Devices for and methods of sample preparation for imaging target molecules

Abstract

Devices and methods of sample preparation for imaging target molecules via atomic force microscopy are provided. Accordingly, the devices include a sample support chip that has an atomically flat imaging surface of a support substrate. A bounded sample segment is defined on the imaging surface via a surrounding hydrophobic barrier. The bounded sample segment includes an imaging region that is substantially devoid of contaminants and a fluidically coupled collection region. The target molecule is electrostatically bonded to the imaging surface in the imaging region in a substantially unperturbed state.

Description

Attorney Docket No. 1147-OOl-OlWODEVICES FOR AND METHODS OF SAMPLE PREPARATION FOR IMAGING TARGET MOLECULESCross-Reference to Related Applications

[0001] This application claims priority to and the filing date benefit of U.S. Provisional PatentApplication No. 63 / 715,206, entitled "‘Devices for and Methods of Sample Preparation for Imaging Target Molecules,” filed November 1, 2024, the disclosure of which is incorporated herein by reference in its entirety.Background

[0002] The embodiments described herein relate to atomic force microscopy. More particularly, the embodiments described herein relate to devices for and methods of sample preparation for imaging target molecules via atomic force microscopy.

[0003] In the fields of molecular biology and biochemistry, DNA sizing is a critical step in known analytical methods. For example, DNA sizing is a core method for genomic analysis in research, healthcare, and / or forensic applications. In some known systems, the DNA sizing methods rely on measuring the propagation time of DNA molecules in liquid solution through a porous matrix. However, in so far as different matrices are required depending on the absolute size of the molecules, no one matrix works across a broad range of sizes. Additionally, known systems have an upper limit to the size of molecules measurable via this technique, which is below the size of the largest molecules that may be technologically relevant. Additionally, unavoidable variations in the matrix material, variations in the ionic strength of the solutions, and other factors that affect the propagation speed of the molecules require internal standards of known size in order to determine DNA size accurately. The combination of these features known systems undesirably reduces the speed at which various samples can be processed.

[0004] Moreover, some known systems and methods of DNA sizing require that the target molecule be stretched and / or aligned to achieve the desired accuracy of the method. For example, some methods of DNA imaging include attaching one end of the target molecule on a solid surface and applying forces (e.g., surface tension or electrical forces) to the other end to stretch the targetAttorney Docket No. 1147-OOl-OlWO molecule. Such procedures can be time consuming and require additional procedures (which can increase the likelihood of contamination or error).

[0005] In addition to the above limitations, known approaches for DNA sizing often require substantial manipulation of the target molecule in preparation for imaging. This manipulation can, for example, include stretching, aligning, dying, or otherwise affecting the target molecule prior to the imaging. Such manipulation can negatively affect the speed and / or accuracy of the DNA sizing.

[0006] Accordingly, there exists a need in the art for improved devices for and methods of sample preparation for imaging target molecules, such as in DNA sizing.Summary

[0007] This summary introduces certain aspects of the embodiments described herein to provide a basic understanding. This summary is not an extensive overview of the inventive subject matter, and it is not intended to identify key or critical elements or to delineate the scope of the inventive subject matter.

[0008] In some embodiments, the present disclosure is directed to a chip to support a sample for analysis via atomic force microscopy. The chip can include a support substrate that has an imaging surface. The chip also includes a bounded sample segment defined by a hydrophobic boundary on the imaging surface. The bounded sample segment includes an imaging region and a collection region.

[0009] In some embodiments, the present disclosure is directed to a method of manufacturing a chip to support a sample for analysis via atomic force microscopy. The method can include exposing an atomically flat imaging surface of a support substrate. The method can also include forming a hydrophobic boundary on the imaging surface to define a bounded sample segment. Additionally, the method can include sealing the imaging surface within a vapor impermeable container following the forming of the hydrophobic boundary.Attorney Docket No. 1147-OOl-OlWO

[0010] In some embodiments, the present disclosure is directed to a method of preparing a chip to support a sample for analysis via atomic force microscopy. The method can include conveying a sample solution into a bounded sample segment on an imaging surface of the chip. The sample solution can include a target molecule. The bounded sample segment is surrounded by a hydrophobic boundary. The method can also include executing a dwell interval following the conveying of the sample solution into the bounded sample segment. The method can further include flowing a wash fluid across the imaging surface after executing the dwell interval. Additionally, the method can include drying the imaging surface at a first temperature, after flowing the wash fluid. After the drying, the method can include executing a heating interval at a second temperature to remove a molecular layer of the wash fluid from the imaging surface. Finally, the method can include positioning the chip in an environmental chamber.

[0011] In some embodiments, the present disclosure is directed to a sample support chip for use with atomic force microscopy. The chip can include an imaging region of a bounded sample segment defined on an imaging surface of a support substrate. The imaging surface is atomically flat, and the imaging region is substantially devoid of contaminants. Tire chip can also include a target molecule electrostatically bonded to the imaging surface in the imaging region, with the target molecule being in a substantially unperturbed state.

[0012] These and other inventive features, aspects, and advantages will become better understood with reference to the following description and drawings.Brief Description of the Drawings

[0013] FIG. l is a schematic view of a system for imaging a target molecule according to an embodiment.

[0014] FIG. 2 is a top view of a chip for analysis via the system of FIG. 1.

[0015] FIG. 3 is a side view of the chip of FIG. 2 depicting a raised hydrophobic boundary according to an embodiment.Attorney Docket No. 1147-OOl-OlWO

[0016] FIG. 4 is a side view of the chip of FIG. 2 depicting a recessed hydrophobic boundary according to an embodiment.

[0017] FIG. 5 is an enlarged view of a portion of the chip of FIG. 2 depicting a bounded sample segment according to an embodiment.

[0018] FIG. 6 is an enlarged view of a portion of the chip of FIG. 2 depicting a target molecule positioned within an imaging region according to an embodiment.

[0019] FIG. 7 is an enlarged view of a portion of the chip of FIG. 2 depicting a bounded sample segment according to an embodiment.

[0020] FIG. 8 is a schematic diagram of a portion of a method of manufacturing a chip for analysis via the system of FIG. 1 depicting a positioning of the chip in an environmental chamber according to an embodiment.

[0021] FIG. 9 is a schematic diagram of a portion of a method of manufacturing a chip for analysis via the system of FIG. 1 depicting the exposure of an imaging surface of the chip according to an embodiment.

[0022] FIG. 10 is a schematic diagram of a portion of a method of manufacturing a chip for analysis via the system of FIG. 1 depicting the forming of a hydrophobic boundary according to an embodiment.

[0023] FIG. 11 is a schematic diagram of a portion of a method of manufacturing a chip for analysis via the system of FIG. 1 depicting the sealing of the chip within a vapor impermeable container according to an embodiment.

[0024] FIG. 12 is a top view of a chip for analysis via the system of FIG. 1 according to an embodiment.

[0025] FIG. 13 is an enlarged view of a portion of the chip of FIG. 12 depicting a plurality of bounded sample segments according to an embodiment.Attorney Docket No. 1147-OOl-OlWO

[0026] FIG. 14 is a schematic diagram of a portion of a method for preparing the chip of FIG. 12 depicting the conveying of a sample solution into a bounded sample segment according to an embodiment.

[0027] FIG. 15 is a schematic diagram of a portion of a method for preparing the chip of FIG. 12 depicting the flowing of a wash fluid across the surface according to an embodiment.

[0028] FIG. 16 is a schematic diagram of a portion of a method for preparing the chip of FIG. 12 depicting the flowing of an air jet across the surface according to an embodiment.

[0029] FIG. 17 is a perspective view of a system for preparing the chip of FIG. 12 according to an embodiment.

[0030] FIG. 18 is a perspective view of the system of FIG. 17 with a housing removed for clarity.

[0031] FIG. 19 is a perspective view of a portion of the system of FIG. 17 depicting the chip in a tilted orientation in preparation for the flowing of the wash fluid across the surface according to an embodiment.

[0032] FIG. 20 is a perspective view of a portion of the system of FIG. 17 depicting the chip in a tilted orientation according to an embodiment.

[0033] FIG. 21 is an atomic force microscope according to an embodiment.

[0034] FIG. 22 is a flow chart of a method of manufacturing the chip to support a sample for analysis via atomic force microscopy according to an embodiment.

[0035] FIG. 23 is a flow chart of a method for preparing a chip to support a sample for analysis via atomic force microscopy according to an embodiment.Detailed Description

[0036] Generally, the present disclosure is directed to the features, manufacturing, and preparation of a sample support chip for use in atomic force microscopy. As atomic forceAttorney Docket No. 1147-OOl-OlWO microscopy uses the movement of a stylus relative to a reference surface to assemble an image of a target (e.g., a target molecule such as DNA, RNA, or a polymer), it is desirable that the imaging surface upon which the sample is positioned to be flat and free from contaminants that could otherwise negatively affect the analysis of the target molecule. For example, the chip can include a sheet of mica that is atomically flat. As used herein, an atomically flat surface can have a deviation that is less than 0.5 nanometers across any 10 microns of linear distance. Said another way, a local roughness of the surface, as measured by the standard deviation of the surface height profde, is less than 33% (e.g., less than 25%) a median height of the objects on the imaging surface (e g., a target molecule such as DNA, RNA, or other similar molecule). The local roughness can be measured in a region surrounding the molecule, extending radially outward in a range of between 5 and 10 times an average width of the object.

[0037] As described herein, the chip includes at least one bounded sample segment that is defined by a hydrophobic boundary. The bounded sample segment includes an imaging region and a collection region. The collection region can be tapered and serve as a repository for contaminants removed from the imaging region during the preparation of the chip for imaging. Accordingly, the bounded sample segment can, for example, have an irregular rounded shape, an oval rounded shape, a raindrop shape, and / or any combination thereof.

[0038] During preparation of the chip for imaging via atomic force microscopy, a liquid sample containing the target molecule in solution is positioned within the bounded sample segment where it is contained by the hydrophobic boundary. The target molecule is allowed to settle onto the imaging surface within the imaging region. The settling can, for example be in response to a random diffusion in the liquid followed by short range (e.g., less than four nanometers) attractive molecule-surface interaction. In some embodiments, the settling can be facilitated by gravitational forces and / or centrifugation. Because the target molecule is allowed to settle without any external intervention, the target molecule contacts and is electrostatically adhered to the imaging surface in a substantially unperturbed state. In other words, the target molecule is not stretched, unwound, aligned, oriented, or otherwise manipulated in any substantial manner, thus resulting in the unperturbed state (i.e., a natural state) of the target molecule including various curves, loops, crossings, overlaps, or other features of the target molecule. In this manner, the methods of sample preparation described herein do not require any additional steps of manipulating the targetAttorney Docket No. 1147-OOl-OlWO molecule that may be required in conventional analysis methods, and thus provide an efficient, rapid method for preparing samples for analysis.

[0039] Once the target molecule is electrostatically adhered to the imaging surface within the imaging region of the bounded sample segment, a wash fluid can be directed across the imaging surface. The wash fluid is directed to flow from the imaging region toward the collection region to move contaminants from the imaging region. For example, as described herein, the chip can be tilted so that the collection region is gravitationally below the imaging region prior to the wash fluid being directed across the chip. As the target molecule is electrostatically bonded with the imaging surface, the target molecule is unaffected by the flow of the wash fluid and remains in the unperturbed state.

[0040] If the chip were permitted to dry under ambient conditions (i.e. via evaporation of the wash fluid), then various deposits would likely form, such via crystalline growth, on the imaging surface. The presence of these deposits would negatively affect the analysis of the target molecule via atomic force microscopy. Therefore, it is desirable that the imaging region be maintained in a state in which only the target molecule is adhered (e.g., coupled or bonded) to the imaging surface within the imaging region. Similarly stated, it is desirable that the imaging region be maintained in a state that minimizes or eliminates the formation of deposits. To that end, the imaging surface can be dried following the flowing of the wash fluid. The drying can, for example, be accomplished by flowing an air j et that is within 5°C of the ambient temperature across the imaging surface. The drying can also be accomplished via a centrifuge that is operated within 5° of the ambient temperature.

[0041] Although the drying can remove a majority of the wash fluid from the imaging surface, a molecular layer of the wash fluid can remain. Accordingly, after the drying, the chip can be subjected to a heating interval to remove the remaining molecular layer from the imaging surface without disrupting the target molecule in the unperturbed state. By removing the molecular layer of the wash fluid, the methods described herein reduce or eliminate the possibility that the molecular layer could distort the analysis of the target molecule either directly or through a resultant crystalline growth.Attorney Docket No. 1147-OOl-OlWO

[0042] As described herein, the chip can include multiple bounded sample segments that are separate from one another and that, in some embodiments, can each contain a different sample. For example, up to 96 bounded sample segments can be defined on the imaging surface for some chips described herein. The presence of multiple bounded sample segments on a single chip can significantly decrease the aggregate time required to process the required samples. For example, analyzing multiple samples in the multiple bounded sample segments on a single chip precludes necessity to prepare an independent chip for each sample. Additionally, having multiple samples on a single chip in independent bounded sample segments precludes the necessity to remove and replace the chip from the atomic force microscope following the analysis of each sample. Thus, the method of sample preparation described herein (and the methods of imaging of the target molecule described in U.S. provisional application docket no. 1147-002-00US, entitled “Systems and Methods for Analyzing Target Molecules,” fded on the same date herewith, the disclosure of which is incorporated herein in its entirety, allow for efficient, cost-effective, and rapid analysis ofDNA.

[0043] During analysis via the atomic force microscope, it is desirable to determine the position of the imaging surface of the chip with respect to a probe of the atomic force microscope to within a few microns (e.g., less than 10 microns in an axis normal to the imaging surface). This determination can be accomplished via optical means. Therefore, it is desirable to obtain a sharp reflection from the imaging surface of the chip. However, as a support substate (e.g., a sheet of mica) can be thin (e.g., less than 0.5 millimeters) and transparent, it is desirable to suppress the reflection from the bottom surface (i.e., the surface opposite the imaging surface) of the support substrate. Without the suppression, top and bottom reflections from the chip can merge, which can reduce the accuracy of the measurement. To that end, the reflection can be suppressed by using a thicker support substrate (e.g., thicker mica) to move the bottom surface away from the imaging surface so that the two reflections do not merge. Alternatively, the reflection can be suppressed on a relatively thin support substrate by adding an absorbative or anti -refl ection coating on the bottom of the support substrate or by minimizing the change in index of refraction between the bottom of the support substrate and a backing substrate, thereby allowing light to pass thorough with minimal reflection. In some embodiments, a bright diffuse background is useful for observing chips or cracks in the support substrate, which thereby renders the use an absorbative or ‘black’Attorney Docket No. 1147-OOl-OlWO coating on the bottom surface suboptimal. Instead, an approximate index match can be achieved by bonding the support substrate to the backing substrate (e.g., glass or similar) using a transparent adhesive with a similar index of refraction as the support substrate and the backing substrate. The bonding can, for example, be accomplished through the use of a relatively thick layer of indexmatching adhesive or a sandwich of index-matching adhesive, and glass. This results in a small change in refractive index going along a support substrate-adhesive-backing substrate path, and results in a relatively weak reflection from the interfaces.

[0044] As used herein, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10 percent of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55. Similarly, the language “about 5” covers the range of 4.5 to 5.5.

[0045] Specific words chosen to describe one or more embodiments and optional elements or features are not intended to limit the invention. For example, spatially relative terms — such as “beneath”, “below”, “lower”, “above”, “upper”, “proximal”, “distal”, and the like — may be used to describe the relationship of one element or feature to another element or feature as illustrated in the figures. These spatially relative terms are intended to encompass different positions (i.e., translational placements) and orientations (i.e., rotational placements) of a device in use or operation in addition to the position and orientation shown in the figures. For example, if a device in the figures is turned over, elements described as “below” or “beneath” other elements or features would then be “above” or “over” the other elements or features. Thus, the term “below” can encompass both positions and orientations of above and below. A device may be otherwise oriented (e.g., rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Likewise, descriptions of movement along (translation) and around (rotation) various axes include various spatial device positions and orientations. The combination of a body’s position and orientation define the body’s pose (e.g., a kinematic pose).

[0046] Similarly, geometric terms, such as “parallel”, “perpendicular”, “round”, or “square”, are not intended to require absolute mathematical precision, unless the context indicates otherwise. Instead, such geometric terms allow for variations due to manufacturing or equivalent functions. For example, if an element is described as “round” or “generally round,” a component that is notAttorney Docket No. 1147-OOl-OlWO precisely circular (e.g., one that is slightly oblong or is a many-sided polygon) is still encompassed by this description.

[0047] In addition, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context indicates otherwise. The terms “comprises”, “includes”, “has”, and the like specify the presence of stated features, steps, operations, elements, components, etc. but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, or groups.

[0048] The terms “nucleic acid molecule” or “nucleic acid” may be used interchangeably herein, and may refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (including known analogs or a combination thereof), and / or RNA molecules derived from DNA and RNA sequencing prep chemistries and processes unless otherwise indicated. Nucleic acid molecules to be manipulated and / or measured as described herein can be obtained from any source of nucleic acid. The nucleic acid molecule can be single-stranded or double-stranded. In some cases, the nucleic acid molecules are DNA. The DNA can be mitochondrial DNA, complementary DNA (cDNA), or genomic DNA. In some cases, the nucleic acid molecules are genomic DNA (gDNA). The DNA can be plasmid DNA, cosmid DNA, bacterial artificial chromosome (BAC), or yeast artificial chromosome (YAC). The DNA can be derived from one or more chromosomes. For example, if the DNA is from a human, the DNA can be derived from one or more of chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, or Y. In some cases, the nucleic acid molecules are RNA can include, but is not limited to, mRNAs, tRNAs, snRNAs, rRNAs, retroviruses, small non-coding RNAs, microRNAs, polysomal RNAs, pre-mRNAs, intronic RNA, viral RNA, cell free RNA and fragments thereof. The non-coding RNA, or ncRNA can include snoRNAs, microRNAs, siRNAs, piRNAs and long nc RNAs. The source of nucleic acid for use with the systems, chips, and methods described herein can be a sample comprising the nucleic acid.

[0049] As used herein, the term “stiffness” relates to an object’s resistance to deflection, deformation, and / or displacement produced by an applied force, and is generally understood to be the opposite of the object’s “flexibility.” For example, a layer or portion of a chip with greater stiffness is more resistant to deflection, deformation and / or displacement when exposed to a forceAttorney Docket No. 1147-OOl-OlWO than is a layer or structure of the container having a lower stiffness. Similarly stated, a portion of a chip (e.g., a layer) having a higher stiffness can be characterized as being more rigid than another portion of a chip (e.g., another layer) having a lower stiffness. Stiffness can be characterized in terms of the amount of force applied to the object and the resulting distance through which a first portion of the object deflects, deforms, and / or displaces with respect to a second portion of the object. When characterizing the stiffness of an object, the deflected distance maybe measured as the deflection of the portion of the object different than the portion of the object to which the force is directly applied. Said another way, in some objects, the point of deflection is distinct from the point where the force is applied.

[0050] Stiffness is an extensive property of the object being described, and thus is dependent upon the material from which the object is formed as well as certain physical characteristics of the object (e g., cross-sectional shape, thickness, boundary conditions, etc.). For example, the stiffness of a chip or portion of a chip can be increased or decreased by selectively including in the chip or portion of the chip a material having a desired modulus of elasticity, flexural modulus and / or hardness. The modulus of elasticity is an intensive property of (i.e., is intrinsic to) the constituent material and describes an object’s tendency to elastically (i.e., non-permanently) deform in response to an applied force. A material having a high modulus of elasticity will not deflect as much as a material having a low modulus of elasticity in the presence of an equally applied stress. Thus, the stiffness of the chip or portion of the chip can be decreased, for example, by introducing into the chip or portion of the chip and / or constructing the chip or portion of the chip of a material having a relatively low modulus of elasticity. Similarly, the flexural modulus is used to describe the ratio of an applied stress on an object in flexure to the corresponding strain in the outermost portions of the object. The flexural modulus, rather than the modulus of elasticity, is often used to characterize certain materials, for example plastics, that do not have material properties that are substantially linear over a range of conditions. An object with a first flexural modulus is more elastic and has a lower strain on the outermost portions of the object than an object with a second flexural modulus greater than the first flexural modulus. Thus, the stiffness of a chip or a portion of a chip can be reduced by including in the object a material having a relatively low flexural modulus.Attorney Docket No. 1147-OOl-OlWO

[0051] FIG. 1 is a perspective view of a system 1000 for imaging a target molecule. The system 1000 can be configured to analyze a target molecule, such as a nucleic acid molecule and more specifically, a strand of DNA, via atomic force microscopy. The system 1000 can be any of the systems shown and described in U.S. provisional application docket no. 1147-002-00US, entitled “Systems and Methods for Analyzing Target Molecules,” filed on the same date herewith, the disclosure of which is incorporated herein in its entirety. As shown, the system 1000 can include an environmental chamber 1600 configured to receive a chip 1100 (e.g., a sample support chip). The environmental chamber 1600 of the system 1000 can be configured to maintain an internal environment that is in a range of 1% to less than 10% of a relative humidity of the internal environment. In some embodiments the environmental chamber 1600 can be configured to maintain the relative humidity of the internal environment within a range of not more than 10% (e.g., between 0.5% and 5%, between 2% and 5%, between 1% and 4%, between 5.5% and 9.5%, between 6% and 9%, in between 6% and 8%). The relative humidity of the internal environment can be maintained by any suitable mechanism, such as, for example, by conveying a dry gas or desiccant into the environmental chamber 1600. In some embodiments, the dry gas can be an inert gas. By minimizing the water content in the internal environment (i.e., by maintaining the relative humidity at a value of not more than 10%), the formation of deposits and / or the collection of contaminants on the chip 1100 can be minimized, thus improving the accuracy of the imaging performed by the system 1000. In some embodiments, the internal volume within the environmental chamber 1600 is minimized to reduce that amount of gas to be conditioned to maintain the relative humidity of the internal environment within the desired range (e.g., between 1% and not more than 10%).

[0052] A displacement sensor 1400 (e.g., an interferometer) can be positioned to detect a position of a stylus (not shown) of a probe (not shown) during the execution of a scan of a sample supported by the chip 1100. A controller (not shown) can analyze the target molecule based at least in part on the output of the displacement sensor 1400. Said another way, the controller can assemble an image of the target molecule based on changes in a distance between a tip of the stylus and an imaging surface of the chip 1100 during the execution of the scan.

[0053] FIG. 2 is a top view of the chip 1100 to support a sample (e.g., the target molecule) for analysis via atomic force microscopy. As depicted, the chip 1100 includes a support substrateAttorney Docket No. 1147-OOl-OlWO1102 that has an imaging surface 1130. The imaging surface 1130 can be on a plane defined by a longitudinal axis AL and an orthogonal lateral axis ALA of the chip 1100. The support substrate 1102 can, for example, be a mica sheet. The imaging surface 1130 of the mica sheet can be atomically flat in order to facilitate analysis of the target molecule via atomic force microscopy. Being atomically flat, the imaging surface 1130 can have a deviation that is less than 0.5 nanometers across any 10 microns of length. Said another way, a local roughness of the surface, as measured by the standard deviation of the surface height profile, is less than 33% (e.g., less than 25%) a median height of the objects on the imaging surface (e.g., a target molecule such as DNA, RNA, or other similar molecule). The local roughness can be measured in a region surrounding the molecule, extending radially outward in a range of between 5 and 10 times an average width of the object. Accordingly, any differences in the changes in the distance between the tip of the stylus and the imaging surface 1130 of the chip 1100 during the execution of the scan via the system 1000 can be attributed to a substance (e.g., the target molecule) on the imaging surface 1130. It is therefore desirable to maintain the imaging surface 1130 free from contamination (e.g., undesirable crystalline growth, particle adherence or the like) during both manufacture of the chip 1100 and the imaging process, which is facilitated by the systems and methods described herein.

[0054] The chip 1100 also includes at least one bounded sample segment 1110. The bounded sample segment 1110 is defined (e.g., surrounded) by a hydrophobic boundary 1120 on the imaging surface 1130. Although the bounded sample segment 1110 is shown as being completely and / or continuously surrounded by the hydrophobic boundary 1120, in other embodiments, any of the bounded sample segments described herein can be substantially, but only partially surrounded by the hydrophobic boundary. For example, in some embodiments, any of the bounded sample segments described herein can be defined by a hydrophobic boundary that has short discontinuities (e.g., recesses or absences) along the length of the hydrophobic boundary. Such discontinuities can make up less than 15% of the overall boundary length.

[0055] As depicted, the bounded sample segment 1110 includes an imaging region 1112 and a collection region 1114. As depicted in FIG. 6, the imaging region 1112 is configured to support the target molecule TM in an unperturbed state SUP. Being in an unperturbed state (i.e., a natural state), the target molecule TM can exhibit various curves, loops, crossings, or overlaps. Similarly stated, using the methods described herein, the chip 1 100 can be prepared for imaging with theAttorney Docket No. 1147-OOl-OlWO target molecule TM supported within the imaging region 1112 without any elongation, stretching or other manipulation of the target molecule TM. Thus, the prepared chip 1100 and the methods described herein are efficient and eliminate time consuming and potentially error-inducing operations of manipulating (i.e., straightening, stretching or elongating) the target molecule TM. The resulting chip (with the target molecule TM thereon in an unperturbed state) is compatible for imaging via atomic force microscopy using the systems and methods shown and described in U.S. provisional application docket no. 1147-002-00US, entitled “Systems and Methods for Analyzing Target Molecules,” filed on the same date herewith, the disclosure of which is incorporated herein in its entirety. For example, such imaging methods can accurately determine the length and / or other characteristics of the target molecule in the unperturbed state. As depicted in FIGS. 2, and 5-7, the imaging region 1112 is positioned to be in fluid communication with the collection region 1114. In some embodiments, the hydrophobic boundary 1120 is shaped to define the imaging region 1112 and the collection region 1 114. For example, the hydrophobic boundary 1 120 can include a tapered portion 1122 that defines the collection region 1114. Being thus tapered, the collection region 1114 functions as a waste chamber that can serve as a repository for contaminants removed from the imaging region 1112 during preparation of the chip 1100 for imaging / analysis.

[0056] As depicted in FIGS. 3 and 4, the chip 1100 includes an axis Az that is orthogonal to the plane defined by the longitudinal axis ALO and the lateral axis ALA. The axis Az can, for example, be a vertical axis on a condition that the imaging surface 1130 is in a horizontal orientation. As depicted in FIG. 3, in some embodiments, the hydrophobic boundary 1120 can have a height H that extends from the imaging surface 1130 along the axis Az. The height H of the hydrophobic boundary 1120 can, for example, extend from the imaging surface 1130 in a range of between 0.1 microns and 100 microns (e g. between 5 microns and 10 microns). In other embodiments, the height H of the hydrophobic boundary 1120 can extend from the imaging surface 1130 in a range of between 1 micron and 10 microns, between 1 micron and 5 microns, between 2 microns and 5 microns, and any suitable range therebetween. The height H can be minimized to preclude interference with (e g., a deflection of) the stylus and / or the probe of the measurement system 1000. Accordingly, in some embodiments, as depicted in FIG. 4, the hydrophobic boundary 1120 can be recessed into the imaging surface 1130. For example, the hydrophobic boundary 1120 can, in some embodiments, be an etched hydrophobic boundary having a depth DAttorney Docket No. 1147-OOl-OlWO extending between 5 microns and 10 microns into the imaging surface 1130. In other embodiments, the depth D of the hydrophobic boundary 1120 can extend into the imaging surface 1130 in a range of between 1 micron and 10 microns, between 1 micron and 5 microns, between 2 microns and 5 microns, and any suitable range therebetween.

[0057] As further depicted in FIG. 3 and 4, in some embodiments, a backing substrate 1104 can be coupled to or formed with the support substrate 1102 opposite the imaging surface 1130. The backing substrate 1104 can have a stiffness or rigidity that is greater than a stiffness or rigidity of the support substrate 1102. In some embodiments, the backing substrate 1104 can have a stiffness or rigidity that is between 1.5 and lO times greater than a stiffness or rigidity of the support substrate 1102. To facilitate maintaining the imaging surface 1130 as atomically flat, the backing substrate 1104 can, in some embodiments, have a surface deviation that is less than 0.5 microns across any 10 microns. For example, the mica sheet can be coupled (e.g., adhered) to a glass backing substrate 1104 (e.g., a microscope slide). In some embodiments, the backing substrate 1104 can be a plastic having a thickness sufficient to support the support substrate 1102 in a substantially rigid manner.

[0058] During scanning, a static electric charge can build on or about the imaging surface 1130. The accumulation of the static electric charge during the imaging can interfere with the analysis via the atomic force microscope. Accordingly, it may be desirable to mitigate or eliminate an accumulation of a static electric charge on or about the imaging surface 1130 during an imaging run. Therefore, in some embodiments, the chip 1100 can be formed to be electrically coupled to a ground such that the static electric charge can be dissipated. For example, the backing substrate 1104 can electrically couple the chip 1100 to a motorized stage assembly (e g., motorized stage assembly 4500 (FIG. 21)) and from there to an electrical ground of the power supply circuit of the system (e.g., atomic force microscope 4000). To that end, in some embodiments, the backing substrate 1104 can be formed from a conductive material that has a stiffness or rigidity that is greater than the stiffness or rigidity of the support substrate 1102. The electrically-conductive backing substrate 1104 can, in some embodiments, have a surface deviation that is less than 0.5 microns across any 10 microns. In embodiments wherein the backing substrate 1104 is an insulator, such as a glass backing substrate, the backing substrate 1104 can be coated with an electrically conductive paint to form an electrically-conductive backing substrate 1104.Attorney Docket No. 1147-OOl-OlWO

[0059] It should be appreciated that to mitigate or eliminate the static electric charge, electrically-conductive backing substrate 1104 can be positioned in electrical contact with the stage assembly. In some embodiments, a resistance between the electrically-conductive backing substrate 1104 and the electrical ground of the system can be less than 5 ohms. In some embodiments, the resistance can be in a range of 0.1 ohms to 1.0 ohm (e.g., 0.2 to 0.9 ohms, 0.5 to 0.9 ohms, or 0.2 to 0.5 ohms).

[0060] Being that during analysis via the atomic force microscope, it is desirable to determine the position of the imaging surface 1130 of the chip 1100 with respect to a probe (e.g., probe 4700 (FIG. 21)) of the atomic force microscope to within a few microns (e.g., less than 10 microns in an axis normal to the imaging surface 1130). This determination can be accomplished via optical means such as via the reflection of a laser spot focused on the imaging surface 1130. However, as a support substate 1102 can be thin (e.g., less than 0.5 millimeters) and substantially transparent, it is desirable to suppress the reflections from the bottom surface (i.e., the surface opposite the imaging surface on 30) of the support substrate 1102 and / or other material below the support substrate 1102 (e.g., an adhesive and / or the backing substrate 1104). Without the suppression, false reflections of the laser spot may be generated from locations below the imaging surface 1130, which can reduce the accuracy of the measurement. Deviations in the accuracy of the measurement can, for example, result in unintended contact between the probe in the imaging surface 1130. To that end, any false reflections can be suppressed, in some embodiments, by using a thicker support substrate 1102 (e.g., thicker mica) to move the bottom surface away from the imaging surface 1130 so that two reflections do not merge. However, in some embodiments, any false reflections can be suppressed on a relatively thin support substrate 1102 by adding an absorbative or antireflection coating on the bottom of the support substrate 1102 or by minimizing the change in index of refraction between the bottom of the support substrate 1102 and an underlying material (e.g., an adhesive and / or the backing substrate 1104), thereby allowing light to pass thorough with minimal reflection. In some embodiments, a bright diffuse background is useful for observing chips or cracks in the support substrate 1102, which thereby renders the use an absorbative or ‘black’ coating on the bottom surface suboptimal. Instead, an approximate index match can be achieved by bonding the support substrate 1102 to the backing substrate 1104 (e.g., glass or similar) using a transparent adhesive with a similar index of refraction as the support substrateAttorney Docket No. 1147-OOl-OlWO1102 and the backing substrate 1104. The bonding can, for example, be accomplished through the use of a relatively thick layer of index-matching adhesive or a sandwich of index-matching adhesive, and glass. This results in a small change in refractive index going along a support substrate-adhesive-backing substrate path, and results in a relatively weak reflection from the interfaces.

[0061] FIG. 5 is an enlarged view of a portion of the chip 1100 depicting an example bounded sample segment 1110. As depicted, in some embodiments, the bounded sample segment 1110 can have an irregular rounded shape with a tapered portion 1122 of the hydrophobic boundary 1120 defining the collection region 1114. However, in other embodiments, the plan form of the bounded sample segment 1110 can have other enclosed shapes. For example, as depicted in FIG. 7, the bounded sample segment 1110 can be generally elliptical. In some embodiments, the bounded sample segment 1110 can, for example, have a raindrop shape, such as depicted in FIGS. 12-16 with reference to the bounded sample segment 2110. By bounding the collection region 1114 with a tapered or narrowing shape, the contaminants from the sample preparation methods described herein can be directed towards and contained in a small region of the sample segment 1110. For example, in some embodiments, the area of the collection region 1114 is less than 30% of the area of the bounded sample segment 1110. In other embodiments, the areas of the collection region 1114 is between 5% and 30% of the area of the bounded sample segment 1110, between 10% and 25% of the area of the bounded sample segment 1110, between 10% and 20% of the area of the bounded sample segment 1110, and any ranges therebetween.

[0062] As depicted in FIGS. 5-7, the bounded sample segment 1110 has a first segment axis Asi and a second segment axis As2. The second segment axis As2 is orthogonal to the first segment axis Asi. The second segment axis As2 can, for example, be parallel to the lateral axis ALA of the chip 1100, while the first segment axis Asi extends parallel to the longitudinal axis ALO of the chip 1100. As depicted in FIGS. 5-7, the bounded sample segment 1110 is elongated about the second segment axis As2. In some embodiments, the second segment axis As2, can correspond to an axis of orientation extending from the imaging region 1112 to the collection region 1114.

[0063] FIGS. 8-11 are schematic diagrams of portions of a method of manufacturing the chip 1100 for analysis via atomic force microscopy. Broadly speaking, chip 1100 can be manufacturedAttorney Docket No. 1147-OOl-OlWO by exposing an atomically flat imaging surface 1130 of the support substrate 1102. The hydrophobic boundary 1120 can then be formed on the imaging surface 1130 to define at least one bounded sample segment 1110. Following the formation of the hydrophobic boundary 1120, the imaging surface 1130 (e.g., the chip 1100) can be sealed within a vapor impermeable container 1202 (FIG. 11).

[0064] As depicted in FIG. 8, to manufacture the chip 1100, the support substrate 1102 can be positioned in an environmental chamber 1204. The environmental chamber 1204 used during manufacturing is different than the environmental chamber 1600 (described above) that maintains an internal environment that is in a range of 1% to less than 10% of a relative humidity of the environment surrounding the environmental chamber 1600 during an analysis of the chip 1100 (with the target molecule TM thereon). Rather, the environmental chamber 1204 establishes a suitable environment for manufacturing the chip 1100 as described herein (e.g., prior to conveying a sample or completing any sample preparation steps). When positioned in the environmental chamber 1204, the chip 1100 can include the support substrate 1102, the backing substrate 1104, and an outermost layer 1106, which may have been previously exposed to environment conditions. The outermost layer 1106 can, for example, be an outermost layer of a mica substrate.

[0065] In some embodiments, the environmental chamber 1204 can establish a humidity level therein that is within a range of 0% to 10% (e.g., 1% to 5%, 1% to 8%, 6% to 8%, and 7% to 9%) prior to the introduction of the chip 1100. Establishing the humidity level within the specified range can limit the development of crystals or other contaminants on the imaging surface 1130 during the manufacturing of the chip 1100. In some embodiments, an inert (or noble) gas can be conveyed into the environmental chamber 1204 prior to the introduction of the chip to establish an inert environment therein. The establishment of an inert environment can limit the development of surface contamination on the imaging surface 1130 during the manufacturing process.

[0066] FIG. 9 depicts the exposure of the atomically flat imaging surface 1130 of the support substrate 1102 by the removal (or cleaving) of the outermost layer 1106 of the support substrate 1102. As shown, the removal of the outermost layer 1106 can be accomplished with the chip 1100 positioned within the environmental chamber 1204 such that the imaging surface 1130 is shielded from exposure to ambient conditions. In some embodiments, a removal member 1206 can beAttorney Docket No. 1147-OOl-OlWO coupled to (e.g., adhered to) the outermost layer 1106 (e.g., an outermost layer of a mica substrate). The removal member 1206 can include any suitable structure or mechanism for being adhered to the outermost layer 1106, such as an adhesive layer. With the removal member 1206 coupled thereto, the outermost layer 1106 can be separated from the remainder of the support substrate 1102 (which can have the backing substrate 1104 coupled thereto) via an upward motion and / or a rotational motion such as in the direction indicated by arrow Ai. The movement of the outermost layer 1106 away from the atomically flat imaging surface 1130 can expose the atomically flat imaging surface 1130 to the environmental conditions within the environmental chamber 1204 but not to the ambient environmental conditions surrounding the environmental chamber 1204.

[0067] As depicted in FIG. 10, following the exposure of the atomically flat imaging surface 1130, the hydrophobic boundary 1120 can be formed on the imaging surface 1130 to define at least one bounded sample segment 1110. In some embodiments, forming the hydrophobic boundary 1120 can include etching the imaging surface 1130 to produce a depth extending into the imaging surface 1130 in a range of between 5 microns and 10 microns. In other embodiments, the depth of the hydrophobic boundary 1120 can extend into the imaging surface 1130 in a range of between 1 micron and 10 microns, between 1 micron and 5 microns, between 2 microns and 5 microns, and any suitable range therebetween. In further embodiments, the hydrophobic boundary 1120 can be formed by depositing the hydrophobic boundary 1120 on the imaging surface 1130. The hydrophobic boundary 1120 can be formed with a height extending away from the imaging surface 1130 in a range of between 5 microns and 10 microns. In other embodiments, the height of the hydrophobic boundary 1120 can extend from the imaging surface 1130 in a range of between 1 micron and 10 microns, between 1 micron and 5 microns, between 2 microns and 5 microns, and any suitable range therebetween. In some embodiments, the hydrophobic boundary 1120 can be formed by depositing a hydrophobic ink on the imaging surface 1130. The hydrophobic ink can be of any suitable formulation that minimizes any diffusion or “bleeding” into the imaging region (which could negatively impact the measurements). For example, the hydrophobic ink can be deposited via deposition assembly 1208 (e.g., a stamp, a roller, a brush, a felt, or a rod). When configured as a stamp, the deposition assembly 1208 can be moved in the direction indicated by arrow A2. In some embodiments, the hydrophobic boundary 1120 can be formed by positioning a gasket member on the imaging surface 1130.Attorney Docket No. 1147-OOl-OlWO

[0068] As depicted in FIG. 11, the chip 1100 can be sealed within a vapor impermeable container 1202 prior to removal from the environmental chamber 1204. By sealing the chip 1100 within the vapor impermeable container 1202 following the formation of the hydrophobic boundary 1120, the isolation of the imaging surface 1130 from water vapor and / or carbon dioxide is maintained. Instead, packaging the chip 1100 within the vapor impermeable barrier within the environmental chamber 1204 limits or precludes the formation of precipitates that could otherwise form if the newly exposed atomically flat imaging surface 1130 were exposed to water vapor and / or carbon dioxide.

[0069] Due to the potentially undesirable effects of exposure to water vapor and / or carbon dioxide, in some embodiments the chip 1100 includes a sensor element 1108 coupled to the support substrate 1102 (see FIG. 4). The output of the sensor element 1108 can, for example, be indicative of a humidity exposure. For example, the sensor element can have a first indicia (e.g., color) on a condition that the exposure of the chip 1100 has been maintained below a threshold and a second indicia (e.g., color) on a condition that the chip 1100 has been exposed to water vapor and / or carbon dioxide in excess of the threshold.

[0070] FIGS. 12 and 13 depict a chip 2100 to support a sample (e.g., the target molecule) for analysis via atomic force microscopy according to some embodiments. The chip 2100 has a plurality 2140 of bounded sample segments 2110. The chip 2100 (with the target molecules adhered thereto) can be analyzed using the system 1000 and any of the systems and methods shown and described in U.S. provisional application docket no. 1147-002-00US, entitled “Systems and Methods for DNA Imaging,” fded on the same date herewith, the disclosure of which is incorporated herein in its entirety. Each bounded sample segment 2110 can include any of the elements and / or features described herein with reference to the bounded sample segment 1110. In some embodiments, the plurality 2140 of bounded sample segments 2110 includes between four and ninety-six noncontiguous (i.e., separate) bounded sample segments 2110. For example, as depicted in FIG. 12, the plurality 2140 of bounded sample segments 2110 can include sixteen bounded sample segments. Each bounded sample segment 2110 of the plurality 2140 of bounded sample segments 2110 is configured to receive a separate sample SS, which may contain a different target molecule (FIG. 14). Accordingly, the chip 2100 can be prepared for and multiple samples can be analyzed via atomic force microscopy without the necessity to use multiple different chipsAttorney Docket No. 1147-OOl-OlWO within the system 1000 or otherwise load or exchange between sample support chips during analysis. Said another way, the amount of time required to analyze the totality of the samples via atomic force microscopy is reduced by supporting multiple independent samples on a single chip (e.g., the chip 2100) in accordance with the methods described herein. In contrast, supporting only a single sample per mica sheet increases the amount of time required as the chip must be swapped between scans.

[0071] As depicted, the chip 2100 includes a support substrate 2102 that has an imaging surface 2130. The imaging surface 2130 can be on a plane defined by a longitudinal axis ALO and an orthogonal lateral axis ALA of the chip 2100. The support substrate 2102 can, for example, be a mica sheet. The imaging surface 2130 of the mica sheet can be atomically flat in order to facilitate analysis of the target molecule via atomic force microscopy. Being atomically flat, the imaging surface 2130 can have a deviation that is less than 0.5 nanometers across any 10 microns of length. Accordingly, any differences in the changes in the distance between the tip of the stylus and the imaging surface 2130 of the chip 2100 during the execution of the scan (e.g., via the system 1000) can be attributed to a substance (e.g., the target molecule) on the imaging surface 2130.

[0072] Each bounded sample segment 2110 of the plurality 2140 of bounded sample segments 2110 is defined (e.g., surrounded) by a hydrophobic boundary 2120 of a plurality of hydrophobic boundaries 2120, each of which can include any of the features and / or elements of the hydrophobic boundaries 1120. Each hydrophobic boundary 2120 can be noncontiguous with every other hydrophobic boundary 2120 of the chip 2100. However, in some embodiments, the portions of the plurality of hydrophobic boundaries 2120 can be interconnected while each bounded sample segment 2110 is maintained as separate from and / or fluidically isolated (within the plane defined by the longitudinal axis ALO and the orthogonal lateral axis ALA) from every other bounded sample segment 2110.

[0073] As depicted, the bounded sample segments 2110 each include an imaging region 2112 and a collection region 2114. Each imaging region 2112 is configured to support a separate target molecule TM (FIGS. 15 and 16) in an unperturbed state. Being in an unperturbed state (i.e., a natural state), the target molecule TM can exhibit various curves, loops, crossings, or overlaps. Similarly stated, using the methods described herein, the chip 2100 can be prepared for imagingAttorney Docket No. 1147-OOl-OlWO with the target molecule TM supported within each imaging region 2112 without any elongation, stretching or other manipulation of the target molecule TM. Thus, the prepared chip 2100 and the methods described herein are efficient and eliminate time consuming and potentially errorinducing operations of manipulating (i.e., straightening, stretching or elongating) the target molecule TM. The resulting chip (with the target molecule TM thereon in an unperturbed state) is compatible for imaging via atomic force microscopy using the systems and methods shown and described in U.S. provisional application docket no. 1147-002-00US, entitled “Systems and Methods for Imaging Target Molecules,” filed on the same date herewith, the disclosure of which is incorporated herein in its entirety. For example, such imaging methods can accurately determine the length and / or other characteristics of the target molecule in the unperturbed state. As depicted in FIGS. 2, and 5-7, the imaging region 2112 is positioned to be in fluid communication with the collection region 2114. In some embodiments, the hydrophobic boundary 2120 is shaped to define the imaging region 21 12 and the collection region 21 14. For example, the hydrophobic boundary 2120 can include a tapered portion 2122 that defines the collection region 2114. Being thus tapered, the collection region 2114 functions as a waste chamber that can serve as a repository for contaminants removed from the imaging region 2112 during preparation of the chip 2100 for imaging / analysis. Each of the bounded sample segments 2110 can, as depicted in FIGS. 12-16, have a raindrop shape, with the imaging region 2112 being within the rounded portion (e.g., head) of the raindrop shape and the collection region being within the tapered portion (i.e., tail) of the raindrop shape.

[0074] As depicted in FIG. 12-16, each bounded sample segment 2110 of the plurality 2140 of bounded sample segments 2110 can have the same orientation relative to the longitudinal axis ALO and the lateral axis ALA of the chip 2100. For example, as depicted in FIG. 12, the tapered portion 2122 the hydrophobic boundary 2120 of each bounded sample segment 2110 can be directed toward a first lateral edge 2132 (e.g., a first lateral side edge) of the imaging surface 2130. Similarly, the imaging region 2112 of each bounded sample segment 2110 is positioned between the collection region 2114 and a second lateral edge 2134 (e.g., a second lateral side edge) of the imaging surface 2130. The second lateral edge 2134 is opposite the first lateral edge 2132. As described in more detail below, being thus arranged, facilitates positioning the collection regionAttorney Docket No. 1147-OOl-OlWO condition that the chip 2100 is rotated about the longitudinal axis ALO. The relative positioning of the imaging regions 2112 and the collection regions 2114 facilitates the movement of contaminants from the imaging regions 2112 toward the collection regions 2114 in response to a flowing of a wash fluid across the imaging surface 2130.

[0075] As depicted in FIG. 13, which is an enlarged view of a portion of the imaging surface 2130 depicted in FIG. 12, each bounded sample segment 2110 of the plurality 2140 of bounded sample segment 2110 can, in some embodiments, define an orientation axis AOR that extends from the imaging region 2112 to the collection region 2114 of each bounded sample segment 2110. The orientation axis AOR can, for example, be parallel to the second segment axis As2 (see, e.g., FIG. 6). In some embodiments, each orientation axis AOR is parallel to every other orientation axis AOR defined by the bounded sample segments 2110 the plurality 2140 of bounded sample segments 2110. Accordingly, the positional relationship between the imaging region 2112 and the collection region 2114 of each bounded sample segment 2110 can be the same and can facilitate the preparation of the chip 2100 for analysis via atomic force microscopy.

[0076] In some embodiments, each bounded sample segment 2110 is positioned at a different longitudinal position LP along the imaging surface. Said another way, the orientation axis AOR for each bounded sample segment 2110 of the plurality 2140 of bounded sample segments 2110 can be at a different longitudinal position LP. Said yet another way, the vertex of the tapered portion 2122 of each of the bounded sample segments 2110 is at a different longitudinal position LP. This positioning can, for example, limit or preclude portions of the separate sample SS from one bounded sample segment 2110 from being washed or otherwise moved into a second bounded sample segment 2110, which advantageously reduces or eliminates a contamination potential during processing.

[0077] As depicted in FIGS. 12 and 13, in some embodiments, the imaging regions 2112 of a first portion 2142 of the plurality 2140 of bounded sample segments 2110 are positioned at different longitudinal positions LP along the imaging surface 2130. The imaging regions 2112 of the first portion 2142 can also be aligned at a first lateral location LAI along the imaging surface 2130. Similarly, the imaging regions 2112 of a second portion 2144 of the plurality 2140 of bounded sample segments 2110 are positioned at different longitudinal positions LP along theAttorney Docket No. 1147-OOl-OlWO imaging surface 2130. The imaging regions 2112 of the second portion 2144 can also be aligned at a second lateral location LA2 along the imaging surface 2130. The longitudinal positions LP of the second portion 2144 of the plurality 2140 of bounded sample segments 2110 are longitudinally displaced relative to the longitudinal positions LP of the first portion 2142. Said another way, the first portion 2142 of the plurality 2140 of bounded sample segments 2110 can be longitudinally offset (or staggered) from the second portion 2444. Said yet another way, each bounded sample segment 2110 of the first portion 2142 can be laterally aligned with a space between each of the bounded sample segments 2110 of the second portion 2144. This positioning can, for example, limit or preclude portions of the separate samples SS from the bounded sample segments 2110 of the first portion 2142 from being washed or otherwise moved into the bounded sample segments 2110 of the second portion 2144, which advantageously reduces or eliminates a contamination potential during the sample preparation and methods of processing of the chip 2100 for analysis via atomic force microscopy.

[0078] FIGS. 14-16 are schematic perspective views of portions of a method of preparing the chip 2100 to support a sample (e.g., a target molecule TM) for analysis via atomic force microscopy. Broadly speaking, to prepare the previously manufactured (such as described above with reference to FIGS. 8-11) chip 2100, a sample solution SAs is conveyed (e.g., dropped, deposited, or positioned) into a bounded sample segment 2110 surrounded by a hydrophobic boundary 2120 on the imaging surface 2130. The sample solution SAs includes a target molecule TM (e.g., a nucleic acid). The sample solution SAs can, for example, include the target molecule TM in suspension having the desired chemical constituency. For example, in some embodiments, the target molecule TM can be a nucleic acid in a buffer solution formulated to maintain a desired pH balance and to protect the nucleic acid from degradation and / or damage. The buffer solution can contain, for example, certain acids or salts. A dwell interval can then be executed to allow the target molecule to settle onto the imaging surface 2130. After the completion of the dwell interval, a wash fluid WF can be flowed across the imaging surface 2130. The wash solution can be any suitable solution to remove undesirable particles or contaminates from the target molecule (e.g., the nucleic acid), such as the salts or other constituents of the sample solution SAs. As described herein, the wash fluid can be deionized water or purified water.Attorney Docket No. 1147-OOl-OlWO

[0079] Following the washing, the imaging surface 2130 can be dried at a first temperature (e.g., an ambient temperature). The heating interval can then be executed to expose the imaging surface 2130 to a second temperature, which is greater than the first temperature, to remove a molecular layer of the wash fluid WF from the imaging surface 2130. Following the heating interval, the prepared chip 2100 can be positioned in an environmental chamber, such as the environmental chamber 1600, for imaging via atomic force microscopy. Positioning the chip 2100 in the environmental chamber limits or precludes crystalline growth on the imaging surface 2130.

[0080] During preparation, the chip 2100 can be positioned within and manipulated relative to a reference frame defined by a first axis Ax, a second axis AY, and a third axis Az. Each of the first axis Ax, the second axis AY, and the third axis Az are orthogonal to one another. The first axis Ax can be parallel to a longitudinal axis ALO of the chip 2100, while the second axis AY is parallel to the lateral axis ALA of the chip 2100.

[0081] In some embodiments, the chip 2100 can be removed from a vapor impermeable container (such as the vapor impermeable container 1202) upon the initiation of the preparation methods. For example, as depicted in FIG. 14, the chip 2100 can be removed from the vapor impermeable container and positioned on a plane defined by the first axis Ax and the second axis AY in a substantially horizontal orientation to receive a sample solution SAs. The sample solution SAs containing a target molecule TM can then be positioned within the imaging region 2112 of one of the bounded sample segments 2110 as a drop of liquid. Additional sample solutions SAs containing additional target molecules TM can then be positioned in additional imaging regions 2112 of at least a portion of the remaining bounded sample segments 2110. The sample solution SAs can, for example, be deposited within the imaging region 2112 via a pipette 2220 or other suitable sample transfer device configured to aspirate or withdraw the sample from a sample container and then deliver a desired amount of the sample to each bounded sample segment 2110.

[0082] Once each of the sample solutions SAs are positioned within the respective imaging regions 2112, the dwell interval is initiated. The target molecule TM is in a suspended state within the sample solution SAs in the bounded sample segment 2110 at the initiation of the dwell interval. During the execution of the dwell interval, the target molecule moves into contact with the imaging surface 2130 in the imaging region 2112 of the bounded sample segment 2110. The targetAttorney Docket No. 1147-OOl-OlWO molecule TM can, for example, move along the third axis Az (e.g., the target molecule can settle). The motion along the third axis Az. can, for example, be in response to a random diffusion in the liquid followed by short range (e.g., less than four nanometers) attractive molecule-surface interaction. In some embodiments, the settling can be facilitated by gravitational forces and / or centrifugation. The motion along the third axis Az can continue until the target molecule TM contacts the imaging surface 2130. The dwell interval can, for example, have a duration in a range of 5 minutes to 15 minutes (e.g., 10 minutes + / - 1 minute). In other embodiments, the dwell interval can a duration in a range of 2 minutes to 30 minutes, 5 minutes to 10 minutes, or 7 minutes to 12 minutes. In some embodiments minimizing the dwell interval (i.e., by maintaining the dwell interval to less than 30 minutes), can limit undesirable crystalline growth and / or formation of precipitate on the imaging surface 2130 that might otherwise result from the evaporation of the sample solution SAs under ambient conditions.

[0083] The target molecule TM is, as depicted in FIG. 15, electrostatically adhered to the imaging surface 2130 in the substantially unperturbed state SUP at the conclusion of the dwell interval. Being electrostatically adhered to the imaging surface 2130, the target molecule TM is in a substantially fixed position within the imaging region 2112. Said another way, following the dwell interval, subsequent preparation steps as described herein do not substantially move or otherwise disturb the target molecule TM. As described herein, the target molecule TM supported within the imaging region 2112 without any elongation, stretching or other manipulation of the target molecule TM. Thus, this method of preparing the chip 2100 is devoid of time consuming and potentially error-inducing operations of manipulating (i.e., straightening, stretching or elongating) the target molecule TM.

[0084] The duration of the dwell interval is such that the sample solution SAs remains in a liquid state at the completion of the dwell interval. In other words, the dwell interval has a duration that is sufficient to permit the electrostatic adherence between the target molecule and the imaging surface 2130 but is insufficient to permit the sample solution SAs to fully dry via evaporation. If, in contrast, the dwell interval was of such a magnitude that the sample solution SAs were permitted to substantially evaporate, undesirable precipitates could be formed within the imaging region 2112. As described herein, the presence of the precipitates would negatively affect the analysis of the target molecule via atomic force microscopy. To facilitate maintaining the sample solutionAttorney Docket No. 1147-OOl-OlWOSAs in a liquid state, the sample solution SAs can be maintained at a temperature that is within 10°C of the ambient dewpoint during the dwell interval. Accordingly, the sample solution can be maintained at the temperature for a duration of between 9 minutes and 11 minutes (or any other dwell interval as described herein).

[0085] Following the conclusion of the dwell interval, a portion of the remaining sample solution SAs is removed by flowing the wash fluid WF across the imaging surface 2130 as depicted in FIG. 15. The wash fluid WF can, for example, be deionized water. The flow of the wash fluid WF can have a wash direction that extends from the imaging region 2112 toward the collection region 2114. This wash direction facilitates the movement of contaminants (e.g., non-target molecules or particles) from the imaging region 2112 to the collection region 2114 via the wash fluid (e g., via entrainment). However, as the target molecule TM is electrostatically adhered to the imaging surface 2130 following the dwell interval, the target molecule TM is not perturbed or otherwise moved by the wash fluid WF.

[0086] As depicted in FIG. 15, in some embodiments the chip 2100 can be pivoted to position the collection region 2114 gravitationally below the imaging region 2112 prior to the introduction of the wash fluid WF. For example, the chip 2100 can be pivoted about the first axis Ax in the direction indicated by arrow A3 to an angle of between 30° and 80° from horizontal. As each of the bounded sample segments 2110 have the same orientation, rotating the chip 2100 about the first axis Ax in the direction indicated by arrow A3 positions the collection region 2114 of each bounded sample segment 2110 at a lower position along the third axis Az than the corresponding imaging regions 2114. The tilting of the chip 2100 facilitates the movement of contaminants from within the imaging regions 2112 to within the collection regions 2114. As depicted in FIG. 15, pivoting the chip 2100 as shown also positions the second portion 2144 of the plurality 2140 of bounded sample segments 2110 gravitationally below the first portion 2142. However, since, as described above, each bounded sample segment 2110 of the first portion 2142 is longitudinally offset from each bounded sample segment 2110 of the second portion 2144, the wash fluid WF passing through the bounded sample segments 2110 of the first portion 2142 does not interfere with or otherwise contaminate the bounded sample segments 2110 of the second portion 2144. For example, wash fluid introduced in the vicinity of the second lateral edge 2134 can flow through the imaging regions 2112 of the first portion 2142, from the collection regions 2114, and acrossAttorney Docket No. 1147-OOl-OlWO unoccupied portions of the imaging surface 2130 before leaving the imaging surface 2130 at the first lateral edge 2132. Similarly, wash fluid introduced in the vicinity of the second lateral edge 2134 can flow across unoccupied portions of the imaging surface 2130 before contacting the imaging regions 2112 of the second portion 2144. The combination of the tilt of the chip 2100 and the longitudinal positioning of the bounded sample segments 2110 precludes or limits a possibility of contaminating any of the imaging regions due to the flowing of the wash fluid WF across the imaging surface 2130.

[0087] In some embodiments, the wash fluid WF can be delivered to the imaging surface 2130 via a wash nozzle 2242 of a fluid delivery assembly 2240. In some embodiments, the wash nozzle can extend along the first axis Ax and have a length that corresponds to a length of the chip 2100. However, in some embodiments, the wash nozzle 2242 can have other suitable shapes. For example, the wash nozzle 2242 can be configured as a pipette that is moved along the first axis Ax to deliver the wash fluid to the imaging surface 2130.

[0088] In some embodiments, the wash nozzle 2242 can be configured to deliver the wash fluid WF at a volume that is proportional to a surface area of the imaging surface 2130. For example, the volume of the wash fluid WF flowed across the imaging surface 2130 can be proportional to the surface area of the imaging surface 2130 in a ratio of between 1 :0.04 and 1 :0.12. Delivering the wash fluid WF at volumes within this range ensures that a sufficient quantity of wash fluid WF is flowed across the imaging surface 2130 to move contaminants from the imaging regions 2112 and into the collection regions 2114 while precluding or minimizing any potential impacts on the unperturbed state SUP of the target molecule TM.

[0089] In some embodiments, the wash nozzle 2242 can be configured to deliver the wash fluid WF at a specified shear rate. For example, the wash fluid can be delivered at a shear rate of between 1 reciprocal second and 100 reciprocal seconds. The wash fluid can have a viscosity in a range of between 0.65 millipascal second and 1.6 millipascal seconds. Flowing a wash fluid WF having a viscosity within the specified range at a shear rate within the specified range can move contaminants from the imaging regions 2112 and into the collection regions 2114 while precluding or minimizing any potential impacts on the unperturbed state SUP of the target molecule TM.Attorney Docket No. 1147-OOl-OlWO

[0090] To preclude or minimize the possibility of contaminating the imaging regions 2112 via crystalline growth and / or other precipitate formation that would otherwise result from the evaporation of the wash fluid WF, the chip 2100 is dried after the washing via the wash fluid WF. The drying moves the wash fluid WF molecules from the imaging surface 2130 substantially via either the first lateral edge 2132 or the second lateral edge 2134. The drying of the imaging surface 2130 is accomplished at a first temperature to minimize the evaporation of the wash fluid WF. The first temperature can be within 5°C of the ambient temperature. This drying moves substantially all remaining wash fluid from the imaging surface 2130. In some embodiments, the drying can be accomplished via rotating the chip 2100 via a centrifuge.

[0091] As depicted in FIG. 16, in some embodiments, the imaging surface 2130 can be dried by flowing an air jet AJ across the imaging surface 2130. The air jet AJ can have a temperature that is within 5°C of an ambient temperature. The air jet AJ can be directed onto and across the imaging surface 2130 via an air nozzle 2250. In some embodiments, the air jet AJ can be directed across the imaging surface 2130 at an angle in a range of 15° to 45° relative to the lateral axis ALA of the imaging surface 2130 or the second axis AY. In some embodiments, the chip 2100 can be maintained at the same angle from horizontal that was established during the flowing of the wash fluid WF. The air jet AJ can be configured to have a lineal impact in a range of 0.01 kilograms per centimeter and 0.1 kilograms per centimeter. Delivering a jet of air having a lineal impact within the specified range at an angle within the specified range facilitates the drying of the imaging surface 2130 via the moving of the remaining wash fluid from the imaging surface 2130 while also precluding or minimizing any potential impacts on the unperturbed state SUP of the target molecule TM.

[0092] While the drying of the imaging surface 2130 moves substantially all the wash fluid WF from the imaging surface 2130, nevertheless, a molecular layer of wash fluid WF can remain within the imaging regions 2112. Accordingly, a heating interval can be executed following the drying of the imaging surface 2130. The heating interval can be executed at a temperature that is greater than the first temperature of the drying. For example, the heating interval can include heating the imaging surface 2130 at a temperature in a range of 115°C to 125°C. In some embodiments, the heating interval can have a duration of between 9 minutes and 11 minutes. In other embodiments, duration can be in a range of 2 minutes to 30 minutes, 5 minutes to 10 minutes,Attorney Docket No. 1147-OOl-OlWO or 7 minutes to 12 minutes. Removing the molecular layer of the wash fluid WF from the imaging regions 2112 can facilitate the analysis of the target molecule TM via atomic force microscopy by reducing or eliminating a friction force that would be developed between the molecular layer and the stylus during a scan of the target molecule TM as such a friction force can negatively affect the analysis.

[0093] As previously described, following the removal of the molecular layer of wash fluid WF via the heating interval, the prepared chip 2100 can be positioned in an environmental chamber, such as the environmental chamber 1600, for imaging via atomic force microscopy. However, prior to positioning the prepared chip 2100 in the environmental chamber, a humidity level within the environmental chamber can be established in a range configured to limit preclude crystalline growth on the imaging surface 2130. For example, the relative humidity within the environmental chamber can be within a range of not more than 10% (e.g., between 0.5% and 5%, between 2% and 5%, between 1% and 4%, between 5.5% and 9.5%, between 6% and 9%, in between 6% and 8%). The relative humidity of the internal environment can be maintained by any suitable mechanism, such as, for example, by conveying a dry gas or desiccant into the environmental chamber 1600. In some embodiments, the dry gas can be an inert gas. By minimizing the water content in the internal environment (i.e., by maintaining the relative humidity at a value of not more than 10%), the formation of deposits and / or the collection of contaminants on the chip 1100 can be minimized, thus improving the accuracy of the imaging performed by the system 1000.

[0094] The methods described above with reference to FIGS. 14-16, can be used to prepare the sample support chip 2100 for use with atomic force microscopy. The prepared chip 2100 can, as described herein, include an imaging region 2112 of a bounded sample segment 2110 defined on an imaging surface 2130, which is atomically flat, of a support substrate 2102. As a result of the methods described, the imaging region 2112 is substantially devoid of contaminants, and the target molecule TM is electrostatically bonded to the imaging surface 2130 in the imaging region 2112 in a substantially unperturbed state SUP.

[0095] In some embodiments, the target molecule TM, which can be a strand of DNA, in the unperturbed state SUP can occupy an area having a maximal length of less than 1 millimeter (e g.Attorney Docket No. 1147-OOl-OlWO less than 25 microns, 15 microns or less, and / or between 4.5 microns and 5.5 microns) and a maximal width of less than 1 millimeter (e.g. less than 25 microns, 15 microns or less, and / or between 4.5 microns and 5.5 microns). If the target molecule TM were perturbed, the target molecule TM would have a length that is at least 10% greater than the maximal length of the area occupied in the unperturbed state SUP. Accordingly, the target molecule TM could not be contained within the specified area in the absence of curves, loops, crossings, overlaps, or bends. Therefore, the target molecule TM in the unperturbed state SUP (e.g., a natural state) has at least one loop. In some embodiments, the target molecule TM in the substantially unperturbed state SUP has an elongation of 10% or less of a maximal length of the target molecule TM.

[0096] FIG. 17 is a perspective view of a chip preparation system 3200. The chip preparation system 3200 can be used to implement any of the methods or procedures described herein with reference to FIGS. 14-16 to prepare a chip 3100 (FIG. 18) for analysis via atomic force microscopy. As depicted, the chip preparation system 3200 can include a preparation chamber 3210. The preparation chamber 3210 is configured to minimize contamination or disruption potential during the preparation process. In some embodiments, the environment within the preparation chamber 3210 is substantially the same as the ambient environment.

[0097] The chip preparation system 3200 can include a gantry 3212. The gantry 3212 can be computer-controlled and configured to position portions of the chip preparation system 3200 during the preparation of the chip 3100. For example, at least one computer-controlled pipette 3220 can be supported by the gantry and the movable between various positions within the preparation chamber 3210 including an alignment with one of a plurality of trays 3222 and / or the chip 3100.

[0098] The computer-controlled pipette(s) 3220 can, in some embodiments, be used in the preparation of the sample solution described above. For example, the computer-controlled pipette(s) 3220 can aspirate an initial sample from a tray 3222. The initial sample can then be diluted, for example by a factor of 100, to produce the sample solution. By diluting the initial sample, the concentration of the target molecules can be controlled to ensure that a desired amount of the target molecules are within the imaging region (e.g., imaging region 2112). For example, if the sample solution SAs conveyed into the imaging region 2112 is too concentrated (i.e. containsAttorney Docket No. 1147-OOl-OlWO too many target molecules), the resulting imaging may produce results that are indistinguishable. In contrast, if the sample solution SAs conveyed into the imaging region 2112 is too diluted (i.e. contains too few target molecules), then multiple imaging scans may be required to produce accurate results. Additional imaging scans will result in long analysis times and less efficient processes. In some embodiments, the initial sample can be diluted by any other suitable factor, such as between 50 and 150, 75 and 125, 90 and 110, and about 100. The sample solution can, for example, have a concentration of between 25 pg / mL and 75 pg / mL, between 40 pg / mL and 60 pg / mL, or about 50 pg / mL. In some embodiments, the computer-controlled pipette(s) 3220 can also be used to introduce an ionic exchange material following dilution.

[0099] As further depicted in FIG. 17, the chip preparation system 3200 can include a fluid delivery assembly 3240. The fluid delivery assembly 3240 can be used to flow a wash fluid across the chip 3100 as described herein. The fluid delivery assembly 3040 can include a wash nozzle (not shown) that is fluidically coupled to a wash fluid reservoir 3244 positioned externally to the preparation chamber 3210. The wash nozzle can be supported by the gantry 3212 so as to be movable relative to the chip 2100 thereby facilitating the delivery of the wash fluid during the preparation of the chip 3100 for analysis via atomic force microscopy.

[0100] FIGS. 18-20 depicts portions of the chip preparation system 3200 with the preparation chamber 3210 removed for clarity. The chip preparation system 3200 can include a support assembly 3230 configured to support, secure, and position the chip 3100 during the preparation process. The support assembly 3230 can, for example, be configured to position the ship at an angle relative to horizontal in preparation for the flowing of the wash fluid and / or the air jet across the imaging surface of the chip. Accordingly, the support assembly 3230 can be configured to move the chip 3100 from a substantially horizontal position as depicted in FIG. 18 to an angled position as depicted in FIGS. 19 and 20.

[0101] In some embodiments, the chip preparation system 3200 can include an air nozzle 3250. The air nozzle 3250 can be coupled to an air supply (not shown), such as a pressurized bench air supply. The air nozzle 3250 can be positioned to direct and air jet across the chip 3100 to move wash fluid from the imaging surface of the chip. The air nozzle 3250 can be shaped to direct the air jet at a non-zero angle relative to the lateral axis of the chip.Attorney Docket No. 1147-OOl-OlWO

[0102] In some embodiments, the support assembly 3230 can include a heater 3260. The heater 3260 can be positioned to heat the chip 3100 during a heating interval as described herein. For example, the heater 3260 can be used to heat the chip to a temperature in a range of 60°C to 200°C (e.g., 115°C to 125°C) and maintain the temperature for a duration of between 5 minutes and 30 minutes (e.g., between 9 minutes and 11 minutes).

[0103] FIG. 21 is a perspective view of a representative atomic force microscope 4000 for analyzing a target molecule supported by a sample support chip 4100. The atomic force microscope 4000 can include a displacement sensor 4400 (e g., an interferometer) and a probe 4700. The interferometer 4400 can be configured to detect a change in the position of the probe 4700 during a scan. The sample support chip 4100 can be supported by a motorized stage assembly 4500. The motorized stage assembly 4500 can be configured to move the sample support chip 4100 relative to the probe 4700 during the scan. During a scan, as a stylus (not shown) of the probe 4700 probe contacts the target molecule or feature on the surface of the chip, the displacement sensor 4400 outputs a signal corresponding to a change of position (e.g., a change in the vertical position) of the probe 4700. A coupled controller (not shown) can assemble an image of the target molecule based various changes in the position of the probe 4700 during the scan. The atomic force microscope 4000 can be included within any suitable system or can be similar to any of the atomic force microscope systems described in U.S. provisional application docket no. 1147-002-00US, entitled “Systems and Methods for Analyzing Target Molecules,” filed on the same date herewith, the disclosure of which is incorporated herein in its entirety.

[0104] FIG. 22 is a flow chart of a method 20 of manufacturing a chip to support a sample for analysis via atomic force microscopy according to an embodiment. The method 20 can, for example be used to manufacture the chip 1100, the chip 2100, the chip 3100, and / or the chip 4100 as described herein.

[0105] In some embodiments, as depicted at 22, the method 20 includes exposing an atomically flat imaging surface of a support substrate. As depicted at 24, the method 20 includes forming a hydrophobic boundary on the imaging surface to define a bounded sample segment. Additionally, as depicted at 26, the method 20 includes sealing the imaging surface within a vapor impermeable container following the forming of the hydrophobic boundary.Attorney Docket No. 1147-OOl-OlWO

[0106] FIG. 23 is a flow chart of a method 30 for preparing a chip to support a sample for analysis via atomic force microscopy according to an embodiment. The method 30 can, for example be used to manufacture the chip 1100, the chip 2100, the chip 3100, and / or the chip 4100 as described herein.

[0107] In some embodiments, as depicted at 31, the method 30 includes conveying a sample solution into a bounded sample segment on an imaging surface of the chip. The sample solution includes a target molecule, and the bounded sample segment is surrounded by a hydrophobic boundary. As depicted at 32, the method 30 includes executing a dwell interval following the positioning of the sample solution into the bounded sample segment. As depicted at 33, the method 30 includes flowing a wash fluid across the imaging surface after executing the dwell interval. As depicted at 34, the method 30 includes drying the imaging surface at a first temperature, after flowing the wash fluid. As depicted at 35, the method 30 includes executing a heating interval at a second temperature to remove a molecular layer of the wash fluid from the imaging surface after the drying. As depicted at 36, the method 30 includes positioning the chip in an environmental chamber.

[0108] Although various embodiments have been described as having particular features and / or combinations of components, other embodiments are possible having a combination of any features and / or components from any of embodiments as discussed above.

[0109] Although the collection regions (e.g., the collection region 1114) are shown and described herein as being formed by the same hydrophobic boundary that forms the imaging region (e g., the imaging region 1112), in other embodiments, the collection region 1114 need not be defined by the hydrophobic boundary 1120. Similarly stated, in some embodiments, a chip can include an imaging region defined by a hydrophobic boundary having an opening (e.g., along the broken lines shown in FIGS. 2 and 5) into a collection region. In such embodiments, the collection region can be defined (or surrounded by) a separate hydrophobic boundary of the types shown and described herein. In other embodiments, the collection region need not be defined or surrounded by a hydrophobic boundary.

Claims

Attorney Docket No. 1147-OOl-OlWOWhat is claimed is:

1. A chip to support a sample for analysis via atomic force microscopy, the chip comprising: a support substrate having an imaging surface; and a bounded sample segment defined by a hydrophobic boundary on the imaging surface, the bounded sample segment including an imaging region and a collection region.

2. The chip of claim 1, wherein: the bounded sample segment has a first segment axis and a second segment axis orthogonal to the first segment axis; and the bounded sample segment is elongated about the second segment axis.

3. The chip of claim 2, wherein: the first segment axis is parallel to a longitudinal axis of the chip; and the second segment axis is parallel to a lateral axis of the chip.

4. The chip of claim 1, wherein: the bounded sample segment is one bounded sample segment of a plurality of bounded sample segments defined by a plurality of hydrophobic boundaries; and each bounded sample segment of the plurality of bounded sample segments is configured to receive a separate sample.

5. The chip of claim 4, wherein: the collection region of each bounded sample segment of the plurality of bounded sample segments is defined by a tapered portion of the corresponding hydrophobic boundary; the tapered portion of the hydrophobic boundary of each bounded sample segment of the plurality of bounded sample segments is directed toward a first lateral edge of the imaging surface; andAttorney Docket No. 1147-OOl-OlWO the imaging region of each bounded sample segment of the plurality of bounded sample segments is positioned between the collection region and a second lateral edge of the imaging surface opposite the first lateral edge.

6. The chip of claim 4, wherein: each bounded sample segment of the plurality of bounded sample segments defines an orientation axis extending from the imaging region to the collection region of each bounded sample segment of the plurality of bounded sample segments; and each of the orientation axes is parallel to every other orientation axis defined by the bounded sample segments of the plurality of bounded sample segments.

7. The chip of claim 4, wherein: each bounded sample segment of the plurality of bounded sample segments is positioned at a different longitudinal position along the imaging surface.

8. The chip of claim 4, wherein: the imaging regions of a first portion of the plurality of bounded sample segments are positioned at different longitudinal positions along the imaging surface and are aligned at a first lateral location along the imaging surface; the imaging regions of a second portion of the plurality of bounded sample segments are positioned at different longitudinal positions along the imaging surface and are aligned at a second lateral location along the imaging surface; and the longitudinal positions of the second portion of the plurality of bounded sample segments are longitudinally displaced relative to the longitudinal positions of the first portion of the plurality of bounded sample segments.

9. The chip of claim 1, wherein: the hydrophobic boundary has a height extending from the imaging surface that is in a range of between 5 microns and 10 microns.

10. The chip of claim 1, wherein:Attorney Docket No. 1147-OOl-OlWO the hydrophobic boundary is an etched hydrophobic boundary having a depth extending into the imaging surface in a range of between 5 microns and 10 microns.

11. The chip of claim 1, wherein: the support substrate is a mica substrate; and the imaging surface is atomically flat.

12. The chip of claim 1, further comprising: a backing substrate coupled to the support substrate opposite the imaging surface, the backing substrate having a stiffness that is greater than a stiffness of the support substrate.

13. The chip of claim 12, wherein: the backing substrate is electrically conductive.

14. The chip of claim 1, further comprising a sensor element coupled to the support substrate, an output of the sensor element being indicative of a humidity exposure.

15. A method of manufacturing a chip to support a sample for analysis via atomic force microscopy, the method comprising: exposing an atomically flat imaging surface of a support substrate; forming a hydrophobic boundary on the imaging surface to define a bounded sample segment; and sealing the imaging surface within a vapor impermeable container following the forming of the hydrophobic boundary.

16. The method of claim 15, further comprising: positioning the support substrate in an environmental chamber prior to exposing the atomically flat imaging surface.

17. The method of claim 16, further comprising:Attorney Docket No. 1147-OOl-OlWO before positioning the support substrate, establishing a relative humidity level within the environmental chamber in a range of 1% to 10% of a relative humidity surrounding the environmental chamber.

18. The method of claim 16, further comprising: conveying a noble gas into the environmental chamber prior to positioning the support substrate in the environmental chamber.

19. The method of claim 15, wherein: the support substrate is a mica substrate; and exposing the atomically flat imaging surface includes removing an outermost layer of the mica substrate.

20. The method of claim 15, wherein exposing the atomically flat imaging surface includes: adhering a removal member to an outermost layer of the support substrate; and separating the outermost layer from a remainder of the support substrate via the removal member to expose the atomically flat imaging surface.

21. The method of claim 15, wherein: forming the hydrophobic boundary includes defining an imaging region and a collection region of the bounded sample segment.

22. The method of claim 15, wherein: forming the hydrophobic boundary includes depositing the hydrophobic boundary on the imaging surface; and the hydrophobic boundary has a height extending away from the imaging surface in a range of between 5 microns and 10 microns.

23. The method of claim 22, wherein: depositing the hydrophobic boundary on the imaging surface includes depositing a hydrophobic ink on the imaging surface.Attorney Docket No. 1147-OOl-OlWO24. The method of claim 22, wherein forming the hydrophobic boundary includes etching the imaging surface to produce a depth extending into the imaging surface in a range of between 5 microns and 10 microns.

25. The method of claim 15, further comprising: coupling a backing substrate to the support substrate opposite the imaging surface, the backing substrate having a stiffness that is greater than a stiffness of the support substrate.

26. The method of claim 25, wherein: the backing substrate is electrically conductive.

27. A method of preparing a chip to support a sample for analysis via atomic force microscopy, the method comprising: conveying a sample solution into a bounded sample segment on an imaging surface of the chip, the sample solution including a target molecule, the bounded sample segment being surrounded by a hydrophobic boundary; executing a dwell interval following the conveying of the sample solution into the bounded sample segment; flowing, after executing the dwell interval, a wash fluid across the imaging surface; drying, after flowing the wash fluid, the imaging surface at a first temperature; executing, after the drying, a heating interval at a second temperature to remove a molecular layer of the wash fluid from the imaging surface; and positioning the chip in an environmental chamber.

28. The method of claim 27, wherein: the target molecule is electrostatically bonded to the imaging surface in a substantially unperturbed state at a conclusion of the dwell interval.

29. The method of claim 28, wherein,Attorney Docket No. 1147-OOl-OlWO the target molecule is in a suspended state within the sample solution in the bounded sample segment at an initiation of the dwell interval; and the target molecule moves into contact with the imaging surface in the bounded sample segment during the execution of the dwell interval.

30. The method of claim 27, wherein: executing the dwell interval includes maintaining the sample solution at a temperature that is within 10°C of an ambient dewpoint; and the sample solution is maintained at the temperature for a duration of between 5 minutes and 30 minutes.

31. The method of claim 27, further comprising: removing the chip from a vapor impermeable container prior to the conveying of the sample solution into the bounded sample segment.

32. The method of claim 27, wherein: positioning the chip in the environmental chamber limits a crystalline growth on the imaging surface.

33. The method of claim 27, wherein: conveying the sample solution into the bounded sample segment includes positioning the sample solution in an imaging region of the bounded sample segment; the target molecule is electrostatically bonded to the imaging surface within the imaging region in an unperturbed state at a conclusion of the dwell interval; and the bounded sample segment including a collection region in fluid communication with the imaging region.

34. The method of claim 33, wherein: flowing the wash fluid across the imaging surface includes flowing the wash fluid in a wash direction that extends from the imaging region toward the collection region to facilitate movement of a contaminant from the imaging region to the collection region via the wash fluid.Attorney Docket No. 1147-OOl-OlWO35. The method of claim 33, further comprising: pivoting the chip to position the collection region gravitationally below the imaging region.

36. The method of claim 27, wherein: the hydrophobic boundary is one hydrophobic boundary of a plurality of hydrophobic boundaries; the bounded sample segment is one bounded sample segment of a plurality of bounded sample segments; defined on the imaging surface of the chip; each bounded sample segment of the plurality of bounded sample segments is fluidically isolated from every other bounded sample segment via a corresponding hydrophobic boundary of the plurality of hydrophobic boundaries; and conveying the sample solution includes conveying a different sample solution having a different target molecule into each bounded sample segment of the plurality of bounded sample segments.

37. The method of claim 27, wherein: a volume of the wash fluid flowed across the imaging surface is proportional to a surface area of the imaging surface in a ratio of between 1 :0.04 and 1 :0.12.

38. The method of claim 27, wherein: wherein flowing the wash fluid includes flowing the wash fluid at a shear rate of between1 reciprocal second and 100 reciprocal seconds; and the wash fluid has a viscosity in a range of between 0.65 millipascal second and 1.6 millipascal seconds.

39. The method of claim 27, wherein: drying the imaging surface includes flowing an air jet across the imaging surface; and the air jet has a temperature that is within 5°C of an ambient temperature.Attorney Docket No. 1147-OOl-OlWO40. The method of claim 39, wherein: the air jet is directed across the imaging surface at an angle in a range of 15 degrees to 45 degrees relative to a lateral axis of the imaging surface; and the air jet is configured to have a lineal impact in a range of 0.01 kilograms per centimeter and 0.1 kilograms per centimeter.

41. The method of claim 27, wherein: drying the imaging surface includes rotating the chip via a centrifuge.

42. The method of claim 27, wherein: executing the heating interval includes heating the imaging surface at a temperature in a range of 60°C to 200°C; and the heating interval has a duration of between 1 minutes and 20 minutes.

43. The method of claim 27, further comprising: establishing a humidity level within the environmental chamber in a range of 0% to 8% prior to positioning the chip in the environmental chamber.

44. A sample support chip for use with atomic force microscopy, the chip comprising: an imaging region of a bounded sample segment defined on an imaging surface of a support substrate, the imaging surface being atomically flat, the imaging region being substantially devoid of contaminants; and a target molecule electrostatically bonded to the imaging surface in the imaging region, the target molecule being in a substantially unperturbed state.

45. The chip of claim 44, wherein: the target molecule is a strand of DNA or RNA.

46. The chip of claim 44, wherein: the target molecule in the unperturbed state occupies an area having a maximal length of 1 millimeter and a maximal width of 1 millimeter; andAttorney Docket No. 1147-OOl-OlWO on a condition that the target molecule is perturbed, the target molecule has a length that is at least ten percent greater than the maximal length of the area occupied in the unperturbed state.

47. The chip of claim 44, wherein: the target molecule has a plurality of loops in the unperturbed state.

48. The chip of claim 44, wherein: the target molecule in the substantially unperturbed state has an elongation of 10% or less of a maximal length of the target molecule.

49. The chip of claim 44, wherein: the imaging region is one imaging region of a plurality of imaging regions on the imaging surface of the support substrate; the target molecule is one target molecule of a plurality of different target molecules; and each different target molecule is electrostatically bonded in an unperturbed state to the imaging surface in a different imaging region of the plurality of imaging regions.

Images