (e.g., as shown).

[0036] Figure 6B Another exemplary embodiment of a sample probe 650 (e.g., a tissue probe) configured with a sample measurement device 310, such as an attenuated total reflection infrared (ATR-IR) sample measurement device, is shown, including a crystal defining a sample-receiving surface / interface, such as previously described with respect to the sample probe 500. Figure 4 The sample probe 650 includes a distal open end 604, near which is located the sample measurement device 310, with its sample-receiving surface / interface facing distally (i.e., positioned and oriented relative to the distal open end 604 so as to place (press against) sample material advanced through the distal open end on the sample-receiving surface / interface. In this exemplary embodiment, the probe 650 includes a surface / edge 656 configured (shaped) to provide a shield / piercing structure, which is repositionable (as represented by the double-headed arrow 652) between a forward / extended position (protruding distally relative to the distal open end 604 of the probe and providing a barrier over the distal open end) and a retracted position 666 (shown in dashed lines) recessed within the probe housing distal from the distal open end 604 (e.g., as shown). Optionally, in this embodiment, the sample measurement device 310 is configured to be repositionable (as represented by the double-headed arrow 602) between a forward / extended position adjacent the distal end of the probe and a retracted position 610 (shown in dashed lines) recessed within the probe housing distal from the distal open end 604 (e.g., as shown).

[0037] With respect to the repositionable components / component portions of the sample probes, various repositioning mechanisms can be utilized, including manual, spring-biased, spring and / or actuator driven elements, and possibly mechanisms similar to those employed in conjunction with, for example, a guarded scalpel or a surgical implement having a retractable blade on a smaller scale.

[0038] Reference is made to Figure 6A and6B The probes described herein have a probe length L that can vary between 0-5 mm (typically about 1-2 mm) P with a longest probe length L P of about 6 cm. The probes described herein have a probe width W (or diameter) that can be about 1 mm or less P such that the sample material (e.g., tissue) is not substantially damaged during testing.

[0039] Figure 7 is a flowchart illustrating example ATR-IR sample measurement method 700 or steps associated therewith. At 702 (which is optional in some example methods), method 700 includes configuring a spectroscopic system or apparatus to have a probe containing an ATR-IR sample measurement apparatus including a crystal defining a sample receiving surface / interface that is operatively connected (e.g., optically coupled at opposite ends of the optical path of the crystal via associated optics) to an IR source and an IR detector. At 704, method 700 includes advancing the probe into a sample material (e.g., a tissue sample) such that material (tissue) at a selected depth location (denoted as "L", Figure 5 ) within the sample material is in direct contact with the sample receiving surface / interface. Next at 706, method 700 includes employing the IR source to generate an internally reflected IR beam that propagates through the crystal when the material (tissue) at the selected depth location is resting on the sample receiving surface / interface. At 708, method 700 includes using the IR detector to record an attenuated IR beam output (as an interferogram signal) of the crystal. And at 710, method 700 includes using the attenuated IR beam to generate / provide one or more IR spectra (visual representations of). At 712 (which is optional in some example methods), method 700 further includes evaluating the one or more IR spectra to determine whether there is a signal (e.g., at about 1000 cm -1 and 1100 cm -1between the signal peaks). In an example method, the sample material is a tissue sample. In an example method, the fixative is formalin / formaldehyde. In an example method, the selected depth position is about half the thickness of the sample material (or another depth position between the outer / top surface of the sample and a mid-way position in the sample). The step 710 of generating / providing one or more IR spectra using the attenuated IR beam can include generating / providing a visual representation of a spectral pattern that indicates the measurements obtained from measurements taken at different times, respectively. The step 710 of generating / providing one or more IR spectra using the attenuated IR beam can include generating / providing a visual representation of a spectral pattern that indicates the measurements obtained from measurements taken at different times and / or at different positions on or within the sample material. In an example method, the method 700 further includes, during (or after) the roughing of the sample material at the region of interest, obtaining measurements taken at different positions around or near the region of interest. For example, in the case of a large sample, a pathologist / designated person can visually inspect the tissue sample and cut out a region of interest that will be processed for study (this process is referred to as "grossing"). During grossing, the user can scan (assess) the area around the region of interest to determine the degree of fixation, assuming that if the positions around the region of interest exhibit the presence of fixation, then the region of interest has also received its share (sufficient amount) of fixative. In an example method, the step 704 of advancing the probe into the sample material includes obtaining measurements taken at different positions around or near the region of interest of the sample material.

[0040] Example embodiments and implementations relate to instrument usage that is configured or provided to facilitate such assessment of the presence and intensity of fixative at (via) multiple different positions within a sample placement structure / area of a measurement instrument (e.g., to measure at different positions simultaneously). FIG. 3B shows one example of such a sample placement structure / area, namely a sample placement structure 300-2 (e.g., a surface on which to place a tissue sample to be measured) having four (4) different spatially separated sample measurement devices 310, each having a sample receiving surface / interface defined by a crystal such as, for example, an ATR-IR diamond. Figure 3C FIG. 3C shows another example of such a sample placement structure / area, namely a sample placement structure 300-3 (e.g., a surface on which to place a tissue sample to be measured) having nine (9) different spatially separated sample measurement devices 310, each having a sample receiving surface / interface defined by a crystal such as, for example, an ATR-IR diamond.

[0041] Exemplary embodiments relate to a spectroscopic system or apparatus configured with a raised piercing structure containing a plurality of ATR-IR sample measurement devices provided on a spike configured to pierce into a sample material. A sample placement structure / area with a spatial distribution of ATR-IR sample measurement devices such as, for example, shown in FIGS. 3B or 3C can be provided in the form of a raised piercing structure containing a plurality of ATR-IR sample measurement devices provided on a spike configured to pierce into a sample material. Alternatively, a sample placement structure / area can be provided in the form of a raised piercing structure containing a plurality of ATR-IR sample measurement devices, each of the sample measurement devices provided on a separate (individual) spike configured to pierce into a sample material. Further, a sample placement structure / area and spike or other piercing structure can be configured to pierce to different depths within a sample material placed on the sample placement structure / area. In exemplary embodiments, the plurality of ATR-IR sample measurement devices comprise diamond crystals.

[0042] Figure 8 is a flowchart showing an exemplary instrument use design method 800. At 802, method 800 includes providing or configuring a spectroscopic system or apparatus with a probe or raised piercing structure containing one or more attenuated total reflection infrared (ATR-IR) sample measurement devices, each comprising a crystal defining a sample receiving surface / interface operably connected to an IR source and an IR detector.

[0043] Thus, in one exemplary embodiment, an instrument use method includes providing or configuring a spectroscopic system or apparatus with a probe or raised piercing structure containing one or more attenuated total reflection infrared (ATR-IR) sample measurement devices, each comprising a crystal defining a sample receiving surface / interface.

[0044] Accordingly, in one exemplary embodiment, an apparatus for measuring a sample material includes a spectroscopic system or device including or configured to have a structure containing one or more attenuated total reflection infrared (ATR-IR) sample measurement devices each including a crystal defining a sample receiving surface / interface. The sample receiving surface / interface of each ATR-IR sample measurement device is operatively connected to an IR source and an IR detector. In exemplary embodiments and implementations, the spectroscopic system or device includes a probe having a sample measurement device configured to be repositionable between a forward / extended position (adjacent a distal end of the probe) and a backward / retracted position within a recessed probe housing away from the distal end. In exemplary embodiments and implementations, the spectroscopic system or device includes a probe having a piercing structure and / or a cutter at a distal end of the probe. In exemplary embodiments and implementations, the spectroscopic system or device includes a probe having a shield / piercing structure configured to be repositionable between an extended position providing a barrier over a distal end opening of the probe and a retracted position. In exemplary embodiments and implementations, the spectroscopic system or device includes a raised piercing structure containing a plurality of ATR-IR sample measurement devices provided on a (structure) spike configured to pierce into a sample material. For example, the plurality of ATR-IR sample measurement devices includes diamond crystals. In exemplary embodiments and implementations, the one or more ATR-IR sample measurement devices includes a plurality of ATR-IR sample measurement devices configured to measure a sample material at different depths, respectively, within the sample material. In exemplary embodiments and implementations, the one or more ATR-IR sample measurement devices includes a plurality of ATR-IR sample measurement devices configured to measure a sample material at different spatially separated locations, respectively, across the sample material. In exemplary embodiments and implementations, the one or more ATR-IR sample measurement devices includes a plurality of ATR-IR sample measurement devices configured to measure a sample material at different spatially separated locations, respectively, across the sample material. In exemplary embodiments and implementations, the spectroscopic system or device includes a probe having a sample placement structure / region containing a plurality of ATR-IR sample measurement devices. In exemplary embodiments and implementations, the spectroscopic system or device includes a raised piercing structure having a sample placement structure / region containing a plurality of ATR-IR sample measurement devices.

[0045] Figure 9is a flowchart showing an example ATR-IR sample measurement method 900 or steps associated therewith that do not involve (or require) the use of a probe to penetrate a sample material. At 902 (which is optional in some example methods), the method 900 includes configuring a spectroscopic system or apparatus to have an ATR-IR sample measurement apparatus that includes a crystal defining a sample-receiving surface / interface that is operatively connected (e.g., optically coupled at opposite ends of an optical path of the crystal via associated optics) to an IR source and an IR detector. At 904, the method 900 includes placing a sample material (e.g., a tissue sample) on the ATR-IR sample measurement apparatus such that the sample material (e.g., the tissue sample) is in direct contact with the sample-receiving surface / interface. Next at 906, the method 900 includes employing the IR source to generate an internally-reflected IR light beam that propagates through the crystal when the sample material (e.g., tissue) is resting on the sample-receiving surface / interface. At 908, the method 900 includes using the IR detector to record an attenuated IR light beam output (as an interferogram signal) of the crystal. And at 910, the method 900 includes using the attenuated IR light beam to generate / provide one or more IR spectra (of visual representations). At 912 (which is optional in some example methods), the method 900 further includes evaluating the one or more IR spectra to determine whether there is a signal (e.g., a peak between about 1000 cm-1 and 1100 cm-1) indicative of whether a fixative (e.g., formalin) is present in the sample material. In example methods, the sample material is a tissue sample. In example methods, the fixative is formalin / formaldehyde. In example methods, the method 900 further includes, prior to placing the sample material, roughly processing the sample material to expose material at a selected depth location within the sample material, and the step 904 of placing the sample material includes placing the sample material on the ATR-IR sample measurement apparatus such that the exposed material at the selected depth location is in direct contact with the sample-receiving surface / interface. In example methods, the selected depth location is about half the thickness of the sample material (or another depth location between an outer / top surface of the sample and a mid-way location in the sample). In example methods, the method 900 further includes, after roughly processing the sample material and prior to placing the sample material, washing and removing excess fluid on the sample material that includes the exposed material at the selected depth location. The step 910 of using the attenuated IR light beam to generate / provide one or more IR spectra can include generating / providing a visual representation of a spectral pattern that indicates measurement results obtained from measurements taken at different times, respectively. The step 910 of using the attenuated IR light beam to generate / provide one or more IR spectra can include generating / providing a visual representation of a spectral pattern that indicates measurement results obtained from measurements taken at different times and / or at different locations on the sample material or at an exposed surface of the sample material.In the example method, the method 900 further includes obtaining measurements taken at different locations around or near the region of interest during (or after) the coarse processing of the sample material at the region of interest. For example, in the case of a large sample, a pathologist / designated person can visually inspect the tissue sample and cut out a region of interest that will be processed for study (this process is referred to as "grossing"). During grossing, the user can scan (assess) the area around the region of interest to determine the degree of fixation, if the locations around the region of interest exhibit the presence of fixation, it is assumed that the region of interest has also received its share (enough) of the fixative. In the example method, the step 904 of placing the sample material on the ATR-IR sample measurement device includes obtaining measurements taken at different locations around or near the region of interest of the sample material.

[0046] A "computer," "computer system," "computing device," "component," or "computer processor" can be, for example and without limitation, a processor, microcomputer, minicomputer, server, mainframe, laptop computer, personal data assistant (PDA), wireless electronic mail device, smart phone, mobile telephone, electronic tablet, cellular telephone, pager, facsimile machine, scanner, or any other programmable device or computer equipment configured to transmit, process, and / or receive data. The computer systems and computer-based devices disclosed herein can include memory and / or storage components for storing certain software applications used to obtain, process, and communicate information. It can be appreciated that such memory can be internal or external with respect to the operation of the disclosed embodiments. In various embodiments, a "host," "engine," "loader," "filter," "platform," or "component" can include various computers or computer systems, or can include a reasonable combination of software, firmware, and / or or hardware. In certain embodiments, a "module" can include software, firmware, hardware, or any reasonable combination thereof.

[0047] In general, it is apparent to those of ordinary skill in the art that the various embodiments described herein, or components thereof, can be implemented in various different embodiments of software, firmware, and / or hardware, or a combination thereof. Software code or special-purpose control hardware employed in implementing some embodiments of the present application is not limited to the present application. For example, the embodiments described above can be implemented in computer software using, for example, conventional or object-oriented techniques in any suitable computer programming language such as, for example,.NET or HTML. Programming languages of computer software and other computer-implemented instructions can be translated into machine language by a compiler or an assembler, or interpreted by an interpreter, before execution. Examples of assembly languages include ARM, MIPS, and x86; examples of high-level languages include Ada, BASIC, C, C++, C#, COBOL, Fortran, Java, Lisp, Pascal, Object Pascal; examples of scripting languages include Bourne script, JavaScript, Python, Ruby, PHP, and Perl. For example, the various embodiments can be employed in a Lotus Notes environment. Such software can be stored on any type of suitable computer-readable media such as, for example, magnetic or optical storage media.

[0048] Examples of software, engines, and / or modules can include software components, programs, applications, computer programs, application programs, system programs, machine programs, operating system software, middleware, firmware, software modules, routines, subroutines, functions, methods, procedures, software interfaces, application program interfaces (APIs), instruction sets, computing code, computer code, code segments, computer code segments, words, values, symbols, or any combination thereof. Determining whether to implement embodiments using hardware elements and / or software elements can vary depending on, for example, desired computational rate, power level, heat tolerance, processing cycle budget, input data rate, output data rate, memory resources, data bus speed, and other design or performance constraints.

[0049] In some cases, the various embodiments can be implemented as an article of manufacture. The article of manufacture can include a computer-readable storage medium arranged to store logic, instructions, and / or data for performing various operations of one or more embodiments. In various embodiments, for example, the article of manufacture can comprise a disk, optical disk, flash memory, or firmware containing computer program instructions suitable for execution by a processor or special-purpose processor.

[0050] Unless specifically stated otherwise, it can be appreciated that terms such as "processing," "computing," "calculating," "determining," or the like, refer to the action and / or processes of a computer or computing system, or similar electronic computing device, a DSP, ASIC, FPGA, or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein that manipulates and / or transforms data represented as physical quantities (e.g., electronic) within registers and / or memories into other data similarly represented as physical quantities within the memories, registers or other such information storage, transmission or display devices.

[0051] Certain embodiments can be described as using the expressions "coupled" and "connected" and their derivatives. These terms are not necessarily synonyms. For example, a term "connected" and / or "coupled" can be used to indicate that two or more elements are in direct physical or electrical contact with one another. However, the term "coupled" can also mean that two or more elements are not in direct contact with one another, but yet still co-operate or interact with one another. For example, with respect to software elements, the term "coupled" can mean interfaces, message interfaces, application program interfaces (APIs), exchange messages, etc.

[0052] It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the disclosure and are included within its scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the disclosure and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the disclosure as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present disclosure, therefore, is not intended to be limited to the examples shown herein but is to be accorded the full scope consistent with the scope of the claims, wherein reference to an

[0053] While various systems described herein can be embodied in software or code executed by hardware, as discussed above, alternative embodiments can also be realized by dedicated hardware, software / hardware and a combination of the two. If embodied in software or code, the software or code can be executed by any processor or collection of processors, including one or more digital signal processors, ASICs, FPGAs, or other programmable logic devices, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein that are represented as physical quantities within the computer's registers and / or memories and / or other such information storage, transmission or display devices.

[0054] The flow charts and methods described herein show the functionality and operation of various implementations. If embodied in software, each block, step or action can represent a module, segment, or portion of code that comprises program instructions implementing the specified logical function. The program instructions might be embodied in the form of source code that comprises human- readable statements written in a programming language or machine code that comprises numerical instructions recognizable by a suitable execution system such as a processing component in a computer system. If embodied in hardware, each block can represent a circuit or a number of interconnected circuits for performing the specified logical function.

[0055] In the foregoing description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments. It can be apparent, however, to one skilled in the art that one or more other embodiments can be practiced without some of these specific details. The particular embodiments described are not provided to limit the present disclosure but to illustrate it. The scope of the present disclosure is not to be determined by the specific examples provided above but only by the claims below. In other instances, well-known structures, devices, and operations have been shown in block diagram form or have been left out in order to avoid obscuring the understanding of the description. Where considered useful to clarify the description, reference numbers or portions of reference numbers have been repeated among the figures to indicate corresponding or analogous elements, which can optionally have similar characteristics.

[0056] It is also to be understood that the phraseology or terminology employed herein, such as "one embodiment" or "an embodiment" and the like, are used for descriptive purposes to convey the meaning of the present disclosure. It is further noted that the terms "comprise", "comprising", "include", "including", and the like, when used in this specification, can specify the presence of stated features, integers, steps, operations, elements, or components, but do not have a limiting effect. It is to be understood that the terms "including", "comprising", "consisting" and "consisting essentially of" are open-ended, and also include the case of "consisting of". It is to be understood that the phraseology "consisting essentially of" indicates the presence of stated features, integers, steps, operations, elements, or components as well as those that do not materially affect the method or process being described. That is, it covers the case of "consisting of" but also includes additional features, integers, steps, operations, elements, or components that do not materially affect the method or process being described.

[0057] While various embodiments of the present application have been described herein, it should be apparent that various modifications, changes, and substitutions are also possible in light of the foregoing disclosure. Accordingly, the disclosed embodiments are intended to be illustrative only and not limiting of the scope of the application as claimed.

Claims

1. An attenuated total reflection infrared (ATR-IR) sample material measurement method, comprising: configuring a spectroscopic system or apparatus to have an ATR-IR sample measurement device, the sample measurement device including a crystal defining a sample receiving surface / interface operatively connected to an IR source and an IR detector; placing a sample material on the ATR-IR sample measurement device such that the sample material is in direct contact with the sample receiving surface / interface; employing the IR source to generate an internally reflected IR light beam that propagates through the crystal when the sample material is resting on the sample receiving surface / interface; using the IR detector to record attenuated IR light beam output from the crystal; using the attenuated IR light beam to generate / provide an IR spectrum or a plurality of IR spectra; and evaluating the IR spectrum or the plurality of IR spectra to determine whether a signal indicative of the presence of a fixed substance in the sample material is present, wherein the sample material is a tissue sample. the fixed substance is formaldehyde.

2. The attenuated total reflection infrared (ATR-IR) sample material measurement method according to claim 1, wherein, The step of placing a sample material on the ATR-IR sample measurement device includes obtaining measurements taken at different locations around or near a region of interest of the sample material.

3. The attenuated total reflection infrared (ATR-IR) sample material measurement method of claim 1, wherein, 4. The attenuated total reflection infrared (ATR-IR) sample material measurement method of claim 1, further comprising: prior to placing the sample material, roughly treating the sample material to expose material at a selected depth location within the sample material; and wherein the step of placing a sample material includes placing the sample material on the ATR-IR sample measurement device such that the exposed material at the selected depth location is in direct contact with the sample receiving surface / interface. the selected depth location is approximately one-half of a thickness of the sample material.

5. The attenuated total reflection infrared (ATR-IR) sample material measurement method according to claim 4, wherein, 6. The attenuated total reflection infrared (ATR-IR) sample material measurement method of claim 4, further comprising: after roughly treating the sample material and prior to placing the sample material, washing and removing excess fluid on the sample material including the exposed material at the selected depth location. The step of using the attenuated IR light beam to generate / provide an IR spectrum or a plurality of IR spectra includes generating / providing a visual representation of a spectral pattern that respectively indicates measurement results obtained from measurements taken at different times.

7. The attenuated total reflection infrared (ATR-IR) sample material measurement method of claim 1, wherein, The step of using the attenuated IR light beam to generate / provide an IR spectrum or a plurality of IR spectra includes generating / providing a visual representation of a spectral pattern that indicates measurement results obtained from measurements taken at different times and / or at different locations on the sample material or at an exposed surface of the sample material.

8. The attenuated total reflection infrared (ATR-IR) sample material measurement method of claim 1, wherein, 9. An apparatus for measuring a sample material, comprising: a spectroscopic system or apparatus including a probe, the probe including one or more attenuated total reflection infrared (ATR-IR) sample measurement devices, the sample measurement devices each including a crystal defining a sample receiving surface, ​ wherein the sample receiving surface is defined by the crystal and is operatively connected to an IR source and an IR detector at opposite ends of the crystal via associated optics, wherein the sample receiving surface is configured to be positionable between an extended position adjacent a distal end of the probe and a retracted position recessed within the probe housing away from the distal end, and wherein internal reflections of the crystal produce evanescent waves that extend beyond the sample receiving surface of the crystal into the sample in contact with the crystal when the sample is in contact with the sample receiving surface.

10. The apparatus for measuring a sample material according to claim 9, wherein, The sample receiving surface / interface of each of the ATR-IR sample measurement devices is operatively connected to an IR source and an IR detector.

11. The apparatus for measuring sample material of claim 9, wherein the probe includes a piercing structure and / or a cutter at a distal end of the probe.

12. The apparatus for measuring a sample material according to claim 9, wherein, The probe includes a shield / piercing structure configured to be positionable between the extended position and the retracted position, the extended position providing shielding over the distal open end of the probe.

13. The apparatus for measuring a sample material according to claim 9, wherein, The spectroscopic system or device includes a raised piercing structure containing a plurality of ATR-IR sample measurement devices provided on spikes configured to pierce into sample material.

14. The apparatus for measuring a sample material according to claim 13, wherein, The plurality of ATR-IR sample measurement devices includes diamond crystals.

15. The apparatus for measuring a sample material of claim 9, wherein, The one or more ATR-IR sample measurement devices include a plurality of ATR-IR sample measurement devices configured to respectively measure the sample material at different depths within the sample material.

16. The apparatus for measuring a sample material of claim 9, wherein, The one or more ATR-IR sample measurement devices include a plurality of ATR-IR sample measurement devices configured to respectively measure the sample material at different spatially separated locations across the sample material.

17. The apparatus for measuring a sample material of claim 9, wherein, The probe includes a sample placement structure / region containing a plurality of ATR-IR sample measurement devices.

18. The apparatus for measuring a sample material of claim 9, wherein, The spectroscopic system or device includes a raised piercing structure having a sample placement structure / region containing a plurality of ATR-IR sample measurement devices.

19. A method of attenuated total reflection infrared (ATR-IR) sample material measurement, comprising: configuring a spectroscopic system or device to have a probe containing an ATR-IR sample measurement device, the sample measurement device including a crystal defining a sample receiving surface / interface operatively connected to an IR source and an IR detector; advancing the probe into a sample material having material at a selected depth position in direct contact with the sample receiving surface / interface, the sample material being a tissue sample removed from a body; employing the IR source to generate an internally reflected IR beam propagating through the crystal when the material at the selected depth position is resting on the sample receiving surface / interface; using the IR detector to record attenuated IR beam output from the crystal; using the attenuated IR beam to generate / provide an IR spectrum or a plurality of IR spectra; and and evaluating the one / more IR spectra to determine whether a signal indicative of the presence of a fixed substance in the sample material at the selected depth position is present.

20. The attenuated total reflection infrared (ATR-IR) sample material measurement method of claim 19, wherein, The fixed substance is formaldehyde.

21. The attenuated total reflection infrared (ATR-IR) sample material measurement method according to claim 19, wherein, The step of advancing the probe into the sample material includes obtaining measurements taken at different locations around or near the region of interest of the sample material.

22. The attenuated total reflection infrared (ATR-IR) sample material measurement method according to claim 19, wherein, The selected depth position is approximately half the thickness of the sample material.

23. The attenuated total reflection infrared (ATR-IR) sample material measurement method of claim 19, wherein, The step of generating / providing one / more IR spectra using the attenuated IR beam includes generating / providing a visual representation of a spectral pattern, the spectral pattern being indicative of the measurements obtained from the measurements taken at different times, respectively.

24. The attenuated total reflection infrared (ATR-IR) sample material measurement method according to claim 19, wherein, The step of generating / providing one / more IR spectra using the attenuated IR beam includes generating / providing a visual representation of a spectral pattern, the spectral pattern being indicative of the measurements obtained from the measurements taken at different times and / or at different locations on / in the sample material.

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