Systems and methods for analyzing target molecules

Abstract

System and methods for analyzing target molecules via atomic force microscopy are provided. Accordingly, a chip supporting the target molecule is positioned in an environmental chamber of an imaging system. A stylus of the imaging system is positioned in contact with one of the target molecule or an imaging surface of the chip. A scan of a capture region is then executed via the stylus. The capture region has a first length along a first axis and a second length along a second axis, with the first length having a ratio relative to the second length in a range of 1:4 to 1:4100. A feature of the target molecule is determined based on a displacement of the stylus during the scan.

Description

Attorney Docket No. 1147-002-01WOSYSTEMS AND METHODS FOR ANALYZING TARGET MOLECULESCross-Reference to Related Applications

[0001] This application claims priority to and the filing date benefit of U.S. Provisional Patent Application No. 63 / 715,217, entitled “Systems and Methods for Analyzing Target Molecules,” filed November I, 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 the high-speed analysis of 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 target molecule. Such procedures can be time consuming and require additional procedures (which can increase the likelihood of contamination or error).Attorney Docket No. 1147-002-01WO

[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] Some known systems use forms of microscopy for genomic analysis. For example, optical microscopy (i.e., light microscopy) can be used to determine the length of prepared (e.g., fluorescently stained) DNA fragments. While this approach can have advantages over the approaches that use a porous matrix, optical microscopy lacks sufficient resolution to measure a full range of potential target molecules. For example, the resolution of such a system can be diffraction limited at about 200 nanometers. Accordingly, such systems may be unsuited to measure target molecules that have approximately 600 or fewer base pairs. Alternatively, electron microscopy can be used for visualizing the structure of target molecules. However, electron microscopy requires significant sample preparation and complex imaging operations. Accordingly, the analysis of the target molecule via electron microscopy cannot be achieved at a desirable throughput rate.

[0007] Atomic force microscopy (AFM) is an additional form of microscopy that can be used to image the target molecules, such as DNA. AFM systems measure the 3D surface topology of samples on a sub-nanometer scale. Known AFM systems use optical beam defelection to detect changes in the height of a probe (e.g. a stylus) to generate an image of a scan area. Some known AFM systems can resolve single atoms (0.2 nanometer resolution), though resolutions in a range of 2 nanometers to 20 nanometers are more common. To achieve such a high-resolution, AFM systems are configured to minimize potential sources of vibration. For example, in some known systems, the scan area is moved at a slow speed relative to the probe via a pair of piezoelectric motors to minimize distortion of the image resulting from vibrations. This, in turn, limits the size of the area that can be scanned. The slow speed and limited scan have an undesirable negative impact on the throughput of the analysis via known AFM systems.

[0008] Accordingly, there exists a need in the art for improved systems and methods for analyzing target molecules, such as in DNA sizing.Attorney Docket No. 1147-002-01WOSummary

[0009] 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.

[0010] In some embodiments, the present disclosure is directed to a method of analyzing a target molecule via atomic force microscopy. The method can include positioning a chip in an environmental chamber of an imaging system. The chip supporting the target molecule within an imaging region of a bounded sample segment of the chip. The method can include causing the positioning of a contact portion of a stylus of the imaging system in contact with one of the target molecule or an imaging surface within the bounded sample segment of the chip. The method can also include causing the execution of a scan of a capture region via the stylus. The capture region has a first length along a first axis and a second length along a second axis orthogonal to and coplanar with the first axis. The first length has a ratio relative to the second length in a range of 1 :2 to 1 :4100. Additionally, the method can include determining a feature of the target molecule of the sample based on a displacement of the stylus along a third axis orthogonal to the first axis and the second axis.

[0011] In some embodiments, the present disclosure is directed to an apparatus for analyzing a target molecule via atomic force microscopy. The apparatus includes an environmental chamber and a stage assembly positioned within the environmental chamber. The stage assembly is configured to support a chip supporting a target molecule. The apparatus also includes a probe assembly positioned within the environmental chamber. The probe assembly includes a stylus positioned to contact the target molecule. Additionally, the apparatus includes a displacement detector that has a lens assembly positioned at least partially within the environmental chamber. The displacement detector is configured to output an indication of a position of the stylus. Further, the apparatus includes a controller positioned outside the environmental chamber and operablyAttorney Docket No. 1147-002-01WO coupled to the stage assembly and the displacement detector. The controller is configured to analyze the target molecule based at least in part on the output of the displacement detector.

[0012] In some embodiments, the present disclosure is directed to an apparatus for analyzing a target molecule via atomic force microscopy. The apparatus includes a probe assembly with a stylus. The stylus is configured to move in response to contact with a target molecule of a sample. The apparatus also includes a displacement detector optically coupled to the stylus and configured to output an indication of a position of the stylus. Additionally, the apparatus includes a stage assembly configured to position a chip supporting the target molecule. The stage assembly includes a set of motors to move the chip relative to the stylus. Further, the apparatus includes a controller operably coupled to the stage assembly and the displacement detector. The controller is configured to perform a set of operations. The set of operations includes positioning a contact portion of the stylus in contact with an imaging surface of the chip to establish a reference position for the stylus. The set of operations also includes moving the chip relative to the stylus to execute a scan of a capture region via the stylus. The capture region has a first length along a first axis and a second length along a second axis orthogonal to and coplanar with the first axis. The first length having a ratio relative to the second length in a range of 1 :4 to 1 :4100. The set of operations additionally includes determining a magnitude of a movement of the stylus along a third axis relative to the reference position based on an output of the displacement detector on a condition that the stylus is in contact with the target molecule. The third axis is orthogonal to the first axis and the second axis. Further, the set of operations includes determining a feature of the target molecule of the sample based on the magnitude of the movement.

[0013] In some embodiments, the present disclosure is directed to an apparatus that includes a first stage assembly including a first base member, a first slider member movably coupled to the first base member via a first lead screw, and a first motor coupled to the first lead screw. The first slider member is movable relative to the first base member along a first axis. The apparatus also includes a second stage assembly including a second base member coupled to the first slider member, a second slider member movably coupled to the second base member via a second lead screw, and a second motor coupled to the second lead screw. The second slider member is movable relative to the second base member along a second axis orthogonal to the first axis. The second motor is the only motor positioned to drive a movement along the second axis. Additionally, the apparatusAttorney Docket No. 1147-002-01WO includes a third stage assembly including a third base member, a third slider member movably coupled to the third base member via a piezoelectric motor. The third slider member is movable relative to the third base member along the first axis. Further, the apparatus includes a fourth stage assembly coupled between the second slider member and the third base member. The fourth stage assembly includes a fourth motor positioned to move the fourth stage assembly along a third axis orthogonal to the first axis and to the second axis. The apparatus also includes a chip support assembly coupled to the third slider member and configured to receive a chip supporting a target molecule.

[0014] 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

[0015] FIG. l is a schematic view of a system for imaging a target molecule according to an embodiment.

[0016] FIG. 2 is a schematic side view of the system of FIG. 1 depicting the stylus in contact with an imaging surface of the chip that is in a first position.

[0017] FIG. 3 is a schematic side view of the system of FIG. 1 depicting the stylus in contact with a target molecule following the movement of the chip from the first position of FIG. 2 to a second position.

[0018] FIG. 4 is a top view of a portion of the chip of FIG. 1 depicting a scan of a capture region.

[0019] FIG. 5 is a perspective view of a system for imaging a target molecule according to an embodiment.

[0020] FIG. 6 is a perspective view of the environmental chamber of the system of FIG. 5.

[0021] FIG. 7 is a perspective view of the system of FIG. 5 with the environmental chamber removed for clarity.Attorney Docket No. 1147-002-01WO

[0022] FIG. 8 is a perspective view of a displacement detector of the system of FIG. 5.

[0023] FIG. 9 is a perspective view of a portion of the system of FIG. 5 with the environmental chamber removed for clarity.

[0024] FIG. 10 is a perspective view of the probe assembly of the system of FIG. 5.

[0025] FIG. 11 is a perspective view of a stage assembly of the system of FIG. 5.

[0026] FIG. 12 is a partially exploded view of the stage assembly of FIG. 11.

[0027] FIG. 13 is a perspective view of a first stage assembly of the stage assembly of FIG. 11.

[0028] FIG. 14 is a perspective view of the second stage assembly of the stage assembly ofFIG. 11.

[0029] FIG. 15 is a partially exploded view of the second stage assembly of FIG. 14.

[0030] FIG. 16 is a perspective view of the second stage assembly of FIG. 14 with a second slider member depicted as transparent for clarity.

[0031] FIG. 17 is an exploded view of a second base member of the second stage assembly of FIG. 14.

[0032] FIG. 18 is an inverted exploded view of the second slider member of the second stage assembly of FIG. 14.

[0033] FIG. 19 is a schematic view of a spherical sector in contact with an interface block of the second stage assembly of FIG. 14.

[0034] FIG. 20 is an exploded view of a fourth stage assembly of the stage assembly of FIG. 11.

[0035] FIG. 21 is a perspective view of the fourth stage assembly of FIG. 20 coupled to the second slider member of the second stage of FIG. 14.Attorney Docket No. 1147-002-01WO

[0036] FIG. 22A is a perspective view of a third stage assembly of the stage assembly of FIG. 11.

[0037] FIG. 22B is a perspective view of the third stage assembly of FIG. 22A with a third slider member removed for clarity.

[0038] FIG. 23 is a perspective view of a chip support assembly of the stage assembly of FIG. 11.

[0039] FIG. 24 is a schematic view of a chip for use with the system of FIG. 5 depicting the chip in a first position and in a second position corresponding to range of motion limitations of the stage assembly of FIG. 11.

[0040] FIG. 25 is a schematic view of a chip configured to support a target molecule for analysis via the system of FIG. 5.

[0041] FIG. 26 is a schematic view of a controller of the system of FIG. 5.

[0042] FIG. 27 is a flow chart of a method of analyzing a target molecule via atomic force microscopy according to an embodiment.

[0043] FIG. 28 is a flow chart of a set of controller-implemented operations for analyzing a target molecule via atomic force microscopy according to an embodiment.Detailed Description

[0044] Generally, the present disclosure is directed to the analysis of a target molecule via atomic force microscopy. The target molecule can, for example, be a DNA molecule, an RNA molecule, a protein-DNA complex, a polymer, a nanoscale fiber structure (e.g., a nanotube), or a thin film (e.g., a film having a thickness that is less than 10 nanometers). The systems and methods described herein facilitate the rapid measurement of multiple target molecules of various lengths within a short time frame that supports biomolecular processing workflows. For example, the systems and methods can facilitate the measurement of a collection of large target molecules (e.g., up to 500,000 base pairs) and small molecules (e.g., having at least 50 base pairs) during a single measurement operation, with both the large molecules and the small molecules being measuredAttorney Docket No. 1147-002-01WO with the desired resolution. Similarly stated the systems and methods described herein have a very wide dynamic range (i.e., turn down ratio) for measurement of target molecules. Accordingly, during the measurement operation, up to 2000 target molecules of various sizes can be measured per minute. Each, or portions, of the target molecules analyzed during the measurement operation can be from different samples. Therefore, multiple biomolecular processing workflows can be rapidly supported via the single measurement operation.

[0045] As described herein, the target molecule(s) to be analyzed are supported by a chip, which is, in turn, supported by a stage assembly. The stage assembly moves the chip relative to a stylus of a probe assembly to execute a scan of a capture region. The system uses an optical system, such as an interferometer, to measure changes in the vertical position (e.g., a deflection) of the stylus resulting from contact with the target molecule(s). A controller of the system can then determine a feature (e.g., a length) of the target molecule based on the magnitude of the changes in the vertical position of the stylus. Said another way, the controller can assemble an image of the target molecule based on the magnitudes of the deflections of the stylus.

[0046] The systems and methods described herein include various features, processes, steps, and / or elements that alone or in combination increase the throughput of the analysis compared to other known systems. For example, a system that is optimized for determining the length of a target molecule can implement certain operations that increase the speed of the analysis at the expense of resulting in a decreased resolution of the image of the target molecule. By way of illustration, systems that emphasize the resolution of the image can have a scan rate (e.g., the rate at which the chip is moved relative to the stylus) with a relatively low frequency. While the low frequency can facilitate increased resolution, it has a negative effect on the throughput of the system (i.e., the throughput is slower than desired). Conversely, increasing the scan rate to improve the throughput has conventionally been understood to increase the amount of vibration due to the stylus being attracted to (e.g., “sticking” to) the surface of the chip, the sample, and / or residue from the chip preparation. Insofar as the image produced via an AFM system is based on the displacement of the stylus, vibrations can distort or otherwise negatively affect the analysis of the target molecule. This is one factor in the conventionally understood trade-off between high image resolution and high throughput. However, it has been discovered that in the embodiments described herein, this increasing vibratory effect can be halted and even reversed at higher scanAttorney Docket No. 1147-002-01WO rates. In other words, above a certain scan rate, the tendency of the stylus to “stick” can be overcome and the stylus can “glide” across the chip in a known vibratory fashion. Accordingly, in the systems and methods described herein a lower limit of the scan rate can be established at 400 Hz to reduce the interference from the vibration. Said another way, it has been discovered that for scan rates that would otherwise increase the throughput of the system but are below 400 Hz, the analysis of the target molecule(s) is negatively affected by vibration, but at scan rates above 400 Hz the negative effects are minimized or eliminated. While the vibratory effect due to the attraction of the stylus can be reduced by increasing the scan rate, doing so can increase the vibratory effects induced by the mechanical structure supporting the chip and / or the stylus. To that end, in the systems and methods described herein an upper scan rate limit can be established at 5 kHz. Accordingly, to optimize the throughput of the system, the scan rate can have a frequency that is in a range of 400 Hz to 5 kHz (e.g., a range of 500 Hz to 1 kHz) so that the interference from vibration is below an acceptable limit when measuring the target molecule(s). The embodiments described herein include scan rates within this range, which present an acceptable compromise between image resolution and throughput but may not otherwise be acceptable a system optimized to maximize the image resolution. Similarly stated, the embodiments described herein operate in a manner and within a scan frequency that allows for both an image resolution that is sufficient to support the measurement of the target molecule and fast throughput times.

[0047] Maximizing the sub-nanometer scale resolution of AFM systems requires nano-positioning systems that control the relative positions of the chip or the stylus during a scan while minimizing the vibratory impact on the scan. For moving the chip relative to the stylus, the nano-positioning can conventionally be achieved via a stage assembly that includes one piezoelectric motor for each scan axis. Due to the limited travel of piezoelectric motors, a conventional stage assembly can include an additional rotary motor configured to move portions of the stage assembly along each axis to position the chip over a larger range of travel prior to the execution of the scanning via the piezoelectric motors. However, the limited travel of piezoelectric motors used to control the scan along both axes limits the area of the capture region. For example, the capture region for systems employing two piezoelectric motors can be a square that is 10 microns on a side. The limited area of the capture region can increase the amount of time required to analyze the target molecule and / or the maximal size of the target molecule that can be analyzed.Attorney Docket No. 1147-002-01WOTo achieve a higher throughput, in some embodiments, the system described herein can use the combination of a piezoelectric motor and a rotary motor to control the fine and coarse positioning respectively along a first axis but only a single rotary motor coupled to the lead screw to control both the fine and coarse positioning along a second axis. The rotary motor / lead screw assembly has a greater travel than a piezoelectric motor and the corresponding stage is configured to minimize or eliminate the development of vibration during the movement along the second axis. Accordingly, having only the rotary motor / lead screw assembly controlling the positioning along the second axis facilitates the implementation of capture regions that have a greater area than is otherwise possible when the positioning is via the pair of piezoelectric motors. For example, the systems and methods described herein can produce a capture region having a rectangular area in which the long dimension is between 4 times and 100 times greater (e.g., 10 times greater) than the short dimension. The greater area of the capture region produced by the systems and methods described herein facilitates an increase of the rate at which the any target molecules supported by the chip can be analyzed and, by extension, the throughput of the system.

[0048] As the accurate determination of the deflection of the stylus is critical in atomic force microscopy, a calibration procedure is implemented at least as often as each time a chip is first positioned on a stage assembly. The calibration can determine the position of the stylus relative to the chip that is within 50 microns of an actual position in a plane defined by the first axis and the second axis (e.g., a horizontal plane) and within 10 microns along the third axis (e.g., a vertical axis) normal to the plane. Accordingly, as described herein, the chip is configured to support multiple samples such that only a single calibration procedure is required at the initiation of the measurement operation. Said another way, the number of samples (i.e., target molecules) that can be analyzed per calibration procedure is maximized. As multiple separate samples can be supported on a single chip, there is no need to swap chips between the scan of each sample, and, therefore, the number of calibration procedures is reduced to a single instance. The reduction of the number of calibration procedures required to analyze the desired samples increases the throughput of the system as compared to systems that support a single sample (or a limited number of samples) per chip.

[0049] 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 regionAttorney Docket No. 1147-002-01WO 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.

[0050] 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 in response to gravitational forces. 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 target molecule that may be required in conventional analysis methods, and thus provide an efficient, rapid method for preparing samples for analysis.

[0051] 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.

[0052] 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 surfaceAttorney Docket No. 1147-002-01WO 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 airjetthat 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.

[0053] 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.

[0054] 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 individual bounded sample segments can be spaced apart to align with the individual pipettes of a multi-will pipette apparatus. 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.

[0055] As used herein, the term “measurement operation” can encompass the time interval during which the target molecules supported by a single chip are analyzed (e.g., measured). The measurement operation can extend from the instant in which the chip is placed within an environmental chamber of the system to the completion of a scan of the final target molecule supported by the chip.Attorney Docket No. 1147-002-01WO

[0056] 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.

[0057] 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 includes 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).

[0058] 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 not precisely circular (e.g., one that is slightly oblong or is a many-sided polygon) is still encompassed by this description.

[0059] 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.Attorney Docket No. 1147-002-01WO

[0060] The terms “nucleic acid molecule” or “nucleic acid” may be used interchangeably herein, and may refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including known analogs or a combination thereof 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.

[0061] FIGS. 1-3 are schematic views 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. Accordingly, the system 1000 can be an atomic force microscope. Therefore, in some embodiments, the system 1000 can include an environmental chamber 1600 configured to receive a chip 1100 (e.g., a sample support chip). The system 1000, in some embodiments, can include a motorized stage assembly 1500 positioned within the environmental chamber 1600 and configured to support the chip 1100. As an atomic force microscope, the system 1000, in some embodiments, includes a probe assembly 1700, which includes a stylus 1710, and a displacement detector 1400 (e.g., an optical system such as an interferometer), positioned to detect a position of the stylus 1710 during the execution of a scan of a sample supported by the chip 1100 (i.e., a measurement operation). A controller (such as controller 2800 as depicted in FIG. 26) can analyze the target molecule TM based at least in part on the output of the displacement detector 1400. Said another way, the controller can assemble anAttorney Docket No. 1147-002-01WO image of the target molecule TM based on changes in a distance between a tip of the stylus and an imaging surface of the chip 1100 measured by the displacement detector 1400 during the execution of the scan.

[0062] In some embodiments, 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 5% (e.g., between 0% and 2%, between 0.5% and 5%, between 2% and 5%, and between 1% and 4%). 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 less 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 less than 10%). To minimize the internal volume of the environmental chamber 1600, in some embodiments, only a portion of the displacement detector 1400 is positioned within the environmental chamber 1600.

[0063] In some embodiments, the environmental chamber 1600 can be sized to have a footprint that is in a range of 4.5m2and 6.0m2. Being thus sized, the system 1000 can be supported by a lab bench or other similar structure. Additionally, the footprint being in the range of 4.5m2and 6.0m2minimizes the internal volume. This, in turn, minimizes the amount of time required to achieve the desired internal environment. For example, the environmental chamber 1600 can be sized such that the humidity of the internal environment can be reduced from ambient conditions to less than 10% of the humidity of ambient conditions in less than 1 minute.

[0064] In addition to supporting the chip 1100, the motorized stage assembly 1500 can, in some embodiments, be configured to move the chip 1100 relative to the stylus 1710. In some embodiments, this movement can correspond to the execution of a scan SC (FIG. 4). To move theAttorney Docket No. 1147-002-01WO chip 1100, the motorized stage assembly 1500 can be operably coupled to a controller (e.g., the controller 2800) and can include at least a first stage assembly 1510 and a second stage assembly 1520. The first stage assembly 1510 can include a first motor 1518, and the second stage assembly 1520 can include a second motor 1540. The first stage assembly 1510 can be configured to move the second stage assembly 1520 and the supported chip 1100 along a first axis Ai (e.g., an x-axis of the system 1000) which, in some embodiments, can be parallel to a longitudinal axis of the chip 1100. The second stage assembly 1520 can be configured to move the supported chip along a second axis A2 (e.g., a y-axis of the system 1000), which, can be parallel to a lateral axis of the chip 1100. The first axis Ai and the second axis A2 can be orthogonal to one another and can define a plane (e.g., a horizontal plane) corresponding to an imaging surface 1130 of the chip 1100 (see FIG. 4). It should be appreciated that in some embodiments, the movement of the chip 1100 by the motorized stage assembly 1500 can position the chip 1100 and then the stylus 1710 can be moved relative to the chip 1100 to execute the scan.

[0065] FIG. 2 depicts the chip 1100 in a first position. The first position can, for example, correspond to the position of the chip at the initiation of a scan SC (FIG. 4). With the chip 1100 in the first position, a contact portion 1712 of the stylus 1710 is in contact with the imaging surface 1130 of the chip 1100. As the imaging surface 1130 can be atomically flat, the contact portion 1712, and thus the stylus 1710 of the probe assembly 1700, can be considered to be at a “zero-position” along a third axis A3 (e.g., a vertical axis or a z-axis of the system 1000), which can be orthogonal to both the first axis Ai and the second axis A2. The position of the stylus 1710 can be determined via a beam 1402 transmitted and received by the displacement detector 1400. The beam 1402 can, for example, be a beam of light generated by a laser emitter and received by an optical detector (e.g., a photodiode) of the displacement detector 1400.

[0066] FIG. 3 depicts the chip 1100 following a movement at least along the first axis Ai by the motorized stage 1500 to a second position. The contact portion 1712 of the stylus 1710 is in contact with the target molecule TM on the condition that the chip 1100 is in the second position, such as during the execution of a scan SC. Being in contact with the target molecule TM, the distance between the contact portion 1712 and the imaging surface 1130 is greater than when the contact portion 1712 is in contact with the imaging surface 1130. The displacement detector 1400Attorney Docket No. 1147-002-01WO can measure this change in position via the beam 1402. The controller can then determine a feature of the target molecule TM based on the displacement of the stylus 1710 along the third axis A3.

[0067] FIG. 4 is a top view of a portion of the chip 1100 depicting a scan SC of a capture region 1750 executed to analyze the target molecule TM (e.g., during a measurement operation). To analyze a target molecule TM, the chip 1100 can be positioned in the environmental chamber 1600. In some embodiments, the chip 1100 can be positioned within the environmental chamber 1600 on the plane defined by the first axis Ai and the second axis A2. The plane can have an angle rotation about the first axis Ai that is in a range of zero degrees to less than two degrees.

[0068] The chip 1100 can include any of the features or elements of the chips described in U.S. provisional application docket no. 1147-001-00US, entitled “Devices for and Methods of Sample Preparation for Imaging Target Molecules,” filed on the same date herewith, the disclosure of which is incorporated herein in its entirety. For example, the chip 1100 can include a support substrate that has an imaging surface 1130. The imaging surface 1130 can be on a plane defined by a longitudinal axis and an orthogonal lateral axis of the chip 1100. The support substrate can a mica that is atomically flat in order to facilitate analysis of the target molecule TM 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 SC via the system 1000 can be attributed to a substance (e.g., the target molecule TM) 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 the imaging process, which is facilitated by the environmental chamber 1600.

[0069] The chip 1100 also includes at least one bounded sample segment 1110. The bounded sample segment 11 10 is defined (e.g., surrounded) by a hydrophobic boundary 1 120 on theAttorney Docket No. 1147-002-01WO 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.

[0070] As depicted, the bounded sample segment 1110 includes an imaging region 1112 and a collection region 1114, with the imaging region 1112 being configured to support the target molecule TM 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, the chip 1100 can be prepared for imaging with the target molecule TM being electrostatically coupled and supported within the imaging region 1112 without any elongation, stretching or other manipulation of the target molecule TM. Thus, the preparation of the chip 1100 is efficient and eliminates 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. In other words, the system 1000 can accurately determine the length and / or other characteristics of the target molecule in the unperturbed state.

[0071] With the chip 1100 positioned within the environmental chamber 1600, executing a measurement operation can include analyzing the target molecule TM within the imaging region 1112 of the bounded sample segment 1110. Analyzing the target molecule TM can include positioning the contact portion 1712 of the stylus 1710 in contact with the target molecule TM (as depicted in FIG. 3) or the imaging surface 1130 (as depicted in FIGS. 2 and 4) within the bounded sample segment 1110 of the chip 1100. The stylus 1710 can be positioned in contact with the target molecule TM or the imaging surface 1130 via a movement of the chip 1100 by the motorized stage assembly 1500, a movement of the stylus by the probe assembly 1700, or a combination thereof. For example, in some embodiments, the positioning can include moving the stylus 1710 and / or chip 1100 a first distance at a first adjustment speed and moving the stylus 1710 and / or the chip 1100 a second distance at a second adjustment speed that is less than the first adjustmentAttorney Docket No. 1147-002-01WG speed. In some embodiments, the positioning is controlled via the controller in response to a user input after positioning the chip 1100 in the environmental chamber 1600.

[0072] With the contact portion 1712 of the stylus 1710 in contact with the target molecule TM or the imaging surface 1130, analyzing the target molecule TM can include causing the execution of a scan SC of a capture region 1750 via the stylus 1710, such as depicted in FIG. 4. The contact portion 1712 can be maintained in contact with the target molecule TM or the imaging surface 1130 throughout the entirety of the scan SC of the capture region 1750.

[0073] In some embodiments, the capture region 1750 can have a rectilinear shape with one dimension that is greater than an orthogonal dimension. Accordingly, the capture region 1750 can have a first length Li along the first axis Ai and a second length L2 along the second axis A2, with the second length L2 being greater than the first length Li. For example, in some embodiments, the first length Li can have a ratio relative to the second length L2 in a range of 1 :2 to 1 :4100 (e.g., 1.4 to 1 : 100, 1.4 to 1 :10, or other suitable range) . In some embodiments, the first length Li along the first axis Ai can have a magnitude in a range of 2 microns to 12 microns. In some embodiments, the second length L2 along the second axis can have a magnitude in a range of 20 microns to 50 microns. In some embodiments, for example, the capture region 1750 can be a rectangle with a first length Li of 6 microns and a second length L2 of 48 microns. The use of a rectangular capture region 1750, which is enabled by the system 1000 (or the system 2000) as described herein, can facilitate the analysis of larger target molecules than is otherwise possible via a single square capture region. Additionally, the use of a single rectangular capture region 1750 can increase the speed of the analysis of the target molecule TM relative to an approach that uses a number of square capture regions to create a mosaic having the desired scan area.

[0074] In some embodiments, executing the scan SC of the capture region 1750 can, as depicted in FIG. 4, include executing a raster scan. The raster scan can, for example, be a sinusoidal raster scan. The raster scan can be implemented by maintaining the probe assembly 1700 in a fixed position while the motorized stage assembly 1500 moves the chip 1100 relative to the stylus 1710. In other words, the stylus 1710 can move along the third axis A3 in response to contact with a feature of the target molecule TM or other substance on the imaging surface 1130 but remains fixed relative to the first axis Ai and the second axis A2. The displacement of theAttorney Docket No. 1147-002-01WO stylus 1710 along the third axis A3 can be measured via the displacement detector 1400 and the output of the displacement detector 1400 can be used to determine a feature of the target molecule TM. As the system 1000 is configured to analyze the target molecule TM based on the displacement of the stylus 1710, the displacement detector 1400 is configured to measure only the position or orientation of the stylus 1710 and does not monitor a force applied along the third axis A3 (e.g., a Z-force). Said another way, the output of the displacement detector 1400 has an absence of indications regarding the force along the third axis A3. Said yet another way, the system 1000 can be employed without a necessity to frequently (e.g. more than once per five seconds) adjust the force along the third axis A3. Analyzing the target molecule TM without requiring an indication and / or the frequent adjustment of the force along the third axis A3 facilitates an increase in the speed of the scan SC.

[0075] As a raster scan, the scan SC can have an amplitude SCA extending along the first axis Ai. The amplitude SC can correspond to the first length Li of the capture region 1750. The raster scan can also have a duration SCD extending along the second axis A2. The duration SCD can correspond to the second length L2 of the capture region 1750 and can be greater than the amplitude SCA. In some embodiments, the amplitude SCA can have a ratio relative to the duration SCD in a range of 2:3 to 1 : 1025. For example, the amplitude SCA can have a magnitude in a range of 0.5 microns to 5.0 microns.

[0076] As depicted in FIG. 4, executing the scan SC of the capture region 1750 can include moving the chip 1100 relative to the stylus 1710 along the first axis Ai and along the second axis A2. A first movement Mi of the chip 1100 along the first axis Ai can be between a first scan boundary 1751 and a second scan boundary 1752 of the capture region 1750. For example, the first movement Mi can correspond to an oscillatory movement of the chip 1100 in which a movement in a first direction along the first axis Ai brings the first scan boundary 1751 toward the contact portion 1712 of the stylus 1710 and a movement in the second direction along the first axis Ai opposite the first direction brings the second scan boundary 1752 toward the contact portion 1712. Accordingly, the first scan boundary 1751 is separated from the second scan boundary 1752 by the first length Li along the first axis Ai.Attorney Docket No. 1147-002-01WO

[0077] As further depicted in FIG. 4, a second movement M2 of the chip 1100 along the second axis A2 can be from a third scan boundary 1753 and toward a fourth scan boundary 1754 of the capture region 1750. For example, the second movement M2 can correspond to a linear movement of the chip 1100 along the second axis A2 in which the fourth scan boundary 1754 is moved toward the contact portion 1712 of the stylus 1710. The third scan boundary 1753 is, therefore, separated from the fourth scan boundary 1754 by the second length L2 along the second axis A2.

[0078] In some embodiments, the first movement Mi of the chip 1100 along the first axis Ai can be at a first velocity, while the second movement M2 of the chip 1100 along the second axis A2 is at a second velocity. The first velocity can be greater than the second velocity. Additionally, in some embodiments, the first velocity can be a variable velocity, while the second velocity is a constant velocity. For example, in some embodiments, the chip 1100 can be moved at a relatively high speed along the first axis Ai, slowed as a change in direction approaches (e.g., as either the first scan boundary 1751 or the second scan boundary 1752 approaches the contact portion 1712 of the stylus 1710), and accelerated again following the change in direction. Said another way, in some embodiments, the first movement Mi of the chip 1100 along the first axis Ai is an oscillatory movement that extends between the first scan boundary 1751 and the second scan boundary 1752. The oscillatory movement can have a frequency in a range of at least 0.2 kilohertz and less than 5 kilohertz (e.g., 1.0 kilohertz). Accordingly, the first velocity can be a range of 2,000 to 20,000 microns per second. At velocities within this range, friction between the imaging surface 1130 and the contact portion 1712 of the stylus 1710 and resultant vibrations are minimized. Said another way, velocities along the first axis Ai within the prescribed range support the rapid analysis (e.g., measurement) of the target molecule TM by minimized vibrations that would otherwise result from the attraction between the contact portion 1712 and the imaging surface 1130. Said yet another way, at velocities within the prescribed range, the contact portion 1712 can be said to “glide” along the imaging surface 1130 rather than “dragging,” as is observed during movements at velocities that are less than 2,000 microns per second.

[0079] Contemporaneously with the oscillatory movement along the first axis Ai, the chip 1100 can be moved at a relatively slow speed (e.g., a scanning speed in a range of 0.35 to 0.50 microns per second) along the second axis A2 to move the third scan boundary 1753 away from the stylus 1710 as a distance to the fourth scan boundary 1754 is decreased. The combination ofAttorney Docket No. 1147-002-01WG the oscillatory movement along the first axis Ai and the linear movement along the second axis A2 can result in a single, uninterrupted raster scan SC of the capture region 1750 that extends between the first scan boundary 1751 and the second scan boundary 1752 and from the third scan boundary 1753 to the fourth scan boundary 1754. In some embodiments, the scan SC can have a scan rate that is greater than 144 square microns per minute and less than 500 square microns per minute.

[0080] FIGS. 5-26 depict features, components, and elements of embodiments of a system 2000 for analyzing a target molecule via atomic force microscopy. The system 2000 can include any of the features, components or elements or components described herein with reference to system 1000. The system 2000 can be an atomic force microscopy system configured to analyze target molecules TM (FIGS. 24 and 25) of a set of independent samples supported by a single chip 2100. In some embodiments, the system 2000 can include a controller 2800, an environmental chamber 2600, which can include any of the elements or features described herein with reference to the environmental chamber 1600, and a motorized stage assembly 2500, which can include any of the elements or features described herein with reference to the motorized stage assembly 1500. Additionally, the system 2000 can include a probe assembly 2700 and a displacement detector 2400 (e g. an interferometer), each of which can include any of the elements or features described herein with reference to the probe assembly 1700 and the displacement detector 1400 respectively. The chip 2100 can include any of the features described herein with reference to chip 1100 and any of the features or elements described in U.S. provisional application docket no. 1147-001-00US, entitled “Devices for and Methods of Sample Preparation for Imaging Target Molecules,” filed on the same date herewith, the disclosure of which is incorporated herein in its entirety.

[0081] The system 2000 can be configured to have a footprint that facilitates positioning the system 2000 on a lab bench. For example, the environmental chamber 2600 can be sized to have a footprint that is in a range of 4.5m2and 6.0m2. To minimize the effects of vibration on the analysis of the target molecule TM, at least one vibratory isolation member 2604 can be positioned between the environmental chamber 2600 and a first support surface (e.g., a lab bench), which is, in turn supported by and separated from a second support surface by a height.Attorney Docket No. 1147-002-01WO

[0082] In some embodiments, the environmental chamber 2600 of the system 2000 can be configured to maintain an internal environment that is in a range of 0% to less than 10% (e.g., 1% to less than 9%) of a relative humidity of the internal environment. In some embodiments the environmental chamber 2600 can be configured to maintain the relative humidity of the internal environment within a range of not more than 5% (e.g., between 0.5% and 5%, between 2% and 5%, and between 1% and 4%). 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 2600. 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 less than 10%), the formation of deposits and / or the collection of contaminants on the chip 2100 can be minimized, thus improving the accuracy of the imaging performed by the system 2000.

[0083] To facilitate maintaining the desired internal environment within the environmental chamber 2600, in some embodiments, the system 2000 includes at least one environmental sensor 2620. The environmental sensor(s) 2620 can be positioned within the environmental chamber 2600 to monitor the environment therein. An output of the environmental sensor(s) 2620 can be indicative of the monitored environment. The environmental sensor(s) 2620 can be operably coupled to the controller 2800. Accordingly, the controller 2800 can be configured to establish an environment within the environmental chamber 2600 based at least in part on the output of the environmental sensor(s) 2620.

[0084] In some embodiments, the internal volume within the environmental chamber 2600 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 0% and less than 10%). Minimizing the internal volume of the environmental chamber 2600 minimizes the amount of time required to achieve the desired internal environment. For example, the environmental chamber 2600 can be sized such that the humidity of the internal environment can be reduced from ambient conditions to less than 10% of the ambient conditions in less than 1 minute.

[0085] To facilitate minimizing the internal volume, components, or portions thereof, of the system 2000 can be positioned external to the environmental chamber 2600. For example, theAttorney Docket No. 1147-002-01WO motorized stage assembly 2500 and the probe assembly 2700 can be positioned within the environmental chamber. The controller 2800 can be positioned outside of the environmental chamber 2600. Finally, the displacement detector 2400 can have a lens assembly 2410 (FIG. 7 and 8) positioned at least partially within the environmental chamber 2600 while a laser emitter 2420 and an optical detector 2430 are positioned outside the environmental chamber 2600. To maintain the desired environmental conditions within environmental chamber 2600, a seal member 2610 (FIG. 6) is positioned between the lens assembly 2410 and a wall 2602 of the environmental chamber 2600. Accordingly, though the laser emitter 2420 (FIG. 8) and the optical detector 2430 (FIG. 8) are positioned outside the environmental chamber 2600, they are, nevertheless, optically coupled to a stylus 2710 of the probe assembly 2700 via the lens assembly 2410 while the environmental chamber 2600 maintains the desired environmental conditions within the environmental chamber.

[0086] FIG. 7 is a perspective view of the system 2000 with the environmental chamber 2600 removed for clarity. As depicted, the motorized stage assembly 2500 is configured to position the chip 2100 at a location that is viewable by the displacement detector 2400. The probe assembly 2700 is configured to position the stylus 2710 (FIG. 9) in contact with the chip 2100 (e g., with the target molecule TM (FIG. 24) or the imaging surface 2130 (FIG. 25)). Each of the displacement detector 2400, the probe assembly 2700, and the motorized stage assembly 2500 can be operably coupled to the controller 2800 (FIG. 26). The controller 2800 can be configured to cause the motorized stage assembly 2500 to move the chip 2100 relative to the stylus 2710 to conduct a scan of the capture region 2750 (FIG. 24). The displacement detector 2400 is configured to output an indication of a position of the stylus 2710. The controller 2800 is configured to analyze the target molecule TM based at least in part on the output of the displacement detector 2400.

[0087] The chip 2100 can be formed to support the target molecules TM of multiple independent samples. Each of the independent samples can be associated with a different genomic analysis and / or a different subject. Supporting multiple independent samples on a single chip 2100 can reduce the amount of time required to conduct a measurement operation, which is the time interval during which the target molecules TM supported by a single chip 2100 are analyzed (e.g., measured). Specifically, this arrangement (measuring multiple independent samples on a single chip) allows for measurements to be taken without the need to open the environmental chamberAttorney Docket No. 1147-002-01WO2600 between each scan of independent molecule samples, which would require that the environmental be reestablished between each scan. The measurement operation can extend from the instant in which the chip 2100 is positioned within the environmental chamber 2600 of the system 2000 to the completion of a scan of the final target molecule supported by the chip 2100. For example, more than a million target molecules TM can be supported by a single chip 2100 having an area of 18.75 square centimeters in less than 10 minutes.

[0088] As depicted in FIG. 25, the chip 2100 can have a plurality 2140 of bounded sample segments 2110. 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, 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, which may contain a different target molecule. 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 chips that would otherwise requiring loading or exchange 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 measurement operations.

[0089] 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 and the orthogonal lateral axis of the chip 2100) from every other bounded sample segment 2110.Attorney Docket No. 1147-002-01WO

[0090] 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 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, the chip 2100 can be prepared for imaging 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 chip 2100 can be prepared without 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 system 2000. For example, the system 2000 can be used to accurately determine the length and / or other characteristics of the target molecule TM in the unperturbed state.

[0091] FIG. 8 shows the displacement detector 2400 of the system 2000. The displacement detector 2400 is configured to output an indication of a position of the stylus 2710, which is then used by the operably coupled controller 2800 to analyze the target molecule TM. Accordingly, the displacement detector 2400 can be a heterodyne or homodyne laser displacement detector. Therefore, the displacement detector 2400 can include a laser emitter 2420 and an optical detector 2430. The laser emitter 2420 can be configured to omit a beam 2402 (FIG. 9) of coherent light that is reflected back to a position-sensitive optical detector 2430 by the probe assembly 2700 (e.g., the stylus 2710). A change in the position of the beam 2402 upon contact with the position-sensitive optical detector 2430 is indicative of a change in the position (e.g., a position along the third axis A3, an angular position, or an orientation) of the stylus 2710 resulting from contact with a feature on the imaging surface 2130 of the chip 2100. In some embodiments, the laser emitter 2420 and the optical detector 2430 can be contained within the same housing and directly optically aligned with the lens assembly 2410. However, in some embodiments, the laser emitter 2420 can be positioned in a housing that is separated from the optical detector 2430 such that the beam 2402 can be delivered to the lens assembly 2410 via an optical cable.

[0092] In some embodiments, the displacement detector 2400 can include a camera (not shown) and a lens assembly 2410. The lens assembly 2410 can be an objective lens assembly (e.g., a microscope objective lens). The use of an objective lens assembly can facilitate focusingAttorney Docket No. 1147-002-01WG the beam 2402 on the stylus 2710. Additionally, the use of an objective lens assembly can facilitate positioning the chip 2100 relative to the stylus 2710.

[0093] As depicted in FIG. 8, the displacement detector 2400 can be coupled to a linear actuator 2440. The linear actuator 2440 can be positioned outside the environmental chamber 2600. The linear actuator 2440 can be operably coupled to the controller 2800 and configured to move the displacement detector 2400 along the third axis A3 (FIG. 7). Said another way, the linear actuator 2440 can be configured to affect a separation distance (e.g., a vertical separation distance) between the lens assembly 2410 and the stylus 2710, such as during a focusing operation or calibration operation.

[0094] FIG. 9 depicts an operation of the displacement detector 2400 to measure a movement (e g., deflection or displacement) of the stylus 2710 along the third axis A3. As depicted, the chip 2100 is supported by a chip support assembly 2590 of the motorized stage assembly 2500. The stylus 2710 of the probe assembly 2700 is positioned in contact with the imaging surface 2130 or the target molecule TM. The beam 2402 of the displacement detector 2400 is focused (such as via the lens assembly 2410) on the stylus 2710. The chip 2100 is then moved by the motorized stage assembly 2500 relative to the stylus 2710 to execute a scan. As the stylus 2710 encounters a feature (e.g., a target molecule TM) on the imaging surface 2130, the stylus 2710 deflects along the third axis A3, and a change in the position of a reflected portion of the beam 2402 is indicative of the deflection.

[0095] As previously described, in some embodiments, the chip 2100 is configured with a plurality 2140 of bounded sample segments 2110 to support multiple separate samples on a single imaging surface 2130. Accordingly, the measurement operation can include multiple individual scans. The number of individual scans executed during the measurement operation can correspond to the number of bounded sample segments 2110 into which a separate sample has been deposited. To that end, the chip 2100 can be maintained within the environmental chamber 2600 from prior to the initiation of the first scan (e.g., a scan of the first bounded sample segment 2110 containing the target molecule TM of the first sample) through the completion of the final scan of the samples supported by the chip 2100.Attorney Docket No. 1147-002-01WO

[0096] Although the measurement operation can include multiple individual scans, one and only one calibration or alignment of the system 2000 is required. For example, a single calibration of the system 2000 can be executed following the positioning of the chip 2100 in the environmental chamber 2600 and prior to the execution of a first of the number of scans. The single calibration of the system 2000 can be maintained on a condition that the chip 2100 is maintained in the environmental chamber 2600. Said another way, the system advantageously enables methods with one and only one system calibration being executed during the measurement operation. The calibration can, for example, be a Z-calibration to establish a desired force magnitude between the contact portion of the stylus 2710 and the imaging surface 1130. Additionally, the calibration can include aligning the beam 2402 with the stylus 2710 and focusing the beam 2402 thereon.

[0097] Insofar as the accuracy of the positional relationship between the beam 2402, the stylus 2710, and the chip 2100 is essential to the accuracy of the output of the displacement detector 2400, the calibration procedure must be accomplished each time a chip is first positioned on the motorized stage assembly 2500. However, as the chip 2100 supports multiple separate samples, only a single calibration procedure is required before all the separate samples can be scanned. Similarly stated, all of the separate samples can be scanned without any calibration procedures being performed in between scans of any of the separate scans. Said another way, all of the all of the separate samples can be scanned without any intermediate calibration procedures being performed during the scanning operation. The required accuracy of the positional relationship can result in the calibration procedure being relatively time-consuming. Therefore, the necessity to conduct only a single calibration procedure when the chip 2100 is first positioned on the motorized stage assembly 2500 increases the throughput of the system 2000. In contrast, for systems that utilize a chip that supports only a single sample, the calibration procedures must be repeated with each swapping of the chip. By way of illustration, in an embodiment wherein the chip 2100 supports 96 separate samples, only a single calibration procedure must be implemented to analyze all 96 samples, while 96 calibration procedures would otherwise be required to scan 96 samples supported by 96 individual chips. The number of additional calibration procedures that must be executed decreases the throughput of such a system and increases the amount of time required to analyze the same number of samples that can be analyzed by the system 2000 following a single calibration procedure.Attorney Docket No. 1147-002-01WO

[0098] In some embodiments, executing the number of individual scans (i.e., analyzing multiple separate target molecules of multiple separate samples supported by a single chip 2100) during a single measurement operation can include positioning the stylus 2710 sequentially in contact with each separate target molecule TM or the imaging surface 2130 of the corresponding bounded sample segment 2110. With the stylus 2710 thus positioned, a separate scan can be executed in each bounded sample segment 2110 of the chip 2100. For example, a first scan can be executed of a first bounded sample segment 2110. The chip 2100 can then be moved by the motorized stage assembly 2500 along the first axis Ai and the stylus 2710 positioned in contact with either the target molecule TM or the imaging surface 1130 of a second bounded sample segment 2110. With the stylus 2710 thus positioned, a second scan can then be executed of the second bounded sample segment 2110. This procedure can be repeated to scan the desired number of bounded sample segments 2110 of the single chip 2100. In some embodiments, the contact portion of the stylus 2710 can be aligned with the imaging surface 2130 of the chip 2100 along the third axis A3 prior to the execution of each scan. The alignment of the contact portion with the imaging surface 2130 is merely a return of the stylus 2710 to a zero-position along the third axis A3 established during the one and only calibration procedure and does not reestablish a new zero-position (such as can be established during the calibration procedure). Accordingly, as previously described, a calibration procedure is not required between each of the sequential scans; only prior to the first scan of the sequence. Therefore, the analysis of a large number of samples can be accomplished more quickly than would otherwise be possible if separate samples were supported on separate chips.

[0099] FIG. 10 is a perspective view of the probe assembly 2700 of the system 2000. As depicted, the probe assembly 2700 can include a three-axis positioning assembly 2720. The three-axis positioning assembly 2720 can be positioned within the environmental chamber 2600 and can support the stylus 2710. As depicted, the three-axis positioning assembly 2720 can include a set of motors 2722 (e.g., picomotors). Each motor 2722 can be configured to modify a position of the stylus along one of the first axis Ai, the second axis A2, or the third axis A3. Each motor 2722 can, for example, have at least a 0.5 micron positioning resolution. The controller 2800 can be operably coupled to the three-axis positioning assembly 2720 (i.e., to each motor 2722) to affect a position of the stylus 2710 relative to the chip 2100 supported by the stage assembly 2500.Attorney Docket No. 1147-002-01WO

[0100] FIG. 11 is a perspective view and FIG. 12 is a partially exploded view of the motorized stage assembly 2500 of the system 2000. The motorized stage assembly 2500 is configured to support the chip 2100 on a substantially horizontal plane (e.g., a plane having an angle rotation about the first axis Ai that is in a range of 0 degrees to less than 2 degrees. The motorized stage assembly 2500 is operably coupled to be controlled by the controller 2800. Accordingly, the motorized stage assembly 2500 is configured to move and position the chip 2100 within a coordinate system defined by the first axis Ai, the second axis A2, and the third axis A3.

[0101] To analyze the target molecule TM, the controller 2800 (FIG. 26) is configured to execute a scan of the capture region 2750 (FIG. 24) by moving the chip 2100 relative to the stylus 2710 via the motorized stage assembly 2500. As procedure described with reference to FIG. 4, the motorized stage assembly 2500 can, for example, move the chip 2100 to establish a capture region 2750 having a first length Li along the first axis Ai and a second length L2 along the second axis A2, with the first length Li having a ratio relative to the second length L2 in a range of 1 :4 to 1 : 10.

[0102] In some embodiments, moving the chip 2100 relative to the stylus 2710 via the motorized stage assembly 2500 to execute each scan of the set of individual scans can include moving the chip 2100 along both the first axis Ai and along the second axis A2 while maintaining a position of the chip 2100 along the third axis A3. The controller 2800 can control the motorized stage assembly 2500 to move the chip 2100 along the first axis Ai between a first scan boundary and a second scan boundary of the capture region 2750. The motorized stage assembly 2500 can, for example, move the chip 2100 in an oscillatory manner between the first scan boundary and the second scan boundary. The controller 2800 can also control the motorized stage assembly 2500 to move the chip 2100 along the second axis A2 from a third scan boundary to a fourth scan boundary of the capture region 2750. The motorized stage assembly 2500 can, for example, move the chip 2100 and a linear fashion along the second axis A2. In addition to maintaining the chip 2100 at a single position along the third axis A3 throughout the scan, the controller 2800 can also maintain a contact portion of the stylus 2710 in contact with the imaging surface 2130 or the target molecule TM during the movement of the chip 2100 relative to the stylus 2710 during the execution of each separate scan.Attorney Docket No. 1147-002-01WO

[0103] In some embodiments, the controller 2800 can control the motorized stage assembly 2500 to move the chip 2100 along the first axis Ai at a first velocity and along the second axis A2 at a second velocity. The second velocity can be less than the first velocity. The first velocity can, for example, be sufficient to establish the oscillatory movement of the chip 2100 between the first scan boundary and the second scan boundary at a frequency in a range of 0.2 kilohertz and less than 5.0 kilohertz (e.g., 1.0 kilohertz). The first velocity can, for example, be a scanning speed in a range of 2,000 to 20,000 microns per second , while the second velocity is a scanning speed in a range of 0.2 to 2.0 microns per second (e.g., 0.2 to 0.8 microns per second, 0.35 to 0.50 microns per second, or other suitable ranges).

[0104] The first velocity being in a range of 2,000 to 20,000 microns per second reduce or otherwise minimize friction between the imaging surface 2130 and the stylus 2710 and any resultant vibrations. Said another way, velocities along the first axis Ai within the prescribed range support the rapid analysis (e.g., measurement) of the target molecule TM by minimized vibrations that would otherwise result from the attraction between the stylus 2710 and the imaging surface 2130. Said yet another way, at velocities within the prescribed range, the stylus 2710 can be said to “glide” along the imaging surface 2130 rather than “dragging,” as is observed during movements at velocities that are less than 2,000 microns per second.

[0105] In order to affect the position of the chip 2100 as directed by the controller 2800, the motorized stage assembly 2500, as depicted in FIGS. 11 and 12, includes a first stage assembly 2510, a second stage assembly 2520, a third stage assembly 2560, a fourth stage assembly 2580, and a chip support assembly 2590. The first stage assembly 2510 and the third stage assembly 2560 are configured to affect a position of the chip 2100 only along the first axis Ai. The second stage assembly 2520 is configured to affect a position of the chip 2100 only along the second axis A2. The fourth stage assembly 2580 is configured to affect a position of the chip 2100 along the third axis A3, a rotation of the chip 2100 about the first axis Ai, a rotation of the chip 2100 about the second axis A2 and / or a combination thereof. The motorized stage assembly 2500 is configured to preclude or minimize the development of mechanical vibrations that would otherwise be detected (e.g., measured) by the displacement detector 2400 during the movement of the chip 2100 in the execution of a scan while maximizing the speed of the scan to increase the throughput of theAttorney Docket No. 1147-002-01WG system 2000. Such vibrations would have a negative effect on the analysis of the target molecule based on the displacement of the stylus 2710 as measured by the displacement detector 2400.

[0106] FIG. 13 is a perspective view of the first stage assembly 2510 of the motorized stage assembly 2500. The first stage assembly 2510 includes a first base member 2512. The first base member 2512 can be fixedly coupled to a support structure of the system 2000 and can remain in a stationary position during the execution of a scan. The first base member 2512 can, for example, serve as a foundation for the motorized stage assembly 2500. In some embodiments, the first base member 2512 can be separated from the support structure of the system 2000 via a vibration damping element (not shown).

[0107] The first stage assembly 2510 can include a first slider member 2514. The first slider member 2514 can be movably coupled to the first base member 2512 via a first lead screw 2516. The first slider member 2514 can be movable relative to the first base member 2512 only along the first axis Ai. The movement of the first slider member 2514 can be motivated or produced by a first motor 2518 that is coupled to the first lead screw 2516 and controlled by the controller 2800 the first motor 2518 can, for example, be a stepper motor. The first motor 2518 and the first lead screw 2516 can be configured to control a position of the chip 2100 prior to the execution of a scan. For example, the first motor 2518 can be actuated by the controller 2800 to move the chip 2100 between separate bounded sample segments 2110 (i.e., a coarse movement along the first axis Ai). As the movement of the chip 2100 via the first stage assembly 2510 can occur prior to the initiation of each scan, and vibration produced by the movement of the first slider member 2514 does not impact the analysis of the target molecule.

[0108] In some embodiments, the first stage assembly 2510 has a first travel length TLi (e.g., a range of available travel or a stroke distance) along the first axis Ai that is, as depicted in FIG. 24 be sufficient to align any longitudinal position of the chip 2100 with the stylus 2710. The first travel length TLi of the first stage assembly 2510 is greater than the travel length of any other stage assembly of the motorized stage assembly 2500. In FIG. 24, the chip 2100 is depicted at a travel limit in a first direction along the first axis Ai with a first end aligned with the stylus 2710. The chip 2100’ is depicted at a travel limit in a second direction opposite the first direction with a second end aligned with the stylus 2710. As the chip 2100 can, for example, have a longitudinalAttorney Docket No. 1147-002-01WG length of 75 millimeters, the first travel length TLi of the first stage assembly can be at least 150 millimeters

[0109] FIGS. 14-19 depict aspects of the second stage assembly 2520 of the motorized stage assembly 2500. As depicted in and the perspective view of FIG. 14, the second stage assembly 2520 includes a second base member 2522 and a second slider member 2532. The second base member 2522 is, as depicted in FIG. 11, coupled to the first slider member 2514 of the first stage assembly 2510. The second slider member 2532 is movably coupled to the second base member 2522 via a second lead screw 2542. A single second motor 2540 is coupled to the second lead screw 2542 to move the second slider member 2532 relative to the second base member 2522 only along the second axis Az orthogonal to the first axis Ai. The second motor 2540 can, for example, be a rotational motor, such as a stepper motor, configured to impart a rotational torque to the second lead screw 2542. The second motor 2540 is the one and only motor positioned to drive a movement of the chip 2100 along the second axis A2. Similarly stated, the second motor 2540 includes one and only one device that converts electrical energy (e.g., supplied to the second motor 2540 via the controller 2800) into mechanical energy to produce a movement, as described herein

[0110] In some embodiments, the second stage assembly 2520 has a second travel length TL2 along the first axis A2. In that the second stage assembly 2520 is the only portion of the motorized stage assembly 2500 configured to generate a movement along the second axis A2, the second travel length TL2 can also be the range of travel along the second axis A2 of the motorized stage assembly 2500. The second travel length TL2 can, as depicted in FIG. 24, be sufficient to align any lateral position of the chip 2100 with the stylus 2710. In FIG. 24, the chip 2100 is depicted at a travel limit in a first direction along the second axis A2 with a first side aligned with the stylus 2710 the chip 2100’ is depicted at a travel limit in a second direction along the second axis A2 opposite the first direction with a second side aligned with the stylus 2710. As the chip 2100 can, for example, have a lateral width in a range of 20 millimeters to 30 millimeters, the travel of the second slider member 2532 relative to the second base member 2522 along the second axis A2 can be in a range of 40 millimeters to 60 millimeters.

[0111] The second motor 2540 and the operably coupled second lead screw 2542 can be configured to both position the chip 2100 (e.g., one of the bounded sample segments 2110) alongAttorney Docket No. 1147-002-01WG the second axis A2 prior to the initiation of a scan in a manner similar to the longitudinal positioning driven by the first lead screw 2516 and the first motor 2518. However, unlike the first motor 2518, the second motor 2540 (and the second lead screw 2542) also drive the movement of the chip 2100 the second length L2 along the second axis A2 during the execution of the scan (see, e.g., FIG. 4). In other words, the one and only motor (i.e., the second motor 2540) of the second stage assembly 2520 can be used for both coarse pre-scan positioning movements and fine, low-vibration movements of the scan.

[0112] An alternative approach to the single second motor 2540 being used for both positioning movements and for the scanning movements along the second axis A2 would be to use one motor for the positioning movements and a second, separate motor to execute the scanning movements. This approach is embodied with reference to the first axis Ai movements by the first motor 2518, which is a rotational motor used for coarse positioning movements, and the third motor 2566 (as described below with reference to FIG. 22B), which is a piezoelectric motor. In such an arrangement, the rotational motor, being coupled to a lead screw, can have a greater range of travel than is achievable via a piezoelectric motor but can lack the precision and / or vibratory transmission characteristics of the piezoelectric motor. On the other hand, the piezoelectric motor can be well suited to execute scanning movements but with a limited range of travel. However, to facilitate the increased rate at which the target molecules TM supported by the chip 2100 are analyzed, the controller 2800 is configured to control the motorized stage assembly 2500 to establish a capture region 2750 that has a rectangular area. The rectangular area of the capture region 2750 has a long dimension (i.e., the second length L2 along the second axis A2) that is between 4 times and 10 times greater than the short dimension (i.e., the first length Li along the first axis Ai). Establishing the second length L2 at a magnitude that is between 4 and 10 times greater than the first length Li can exceed the range of travel of a piezoelectric motor. Therefore, the greater travel range coupled with the desired low vibration characteristics (described below) provided by the single second motor 2540 and the second lead screw 2542 facilitates the establishment of the rectangular capture regions 2750 having the desired high-resolution output, which, in turn, increases the throughput of the system 2000. Said another way, the greater range of travel provided by the second motor 2540 and the second lead screw 2542 facilitates moving the chip 2100 relative to the stylus 2710 a greater distance (e.g., the second length L2) along theAttorney Docket No. 1147-002-01WO second axis A2 during a scan than would otherwise be possible via a piezoelectric motor. Said yet another way, the absence of the greater range of travel provided by the second motor 2540 in combination with the second lead screw 2542 would preclude the establishment of the desired rectangular area of the capture region 2750 resulting in an increase in the amount of time required to complete the measurement operation. As an additional benefit, the use of a single motor reduces the complexity of the motorized stage assembly 2500 via the reduction of at least one additional motor (e.g., a piezoelectric motor) and corresponding stage assembly.

[0113] Insofar as the single second motor 2540 and a second lead screw 2542 is used to move the second slider member 2532 (and the supported chip 2100) along the second axis A2 during a scan of the bounded sample segment(s) 2110, it is desirable to minimize any vibration that would otherwise be generated by the movement of the second slider member 2532 relative to the second base member 2522. In the absence of the minimization, any vibrations resulting from friction (e.g., rubbing) between the second slider member 2532 and the second base member 2522 could affect the position of the stylus 2710 along the third axis A3, thereby distorting the visualization of the target molecule TM. To that end, the second stage assembly 2520, as depicted in FIGS. 16, 18, and 19, includes a set of spherical (or partially spherical) sectors 2534. The spherical sectors 2534 are positioned between the second base member 2522 and the second slider member 2532. Each spherical sector 2534 is, as depicted in FIG. 16 in 19, positioned to contact a corresponding interface block 2530. Accordingly, each spherical sector 2534 can be a contact point of a linear bearing defined by each spherical sector-interface block pairing. In some embodiments, the spherical sectors 2534 and / or the interface blocks 2530 can, for example, be metal-polymer bearings that are impregnated with an anti-friction overlay enriched with polytetrafluoroethylene (PTFE) or a suitable low-friction thermoplastic. In some embodiments, the interface blocks 2530 can be formed from a ceramic or a ceramic composite. The moveability (e.g., via sliding) of the second slider member 2532 relative to the second base member 2522 is facilitated by the spherical sector-interface block pairings, which reduce or eliminate the vibrations that would otherwise be produced via the friction between the second slider member 2532 and the second base member 2522 during such a movement. By extension therefore, the positioning of the spherical sector-interface block pairings between the second slider member 2532 and the second baseAttorney Docket No. 1147-002-01WG member 2522 facilitates the use of the single second motor 2540 to both position the chip 2100 prior to scan initiation and move the chip 2100 relative to the stylus 2710 during the scan.

[0114] In FIG. 16, the second slider member 2532 is made transparent for illustrative purposes. As depicted, in some embodiments, each spherical sector 2534 is coupled to the second slider member 2532. Correspondingly, as depicted in FIG. 17, each interface block 2530 is coupled to the second base member 2522. As depicted in FIG. 17, the second base member 2522 can, in some embodiments, include a first face 2524 that is orthogonal to a second face 2526 and a third face 2528, which are parallel to one another. A first portion 2536 of the set of interface blocks 2530 is coupled to the first face 2524, a second portion 2538 is coupled to the second face 2526, and a third portion 2539 is coupled to the third face 2528. Contact between the first portion 2536 of the interface blocks 2530 and a corresponding portion of the spherical sectors 2534 limits a movement of the second slider member 2532 relative to the second base member 2522 along the third axis A3. Said another way, the portion of the spherical sector-interface block pairings positioned along the horizontal face (i.e., the first face 2524) of the second base member 2522 can support the weight of the second slider member 2532 (and any other portions of the motorized stage assembly 2500 supported thereon) and preclude a downward movement of the second slider member 2532. Contact between the second portion 2538 of the interface blocks 2530 and a corresponding portion of the spherical sectors 2534 limits a movement of the second slider member 2532 relative to a second base member 2522 in a first direction along the first axis Ai. Similarly, contact between the third portion 2539 of the interface blocks 2530 and a corresponding portion of the spherical sectors 2534 limits movement of the second slider member 2532 relative to the second base member 2522 in a second direction along the first axis Ai. Said another way, the portion of the spherical sector-interface block pairings positioned along the side face (i.e., the second face 2526 and the third face 2528) of the second base member 2522 preclude the movement of the second slider member 2532 along the first axis Ai in either direction while minimizing or eliminating a friction between the second slider member 2532 and the second base member 2522 during a movement in either direction along the second axis A2.

[0115] As depicted in FIG. 16 and 18, in some embodiments, the second stage assembly 2520 includes a first preload member 2548. The first preload member 2548 is positioned to bias the second slider member 2532 oh away from the second base member 2522. The first preloadAttorney Docket No. 1147-002-01WO member 2548 can, for example, be a magnetic preload member oriented to develop a repulsive force between the second slider member 2532 and the second base member 2522. The biasing of the second slider member 2532 can further reduce or eliminate friction between the second slider member 2532 and the second base member 2522 during a movement along the second axis A2.

[0116] As depicted in FIG. 16 and 18, in some embodiments, the second stage assembly 2520 includes a second preload member 2550. The second preload member 2550 can be positioned between the second slider member 2532 and the third face 2528 of the second base member 2522. The second preload member 2550 can, therefore, be positioned to bias the second slider member 2532 in a first direction along the first axis Ai. Accordingly, the second preload member 2550 can reduce or eliminate undesired spatial clearance (i.e., “slop”) between the second slider member 2532 and the second base member 2522 that could otherwise negatively affect the desired motion along the second axis A2.

[0117] In some embodiments, the combination of the second motor 2540 and the second lead screw 2542 is configured to facilitate fine control of the movement of the second slider member 2532, and by extension, the chip 2100 supported by the motorized stage assembly 2500, over a longer distance than would be achievable via a piezoelectric motor. Accordingly, the second slider member 2532 is movably coupled to the second lead screw 2542 via a ball nut 2544 (FIG. 17). The ball nut 2544 includes a set of ball bearing elements positioned between the internal ball grooves of the ball nut 2544 and the threads of the lead screw 2542. Coupling the second slider member 2532 to the second lead screw 2542 via the ball nut 2544 can reduce the friction (and resultant vibration) associated with the rotation of the lead screw 2542 relative to that that would otherwise be encountered with a coupling via a threaded nut. Additionally, the use of the ball nut 2544 facilitates the desired fine control of the movement of the second slider member 2532. For example, the ball nut 2544 can have an axial play (e.g., backlash) of less than 5 microns along the second axis A2. The axial play can refer to movement in the axial direction that is not attributable to the rotation of the ball nut 2544 or the second lead screw 2542. Therefore, the second motor 2540 and the second lead screw 2542 can accurately affect the position of the chip 2100 relative to the stylus 2710 during a scan without imparting an undesirable vibration to the stylus 2710.Attorney Docket No. 1147-002-01WG

[0118] As depicted in FIGS. 15 and 17, in some embodiments, the second motor 2540 is positioned parallel to and offset from the second lead screw 2542. Accordingly, both the second motor 2540 and the second lead screw 2542 can be positioned within the second base member 2522. The second motor 2540 can be operably coupled to the second lead screw 2542 via a fixed gearing arrangement 2546. The gearing arrangement 2546 can have a fixed gear ratio that is established during the assembly of the motorized stage assembly 2500. In some embodiments, the gearing arrangement 2546 can impart a year reduction on the second motor 2540 and arrange of 1600: 1 to 1700: 1. Such a gearing arrangement can facilitate the movement of the second slider member 2532 at a desired scanning speed along the second axis A2. Said another way, the second motor 2540 and the second lead screw 2542 can be configured to move the second slider member 2532 relative to the second base member 2522 at a scanning speed in a range of 0.35 to 0.50 microns per second during the execution of each scan during a measurement operation.

[0119] As depicted in FIGS. 15 and 17, in some embodiments, the second stage assembly 2520 can include an absolute encoder 2552. The absolute encoder 2552 can be configured to output a signal indicative of a position of the second slider member 2532 along the second axis A2. The absolute encoder 2552 can output a position of the second slider member 2532 even on a condition that the second slider member 2532 is stationary. Said another way, the absolute encoder 2552 does not require a movement of the second slider member 2532 to determine the position of the second slider member 2532. Accordingly, the absolute encoder 2552 can facilitate the positioning of the chip 2100 at a desired location relative to the stylus 2710.

[0120] FIGS. 22A and 22B are perspective views of the third stage assembly 2560. The third stage assembly 2560 is configured to affect the position of the chip 2100 during the execution of a scan. Said another way, the third stage assembly 2560 is configured to cause the chip 2100 to move relative to the stylus 2710 between the first scan boundary and the second scan boundary along the first axis Ai. In some embodiments, the third stage assembly 2560 is configured to develop an oscillatory motion of the chip 2100 relative to the stylus 2710 between the first scan boundary and the second scan boundary along the first axis Al.

[0121] In some embodiments, the third stage assembly 2560 includes a third base member 2562 and a third slider member 2564. The third slider member 2564 is movably coupled to theAttorney Docket No. 1147-002-01WG third base member 2562 via a piezoelectric motor 2566. The third slider member 2564 is movable relative to the third base member 2562 along the first axis Ai. Accordingly, the third stage assembly 2560 can have a third travel length TL3 (e.g., a range of available travel or a stroke distance) along the first axis Ai that is, as depicted in FIG. 24 be sufficient to define (e.g., span) the capture region 2750. The third travel length TL3 (FIG. 4)can, for example, correspond to the distance between the first scan boundary and a second scan boundary (i.e., the first length). The third travel length TL3 can be between 0.001 percent and 0.01 percent of the first travel length TLi.

[0122] In some embodiments, The piezoelectric motor 2566 can operate in conjunction with a preload member 2568 to position the chip 2100 relative to the stylus 2710. The preload member 2568 can, for example, be a preload spring positioned to bias the third slider member 2564 in a direction counter to the motion imparted by the piezoelectric motor 2566. The piezoelectric motor 2566 and the preload member 2568 can develop a movement of the third slider member 2564 along the first axis Ai that has a velocity in a range of 2,000 to 20,000 microns per second.

[0123] FIG. 20 is a perspective view of a fourth stage assembly 2580. The fourth stage assembly 2580 is, as depicted in FIG. 21, movably coupled to the second slider member 2532. The third stage assembly 2560 can be supported by the fourth stage assembly 2580. Said another way, the fourth stage assembly 2580 can be coupled between the second slider member 2532 and the third base member 2562. The fourth stage assembly 2580 can be configured to affect the position of at least a portion of the chip 2100 along the third axis A3. Therefore, the fourth stage assembly 2580 can include a fourth motor 2582 positioned to move the fourth stage assembly 2580 (and the components of the motorized stage assembly 2500 supported thereon) along the third axis A3.

[0124] In some embodiments, the fourth motor 2582 is one of a set of picomotors. Each picomotor is positioned to affect a position of a portion of the fourth stage assembly 2580 along the third axis A3. Affecting the position of a portion of the fourth stage assembly 2580 can affect a pitch and / or a roll of the chip 2100. As depicted, in some embodiments, three picomotors are supported by the second slider member 2532 and operably coupled to the fourth stage assembly 2580 in a triangular arrangement. Additionally, a set of tension members 2584 can be coupled between the fourth stage assembly 2580 and the second slider member 2532 in a similar triangular arrangement. The set of tension members 2584 can bias the fourth stage assembly 2580 towardAttorney Docket No. 1147-002-01WG the second slider member 2532. The arrangement of the three picomotors and the set of tension members 2584 can facilitate the orienting of the chip 2100 by the controller 2800. For example, a portion of the picomotors can be actuated to establish the chip 2100 on a plane defined by the first axis Ai and the second axis A2 that has an angle rotation about the first axis Ai in a range of 0 degrees to less than 2 degrees.

[0125] FIG. 23 is a perspective view of the chip support assembly 2590. The chip support assembly 2590 can be coupled to the third slider member 2564. Accordingly, the chip support assembly 2590 can move in conjunction with the third slider member 2564 along the first axis Ai in response to a force imparted by the piezoelectric motor 2566 and / or the preload member 2568 and in conjunction with the second slider member 2532 along the second axis A2 in response to a force imparted by the second motor 2540 and the second lead screw 2542 to move the chip 2100 relative to the stylus 2710 during the execution of a scan of the capture region 2750. The chip support assembly 2590 is, therefore, configured to receive the chip 2100 supporting a target molecule TM. In some embodiments, the chip support assembly 2590 can be a vacuum chuck. The chip 2100 can be removably coupled to the chip support assembly 2590 is a vacuum chuck without any portion of the chip support assembly 2590 contacting the imaging surface 2130. Securing the chip 2100 via a vacuum chuck renders unnecessary the use of mechanical holding structures that could otherwise interfere with the movement of the chip 2100 during the measurement operation.

[0126] As shown particularly in FIG. 26, a schematic diagram of one embodiment of suitable components that may be included within the controller 2800 is illustrated. In some embodiments, the controller 2800 is positioned externally to but within two meters of the environmental chamber 2600. However, the controller 2800 may also include distributed computing systems wherein at least one aspect of the controller 2800 is at a location which differs from the remaining components of the system 2000 for example, at least a portion of the controller 2800 may be an online controller.

[0127] As depicted, the controller 2800 includes one or more processor(s) 2810 and associated memory device(s) 2820 configured to perform a variety of computer implemented functions (e.g., performing the methods, steps, calculations and the like and storing relevant data as disclosedAttorney Docket No. 1147-002-01WO herein). Additionally, in some embodiments, the controller 2800 includes a communication module 2830 to facilitate communications between the controller 2800 and the various components of the system 2000. The communications module 2830 can also facilitate communications between an operator OP of the system 2000 and the control 2800.

[0128] As used herein, the term “processor” refers not only to integrated circuits referred to in the art as being included in a computer, but also refers to a controller, a microcontroller, a microcomputer, a programmable logic controller (PLC), an application specific integrated circuit, and other programmable circuits. Additionally, the memory device(s) 2820 may generally comprise memory element(s) including, but not limited to, computer readable medium (e.g., random access memory (RAM)), computer readable nonvolatile medium (e.g., a flash memory), a floppy disc, a compact disc read only memory (CD ROM), a magneto optical disc (MOD), a digital versatile disc (DVD) and / or other suitable memory elements. Such memory device(s) 2820 may generally be configured to store suitable computer readable instructions that, when implemented by the processor(s) 2810, configure the controller 2800 to perform various functions.

[0129] As depicted, the communications module 2830 can include an environmental module 2840. The environmental module 2840 can facilitate communication between the controller 2800 and the environmental chamber 2600, including the environmental sensor 2620. The controller 2800 can, therefore, be configured to control the establishment of the environment within the environmental chamber based, at least in part on an output from the environmental sensor 2620. Accordingly, the environmental module 2840 can include an interface (e.g., one or more analog to digital converters) to permit signals transmitted from environmental sensor 2620 to be converted into signals that can be understood and processed by the processors 2810. The environmental sensor 2620 can be communicatively coupled to the communication module 2830 using any suitable means. For example, the environmental sensor 2620 can be coupled to the communication module 2830 via a wired connection and / or via a wireless connection, such as by using any suitable wireless communications protocol known in the art.

[0130] As depicted, the communications module 2830 can include an interferometer module 2850. The displacement detector module 2850 can facilitate communication between the controller 2800 and the displacement detector 2400. The controller 2800 can, therefore, beAttorney Docket No. 1147-002-01WO configured to control the positioning of the displacement detector 2400 and the emission of the beam 2402. Additionally, the controller 2800 can be configured to receive an output from the displacement detector 2400 indicating a position of the stylus 2710. Accordingly, the displacement detector module 2850 can include an interface (e.g., one or more analog to digital converters) to facilitate two-way communication between the displacement detector 1400 and the controller 2800. The displacement detector 2400 can be communicatively coupled to the communication module 2830 using any suitable means. For example, the displacement detector 2400 can be coupled to the communication module 2830 via a wired connection and / or via a wireless connection, such as by using any suitable wireless communications protocol known in the art.

[0131] As depicted, the communications module 2830 can include a probe module 2860. The probe module 2860 can facilitate communication between the controller 2800 and the probe assembly 2700, including, in some embodiments, the three-axis positioning assembly 2720. The controller 2800 can, therefore, be configured to control the positioning of the stylus 2710 via the three-axis positioning assembly 2720. The probe module 2860 can include an interface (e.g., one or more analog to digital converters) to facilitate two-way communication between the probe assembly 2700 and the controller 2800. The probe assembly 2700 can be communicatively coupled to the communication module 2830 using any suitable means. For example, the probe assembly 2700 can be coupled to the communication module 2830 via a wired connection and / or via a wireless connection, such as by using any suitable wireless communications protocol known in the art.

[0132] As depicted, the communications module 2830 can include a stage module 2870. The stage module 2870 can facilitate communication between the controller 2800 and the motorized stage assembly 2500. The controller 2800 can, therefore, be configured to control the positioning of the chip 2100 via the motorized stage assembly 2500. The probe module 2860 can include an interface (e.g., one or more analog to digital converters) to facilitate two-way communication between the motorized stage assembly 2500 and the controller 2800. The motorized stage assembly 2500 can be communicatively coupled to the communication module 2830 using any suitable means. For example, the motorized stage assembly 2500 can be coupled to the communication module 2830 via a wired connection and / or via a wireless connection, such as by using any suitable wireless communications protocol known in the art.Attorney Docket No. 1147-002-01WO

[0133] As described herein, in some embodiments, the controller 2800 is configured to perform a set of computer-implemented operations of operations 80, such as depicted in FIG. 27. The operations 80 can be used to analyze a target molecule via the system 2000 (e.g., via atomic force microscopy).

[0134] As depicted at 82, the operations 80 include positioning a contact portion of the stylus in contact with an imaging surface of the chip to establish a reference position for the stylus. At 84, the operations 80 also include moving the chip relative to the stylus to execute a scan of a capture region via the stylus. The capture region has a first length along a first axis and a second length along a second axis orthogonal to and coplanar with the first axis. The first length has a ratio relative to the second length in a range of 1 :4 to 1 : 10. Additionally, as depicted at 86, the operations 80 include determining a magnitude of a movement of the stylus along a third axis relative to the reference position based on an output of the displacement detector on a condition that the stylus is in contact with the target molecule. The third axis is orthogonal to the first axis and the second axis. Further, as depicted at 88, the operations 80 include determining a feature of the target molecule of the sample based on the magnitude of the movement.

[0135] In some embodiments, wherein the system 2000 includes an environmental chamber, the operations 80 include establishing an environment within the environmental chamber at a humidity level that is in a range of 0% to less than 10% of a relative humidity of the environment surrounding the environmental chamber. Accordingly, in some embodiments, the operations 80 include receiving an output from at least one environmental sensor positioned to monitor the environment within the environmental chamber, and affecting the humidity level based at least in part, on the output from the at least one environmental sensor.

[0136] In some embodiments wherein the probe assembly of the system 2000 includes a three-axis positioning assembly, the operations 80 can include actuating at least one motor of the three-axis positioning assembly to position the contact portion of the stylus in contact with the imaging surface.

[0137] In some embodiments, the operations 80 can include actuating a piezoelectric motor of the stage assembly to move the chip along the first axis between the first scan boundary and theAttorney Docket No. 1147-002-01WO second scan boundary, and actuating a stepper motor of the stage assembly can be actuated to move the chip along the second axis between the third scan boundary and the fourth scan boundary.

[0138] In some embodiments, the operations 80 can include maintaining the contact portion of the stylus in contact with either the imaging surface or the target molecule while the chip is moved relative to the stylus to execute the scan of the capture region.

[0139] In some embodiments, the operations 80 can include digitizing the position of the stylus as a number of pixels. Each pixel is either one nano meter by one nano meter or two nanometers by two nanometers. The pixels can be digitized at a rate in a range of 1.75 and 5 million pixels per second.

[0140] In some embodiments, the operations 80 can include establishing a plane defined by the first axis and the second axis at an angle rotation about the first axis that is in a range of 0 degrees to less than 2 degrees, with the chip being positioned on the plane.

[0141] In some embodiments, the operations 80 can include executing a single calibration to align the displacement detector with the stylus on a condition that the chip is positioned on the stage assembly prior to the execution of a first scan of the plurality of scans. Additionally, the controller 2800 can be configured to maintain the calibration on a condition that the chip is maintained on the stage assembly.

[0142] In some embodiments, the operations 80 can include filtering the output of the displacement detector via a passband filter of 15 kilohertz to 1000 kilohertz.

[0143] In some embodiments, the controller 2800 can be configured to execute a scan that has a scan rate that is greater than 144 square microns per minute and less than 500 square microns per minute.

[0144] In some embodiments, the determining the feature of the target molecule in accordance with the operations 80 can include producing a contiguous image of the capture region with a resolution of at least 2 nanometers. The target molecule can, for example, be a DNA molecule that has a size that is in the range of 100,000 base pairs to 20 million base pairs.Attorney Docket No. 1147-002-01WO

[0145] FIG. 28 is a flow chart of a method 70 of analyzing a target molecule via atomic force microscopy according to an embodiment. The method 70 can, for example be used to analyze the target molecule via the system 1000 or the system 2000 as described herein.

[0146] In some embodiments, as depicted at 72, the method 70 includes positioning a chip in an environmental chamber of an imaging system. The chip supporting the target molecule within an imaging region of a bounded sample segment of the chip. As depicted at 74, the method 70 includes causing the positioning of a contact portion of a stylus of the imaging system in contact with one of the target molecule or an imaging surface within the bounded sample segment of the chip. As depicted at 76, the method includes causing the execution of a scan of a capture region via the stylus. The capture region has a first length along a first axis and a second length along a second axis orthogonal to and coplanar with the first axis. The first length has a ratio relative to the second length in a range of 1 :4 to 1 :4100. As depicted at 78, determining a feature of the target molecule of the sample based on a displacement of the stylus along a third axis orthogonal to the first axis and the second axis.

[0147] 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.

Claims

Attorney Docket No. 1147-002-01WOWhat is claimed is:

1. A method of analyzing a target molecule via atomic force microscopy, the method comprising: positioning a chip in an environmental chamber of an imaging system, the chip supporting the target molecule within an imaging region of a bounded sample segment of the chip; causing the positioning of a contact portion of a stylus of the imaging system in contact with one of the target molecule or an imaging surface within the bounded sample segment of the chip; causing the execution of a scan of a capture region via the stylus, the capture region having a first length along a first axis and a second length along a second axis orthogonal to and coplanar with the first axis, the first length having a ratio relative to the second length in a range of 1:2 to 1 :4100; and determining a feature of the target molecule of the sample based on a displacement of the stylus along a third axis orthogonal to the first axis and the second axis.

2. The method of claim 1, wherein: the target molecule is electrostatically bonded to the imaging surface of the chip in a substantially unperturbed state.

3. The method of claim 1, wherein executing the scan of the capture region includes: moving the chip relative to the stylus along the first axis and along the second axis; the movement of the chip along the first axis is between a first scan boundary and a second scan boundary of the capture region; the first scan boundary is separated from the second scan boundary by the first length along the first axis; the movement of the chip along a second axis is from a third scan boundary toward a fourth scan boundary of the capture region; and the third scan boundary is separated from the fourth scan boundary by the second length along the second axis.Attorney Docket No. 1147-002-01WO4. The method of claim 3, wherein: the movement of the chip along the first axis is at a first velocity; and the movement of the chip along the second axis is at a second velocity that is less than the first velocity.

5. The method of claim 4, wherein: the first velocity is a variable velocity; and the second velocity is a constant velocity.

6. The method of claim 3, wherein: the movement of the chip along the first axis is an oscillatory movement extending between the first scan boundary and the second scan boundary; and the oscillatory movement has a frequency in a range of at least 0.2 kilohertz and less than 5 kilohertz.

7. The method of claim 1, wherein: executing the scan of the capture region includes executing a sinusoidal raster scan; the sinusoidal raster scan has an amplitude extending along the first axis and a duration extending along the second axis; and the duration is greater than the amplitude.

8. The method of claim 7, wherein: the amplitude has a ratio relative to the duration in a range of 2:3 to 1 : 1025.

9. The method of claim 7, wherein: the amplitude has a magnitude in a range of 0.5 microns to 5 microns.

10. The method of claim 1, further comprising: maintaining the stylus in contact with one of the target molecule or an imaging surface through an entirety of the scan.Attorney Docket No. 1147-002-01WO11. The method of claim 1, wherein: the second length has a magnitude in a range of 20 microns to 50 microns.

12. The method of claim 1, wherein: positioning the chip in the environmental chamber includes positioning the chip on a plane defined by the first axis and the second axis; and the plane has an angle rotation about the first axis that is in a range of 0 degrees to less than 2 degrees.

13. The method of claim 1, wherein: the bounded sample segment is one of a plurality of bounded sample segments of the chip; the target molecule is one of a plurality of separate target molecules; and each of the separate target molecules is positioned in a separate bounded sample segment of the plurality of bounded sample segments.

14. The method of claim 13, wherein the scan is one of a plurality of scans, the method further comprising: maintaining the chip in the environmental chamber; positioning the stylus sequentially in contact with each separate target molecule or the imaging surface of the corresponding bounded sample segment of the plurality of bounded sample segments; and executing one scan of the plurality of scans in each bounded sample segment of the plurality of bounded sample segments.

15. The method of claim 14, further comprising: executing a single calibration of the imaging system following the positioning of the chip in the environmental chamber and prior to the execution of a first scan of the plurality of scans; andAttorney Docket No. 1147-002-01WO maintaining the calibration of the imaging system on a condition that the chip is maintained in the environmental chamber.

16. The method of claim 14, further comprising: aligning the contact portion of the stylus with an imaging surface of the chip along the third axis prior to the execution of each scan of the plurality of scans.

17. The method of claim 1, further comprising: establishing an environment within the environmental chamber, the environment having a relative humidity that is in a range of 0% to less than 10%.

18. An apparatus for analyzing a target molecule via atomic force microscopy, the apparatus comprising: an environmental chamber; a stage assembly positioned within the environmental chamber, the stage assembly being configured to support a chip supporting a target molecule; a probe assembly positioned within the environmental chamber, the probe assembly including a stylus positioned to contact the target molecule; a displacement detector having a lens assembly positioned at least partially within the environmental chamber, the displacement detector being configured to output an indication of a position of the stylus; and a controller positioned outside the environmental chamber and operably coupled to the stage assembly and the displacement detector, the controller being configured to analyze the target molecule based at least in part on the output of the displacement detector.

19. The apparatus of claim 18, further comprising: a seal member positioned between the lens assembly and a wall of the environmental chamber.

20. The apparatus of claim 18, wherein: the displacement detector includes a laser emitter and an optical detector;Attorney Docket No. 1147-002-01WO the laser emitter and the optical detector are positioned outside the environmental chamber; and the laser emitter and the optical detector are optically coupled with the stylus.

21. The apparatus of claim 18, wherein: the environmental chamber contains an environment having a relative humidity level that is in a range of 0% to less than 10%.

22. The apparatus of claim 18, further comprising: at least one environmental sensor positioned to monitor an environment within the environmental chamber, an output of the at least one environmental sensor being indicative of the environment within the environmental chamber, the controller being configured to establish an environment within the environmental chamber based at least in part on the output of the environmental sensor.

23. The apparatus of claim 18, wherein: the displacement detector is coupled to a linear actuator positioned outside the environmental chamber; and the linear actuator is configured to affect a separation distance between the lens assembly and the stylus.

24. The apparatus of claim 18, wherein: environmental chamber defines an apparatus footprint in a range of 4.5m2and 6.0m2.

25. The apparatus of claim 18, further comprising: at least one vibratory isolation member positioned between the environmental chamber and a first support surface, the first support surface being supported by and separated from a second support surface by a height.

26. The apparatus of claim 18, wherein:Attorney Docket No. 1147-002-01WO the probe assembly includes a three-axis positioning assembly within the environmental chamber; and the controller is operably coupled to the three-axis positioning assembly to affect a position of the stylus relative to the chip supported by the stage assembly.

27. An apparatus for analyzing a target molecule via atomic force microscopy, the apparatus comprising: a probe assembly including a stylus, the stylus being configured to move in response to contact with a target molecule of a sample; a displacement detector optically coupled to the stylus and configured to output an indication of a position of the stylus; a stage assembly configured to position a chip supporting the target molecule, the stage assembly including a plurality of motors to move the chip relative to the stylus; and a controller operably coupled to the stage assembly and the displacement detector, the controller being configured to perform a plurality of operations comprising: positioning a contact portion of the stylus in contact with an imaging surface of the chip to establish a reference position for the stylus, moving the chip relative to the stylus to execute a scan of a capture region via the stylus, the capture region having a first length along a first axis and a second length along a second axis orthogonal to and coplanar with the first axis, the first length having a ratio relative to the second length in a range of 1 :4 to 1 :4100, determining a magnitude of a movement of the stylus along a third axis relative to the reference position based on an output of the displacement detector on a condition that the stylus is in contact with the target molecule, the third axis being orthogonal to the first axis and the second axis, and determining a feature of the target molecule of the sample based on the magnitude of the movement.

28. The apparatus of claim 27, wherein: the stage assembly and the probe assembly are positioned within an environmental chamber of the apparatus; andAttorney Docket No. 1147-002-01WO the plurality of operations includes establishing an environment within the environmental chamber at a humidity level that is in a range of 0% to less than 10% of a relative humidity of the environment surrounding the environmental chamber.

29. The apparatus of claim 28, wherein the plurality of operations includes: receiving an output from at least one environmental sensor positioned to monitor the environment within the environmental chamber; and affecting the humidity level based at least in part, on the output from the at least one environmental sensor.

30. The apparatus of claim 27, wherein: the probe assembly includes a three-axis positioning assembly; and the plurality of operations includes actuating at least one motor of the three-axis positioning assembly to position the contact portion of the stylus in contact with the imaging surface.

31. The apparatus of claim 27, wherein: moving the chip relative to the stylus to execute the scan of the capture region includes moving the chip relative to the stylus along the first axis and along the second axis; the movement of the chip along the first axis is between a first scan boundary and a second scan boundary of the capture region; the first scan boundary is separated from the second scan boundary by the first length along the first axis; the movement of the chip along a second axis is between a third scan boundary and a fourth scan boundary of the capture region; and the third scan boundary is separated from the fourth scan boundary by the second length along the second axis.

32. The apparatus of claim 31, wherein: the movement of the chip along the first axis is at a first velocity; andAttorney Docket No. 1147-002-01WO the movement of the chip along the second axis is at a second velocity that is less than the first velocity.

33. The apparatus of claim 31, wherein: the movement of the chip along the first axis is an oscillatory movement extending between the first scan boundary and the second scan boundary; and the oscillatory movement has a frequency in a range of at least 0.2 kilohertz and less than 5 kilohertz.

34. The apparatus of claim 31, wherein the plurality of operations includes: actuating a piezoelectric motor of the stage assembly to move the chip along the first axis between the first scan boundary and the second scan boundary; and actuating a stepper motor of the stage assembly to move the chip along the second axis between the third scan boundary and the fourth scan boundary.

35. The apparatus of claim 31, wherein: the second length has a magnitude in a range of 20 microns to 50 microns.

36. The apparatus of claim 27, wherein: the plurality of operations includes maintaining the contact portion of the stylus in contact with either the imaging surface or the target molecule during the moving of the chip relative to the stylus to execute the scan of the capture region.

37. The apparatus of claim 27, wherein: determining a feature of the target molecule of the sample includes digitizing the position of the stylus as a plurality of pixels; each pixel is either one nano meter by one nano meter or two nanometers by two nanometers; and the plurality of pixels are digitized at a rate in a range of 1.75 and 5 million pixels per second.Attorney Docket No. 1147-002-01WO38. The apparatus of claim 27, wherein: the plurality of operations includes establishing a plane defined by the first axis and the second axis at an angle rotation about the first axis that is in a range of 0 degrees to less than 2 degrees; and the chip is positioned on the plane.

39. The apparatus of claim 27, wherein: the sample is a one separate sample of a plurality of separate samples; each separate sample of the plurality of separate samples includes a separate target molecule each separate target molecule is positioned within a separate bounded sample segment of a plurality of noncontiguous bounded sample segments on the imaging surface of the chip; the scan is one of a plurality of scans; the plurality of operations includes moving the chip relative to the stylus to execute a scan of the plurality of scans for each of the separate target molecules; and the chip is positioned continuously between the stage assembly and the stylus from prior to initiation of a first scan of the plurality of scans through completion of a final scan of the plurality of scans.

40. The apparatus of claim 39, wherein: the positioning the contact portion of the stylus in contact with the imaging surface of the chip to establish the reference position for the stylus is repeated between each scan of the plurality of scans.

41. The apparatus of claim 39, wherein the plurality of operations includes: executing a single calibration to align the displacement detector with the stylus on a condition that the chip is positioned on the stage assembly prior to the execution of a first scan of the plurality of scans; and maintaining the calibration on a condition that the chip is maintained on the stage assembly.Attorney Docket No. 1147-002-01WO42. The apparatus of claim 27, wherein: the plurality of operations includes filtering the output of the displacement detector via a passband filter of 15 kHz to 1000 kHz.

43. Apparatus of claim 27, wherein: the scan has a scan rate that is greater than 144 square microns per minute and less than 500 square microns per minute.

44. The apparatus of claim 27, wherein: the moving the chip relative to the stylus to execute a scan includes executing a single uninterrupted raster scan of the capture region.

45. The apparatus of claim 27, wherein: determining the feature of the target molecule of the sample includes producing a contiguous image of the capture region with a resolution of at least 2 nm; and the target molecule is a DNA molecule that has a size that is in a range of 100,000 base pairs to 200 million base pairs.

46. An apparatus, comprising: a first stage assembly including a first base member, a first slider member movably coupled to the first base member via a first lead screw, and a first motor coupled to the first lead screw, the first slider member being movable relative to the first base member along a first axis; a second stage assembly including a second base member coupled to the first slider member, a second slider member movably coupled to the second base member via a second lead screw, and a second motor coupled to the second lead screw, the second slider member being movable relative to the second base member along a second axis orthogonal to the first axis, the second motor being the only motor positioned to drive a movement along the second axis; a third stage assembly including a third base member, a third slider member movably coupled to the third base member via a piezoelectric motor, the third slider member being movable relative to the third base member along the first axis;Attorney Docket No. 1147-002-01WO a fourth stage assembly coupled between the second slider member and the third base member, the fourth stage assembly including a fourth motor positioned to move the fourth stage assembly along a third axis orthogonal to the first axis and to the second axis; and a chip support assembly coupled to the third slider member and configured to receive a chip supporting a target molecule.

47. The apparatus of claim 46, wherein: the chip support assembly is a vacuum chuck.

48. The apparatus of claim 46, wherein: a travel of the second slide member relative to the second base member along the second axis is in a range of 40 mm to 60 mm.

49. The apparatus of claim 46: second stage assembly includes a plurality of spherical sectors positioned between the second base member and the second slide member; each spherical sector is positioned to contact a corresponding interface block of a plurality of interface blocks; and each of the spherical sectors is a contact point of a linear bearing defined by each spherical sector-interface block pairing.

50. The apparatus of claim 49, wherein: each spherical sector of the plurality of spherical sectors is coupled to the second slide member; the second base member includes a first face orthogonal to a second face and a third face, with the second face and the third face being parallel; a first portion of the plurality of interface blocks is coupled to the first face; contact between the first portion of the plurality of interface blocks and a corresponding portion of the plurality of spherical sectors limits a movement of the second slider member relative to the second base member along the third axis a second portion of the plurality of interface blocks is coupled to the second face;Attorney Docket No. 1147-002-01WO contact between the second portion of the plurality of interface blocks and a corresponding portion of the plurality of spherical sectors limits a movement of the second slider member relative to the second base member in a first direction along the first axis; a third portion of the plurality of interface blocks is coupled to the third face; and contact between the third portion of the plurality of interface blocks and a corresponding portion of the plurality of spherical sectors limits a movement of the second slider member relative to the second base member in a second direction along the first axis.

51. The apparatus of claim 50, wherein: the second stage assembly includes a first preload member positioned to bias the second slider member away from the second base member along the third axis; and the second stage assembly includes a second preload member positioned to bias the second slider member in the first direction along the first axis.

52. The apparatus of claim 46, wherein: the second motor is positioned parallel to and offset from the second lead screw; the second motor is operably coupled to the second lead screw via a gearing arrangement; and the gearing arrangement imparts a gear reduction on the second motor in a range ofl 600:1 to 1700: 1.

53. The apparatus of claim 46, wherein: the second motor and a second lead screw are configured to move the second slider member relative to the second base member at a scanning speed in a range of 0.35 to 0.50 microns per second.

54. The apparatus of claim 46, wherein: the second slider member is movably coupled to the second lead screw via a ball nut; and the ball nut includes a plurality of ball bearing elements.

55. The apparatus of claim 46, wherein:Attorney Docket No. 1147-002-01WO the second stage assembly includes an absolute encoder configured to output a signal indicative of a position of the second slider member along the second axis.

56. The apparatus of claim 46, wherein: the fourth motor is one of a plurality of picomotors; and each picomotor of the plurality of picomotors is positioned to affect a position of a portion of the fourth stage assembly along the third axis to affect a pitch and a roll of the chip.

Images