Label-free optical mapping for large-area biomechanical dynamics of multicellular systems

Abstract

A device for label-free optical mapping of biomechanical dynamics of cells is provided. The device includes an optically transparent elastic polymer membrane having a top surface and a bottom surface opposite the top surface wherein the elastic polymer membrane is disposed on an optically transparent rigid substrate. A plurality of diffractive elements are disposed on the elastic polymer membrane, each diffractive element having an opaque occluding element disposed on the top surface of the elastic polymer membrane and an opaque layer disposed on the bottom surface of the elastic polymer membrane, wherein the opaque layer has a hole the same shape as the occluding element. The occluding element is aligned with the hole so that light impinging on the diffractive element normal to the top surface of the elastic polymer membrane is substantially blocked by the occluding element from passing through the hole of the bottom opaque layer.

Description

LABEL-FREE OPTICAL MAPPING FOR LARGE-AREA BIOMECHANICAL DYNAMICS OF MULTICELLULAR SYSTEMSRelated Application

[0001] This Application claims priority to U.S. Provisional Patent Application No. 63 / 532,120 filed on August 11, 2023 which is hereby incorporated by reference. Priority is claimed pursuant to 35 U.S.C. § 119 and any other applicable statute.Technical Field

[0002] The technical field generally relates to devices and methods for profiling real-time biomechanical dynamics of cells, multicellular systems, or tissue made therefrom over large areas with high spatiotemporal resolution. More specifically, the technical field relates to devices and methods that utilize an elastic polymer membrane having a large number of occluding elements disposed on a top surface or layer and an opaque layer disposed on a bottom surface or layer of the elastic polymer membrane wherein the opaque layer includes holes aligned with the occluding elements. The elastic polymer membrane is disposed on an optically transparent substrate and an image sensor is disposed beneath the optically transparent substrate which captures optical diffraction patterns in response to illumination of the cells or tissue disposed on the elastic polymer membrane.Statement Regarding Federally Sponsored Research and Development

[0003] This invention was made with government support under GM127985 awarded by the National Institutes of Health. The government has certain rights in the invention.Background

[0004] Biomechanical properties of cells, such as cellular stiffness and cell-generated force, are direct indications of their physiological states and molecular configurations. For example, the stiffness of infected human red blood cells can increase up to 10-fold compared to their healthy counterparts. In addition, cell-generated forces play essential roles in mechanotransduction, the process that cells sense, adapt and respond to external stimuli in their surrounding microenvironments. Abnormal traction forces in cells are often related todisease states or cellular dysfunctions. For instance, insufficient contractility in cardiac muscle cells (cardiomyocytes) can lead to serious heart diseases. Furthermore, the effectiveness and possible side effects of a drug can be evaluated by observing the induced changes of these biomechanical properties in cell clusters such as abnormal contraction and arrhythmic pattern. Therefore, profiling the biomechanical properties of cell clusters in a biological system can provide valuable information in cell evolution, disease detection, and therapeutic targets. Moreover, biomechanical properties play crucial roles on the collective behaviors in multicellular structures. In a typical multicellular structure, millions of cells need to behave coordinately and coherently to ensure proper biological functions such as migration, proliferation, and differentiation. Chaotic mechanical activities in certain cell clusters can lead to fatal failure of the whole multicellular structures. For example, ventricular fibrillation or atrial fibrillation, caused by the uncoordinated rhythms of lower and upper heart chambers, can lead to deadly cardiac failure. Specifically, cell-generated forces provide means for cells to probe surrounding microenvironments, communicate between cells, and respond to external stimuli accordingly. Hence, studies of cell-generated forces and their roles on various cellular functions can provide informative understanding of diverse physiological events and the keys for multicellular structures to behave in an orchestrated manner. There are different existing techniques to measure and quantify cell-generated forces, spanning different time and length scales and under different scenarios. Such means include but are not limited to traction force microscopy (see, e.g.. Polacheck & Chen (2016) Nat. Meth. 13(5): 415-423), elastic micropillars (micropillar arrays), (see e g. Fu et al. (2010) Nat. Meth. 7(9): 733-736), and molecular sensors (see, e.g., Grashoff et al. (2010) Nature, 466: 263-266; Austen et al. (2015) Nature Cell Biol. 17: 1597-1606).Summary

[0005] Various embodiments provided herein may include, but need not be limited to, one or more of the following:

[0006] Embodiment 1: A device 100 for label free optical mapping, the device including:

[0007] a transparent elastic polymer membrane 104 including a top surface 104a and a bottom surface 104b opposite the top surface 104a wherein the elastic polymer membrane 104 is disposed on an optically transparent rigid substrate 108;

[0008] a plurality of diffractive elements 106 disposed on the elastic polymer membrane 104, each diffractive element 106 including:

[0009] an opaque occluding element 106a disposed on the top surface 104a of the elastic polymer membrane 104; and

[0010] an opaque layer 106b disposed on the bottom surface 104b of the elastic polymer membrane 104 wherein the opaque layer 106b includes a hole 106c having the same shape as the occluding element 106a;

[0011] wherein the occluding element 106a is aligned with the hole 106c and so that light impinging on the diffractive element normal to the top surface 104a of the elastic polymer membrane 104 is substantially blocked by the occluding element 106a from passing through the hole 106c of the bottom opaque layer 106b; and

[0012] wherein the elastic polymer membrane 104 is disposed on the optically transparent rigid substrate 108.

[0013] Embodiment 2: The device 100 of embodiment 1, wherein the hole 106c is the same size and shape as the opaque occluding element 106a.

[0014] Embodiment 3: The device 100 of embodiment 1. wherein the hole 106c is up to 10% smaller than the opaque occluding element 106a and aligned with the opaque occluding element 106a to allow a perimeter of light to pass through.

[0015] Embodiment 4: The device 100 according to any one of embodiments 1-3, wherein the bottom opaque layer 106b is a single continuous layer.

[0016] Embodiment 5: The device 100 according to any one of embodiments 1-3, wherein the bottom opaque layer 106b is a discrete island for each diffractive element 106.

[0017] Embodiment 6: The device 100 according to any one of embodiments 1-5, wherein the elastic polymer membrane 104 is a material selected from the group consisting of PDMS, a polyacrylamide gel, polyethylene glycol (PEG) hydrogel, collagen, extracellular matrix material (ECM), and collagen / ECM combinations.

[0018] Embodiment 7: The device 100 of embodiment 6, wherein the elastic polymer membrane 104 includes PDMS.

[0019] Embodiment 8: The device 100 according to any one of embodiments 1-7, wherein the elastic polymer membrane 104 ranges in thickness from about 2 pm up to about 5 pm.

[0020] Embodiment 9: The device 100 of embodiment 8, wherein the elastic polymer membrane 104 is about 2 pm thick.

[0021] Embodiment 10: The device 100 according to any one of embodiments 1-9, wherein the elastic polymer membrane 104 has a surface area that ranges from about 0.2 cm2up to about 100 cm2.

[0022] Embodiment 11 : The device 100 of embodiment 10, wherein the elastic polymer membrane 104 has a surface area that is about 1 cm2.

[0023] Embodiment 12: The device 100 according to any one of embodiments 1-11, wherein the elastic polymer membrane 104 has a Young's modulus that ranges from about 1 kpa up to about 1 MPa.

[0024] Embodiment 13: The device 100 of embodiment 12, wherein the elastic polymer membrane 104 has a Young's modulus that is about 50kPa.

[0025] Embodiment 14: The device 100 according to any one of embodiments 1-13, wherein the occluding element 106a and / or the bottom opaque layer 106b includes a material selected from the group consisting of a metal, an opaque plastic, an opaque dielectric, a semiconductor, or a composite layer of these materials.

[0026] Embodiment 15: The device 100 of embodiment 14. wherein the occluding element 106a and / or the bottom opaque layer 106b includes a metal.

[0027] Embodiment 16: The device 100 of embodiment 15, wherein the occluding element 106a and / or the bottom opaque layer 106b includes a metal selected from the group consisting of aluminum, aluminum oxide, magnesium, magnesium oxide, palladium, copper, copper oxide, nickel, titanium, titanium dioxide, gold, silver, silver oxide, iron, iron oxide, cobalt, and alloys thereof.

[0028] Embodiment 17: The device 100 of embodiment 16, wherein the occluding element 106a and / or the bottom opaque layer 106b includes aluminum.

[0029] Embodiment 18: The device 100 according to any one of embodiments 1-17, wherein the occluding element 106a and / or the opaque bottom layer 106b ranges in thickness from about 50 nm up to about 5 pm.

[0030] Embodiment 19: The device 100 of embodiment 24, wherein the occluding element 106a and / or the opaque bottom layer 106b have a thickness of about 200 nm.

[0031] Embodiment 20: The device 100 according to any one of embodiments 1-19, wherein the occluding element(s) 106a have a maximum dimension ranging from about 2 pm up to about 205 pm.

[0032] Embodiment 21: The device 100 according to any one of embodiments 1-20, wherein the diffractive elements 106 have an average spacing between diffractive elements 106 ranging from about 20 pm up to about 40 pm.

[0033] Embodiment 22: The device 100 according to any one of embodiments 1-21, wherein the diffractive elements 106 in the elastic polymer membrane 104 are present at a density that ranges from about 40,000 elements / cm2up to about 4,000,000 elements / cm2.

[0034] Embodiment 23: The device 100 of embodiment 22. wherein the diffractive elements 106 in the elastic polymer membrane 104 are present at a density of about 250,000 elements / cm2.

[0035] Embodiment 24: The device 100 according to any one of embodiments 1-23, wherein the device 100 further includes an image sensor 110 configured to detect illumination passing through the device 100 or reflected from the device 100.

[0036] Embodiment 25: The device 100 of embodiment 24, wherein the image sensor 110 includes one or more CCD image sensors.

[0037] Embodiment 26: The device 100 according to any one of embodiments 24-25, wherein the image sensor 110 is disposed against the optically transparent rigid substrate 108.

[0038] Embodiment 27: The device 100 according to any one of embodiments 24-25, wherein the image sensor 110 is disposed in an optical path from the optically transparent rigid substrate 108.

[0039] Embodiment 28: The device 100 according to any one of embodiments 1-27, wherein the device 100 is disposed in a stage-top incubator chamber.

[0040] Embodiment 29: The device 100 according to any one of embodiments 1-28, wherein cells 114 are disposed on the top surface 106a of the elastic polymer membrane 104.

[0041] Embodiment 30: A system 200 for label free optical mapping, the system including:

[0042] a device 100 according to any one of embodiments 1-23;

[0043] a light source 116 configured to produce illumination normal to the top surface 104a of the elastic polymer membrane 104; and

[0044] an image sensor 110 configured to detect illumination passing through the plurality of diffractive elements 106.

[0045] Embodiment 31: The system 200 of embodiment 30, wherein the light source 116 produces monochromatic illumination.

[0046] Embodiment 32: The system 200 of embodiment 31, wherein the light source 116 includes a laser or a light emitting diode (LED).

[0047] Embodiment 33: The system 200 according to any one of embodiments 30-31, wherein the image sensor 110 is a component of a detection system 206.

[0048] Embodiment 34: The system 200 of embodiment 33, wherein the detection system 206 includes one or more of a mirror, a lens, a computer 212, and control circuitry 210.

[0049] Embodiment 35: The system 200 according to any one of embodiments 30-34, wherein the system 200 further includes an incubation chamber 202 in which the device 100 is disposed.

[0050] Embodiment 36: The system 200 of embodiment 35, wherein the incubation chamber 202 is operably coupled to an incubation system 204.

[0051] Embodiment 37: The system 200 of embodiment 36, wherein the incubation system includes one or more of a CO2 module, a humidifier, a CO2 source, a temperature regulator, and a culture medium.

[0052] Embodiment 38: The system 200 according to any one of embodiments 30-37, wherein the system further includes a fluorescence microscopy system 208.

[0053] Embodiment 39: The system 200 of embodiment 38, wherein the fluorescence microscopy system includes a lens or lens system and an imaging device.

[0054] Embodiment 40: The system 200 of embodiment 39, wherein the lens or lens system includes a microscope.

[0055] Embodiment 41 : The system 200 according to any one of embodiment 39-40, wherein the imaging device includes a camera.

[0056] Embodiment 42: The system 200 according to any one of embodiments 30-41, wherein cells are disposed on the top surface 104a of the elastic polymer membrane 104 of the device.

[0057] Embodiment 43: The system 200 of embodiment 42, wherein the cells include mammalian cells.

[0058] Embodiment 44: The system 200 of embodiment 43, wherein the cells include muscle cells.

[0059] Embodiment 45: The system 200 according to any one of embodiments 42-44, wherein the incubator includes cell culture medium.

[0060] Embodiment 46: A method of fabricating a device according to any one of embodiments 1-23, the method comprising

[0061] a) paterning photoresist posts on a substrate using a first photoresist;

[0062] b) paterning a second photoresist to provide a mold comprising a plurality of pillars for molding an elastic polymer membrane 104;

[0063] c) converting the substrate with a material that forms the elastic polymer membrane 104;

[0064] d) removing residue of the elastic polymer membrane 104;

[0065] e) removing the second photoresist;

[0066] f) depositing layers of opaque material on the substrate to form occluding elements 106a and the opaque layer 106b; and

[0067] g) applying the material that forms the elastic polymer membrane 104 again to fill air gaps between the occluding elements.

[0068] Embodiment 47: The method of embodiment 46, wherein the first photoresist is a negative photoresist.

[0069] Embodiment 48: The method of embodiment 47, wherein the first photoresist is SU8 photoresist.

[0070] Embodiment 49: The method according to any one of embodiments 46-48, wherein the second photoresist is a positive photoresist.

[0071] Embodiment 50: The method of embodiment 49, wherein the positive photoresist is AZ5214 photoresist.

[0072] Embodiment 51: The method according to any one of embodiments 46-50, wherein the substrate is clear glass or plastic.

[0073] Embodiment 52: The method according to any one of embodiments 46-51, wherein steps c and g include a stamping process.

[0074] Embodiment 53: The method according to any one of embodiments 46-52, wherein the opaque material includes a metal.

[0075] Embodiment 54: The method of embodiment 53, wherein the metal is selected from the group consisting of aluminum, aluminum oxide, magnesium, magnesium oxide, palladium, copper, copper oxide, nickel, titanium, titanium dioxide, gold, silver, silver oxide, iron, iron oxide, cobalt, and alloys thereof.

[0076] Embodiment 55: The method of embodiment 54, wherein the metal includes aluminum.

[0077] Embodiment 56: The method according to any one of embodiments 46-55, wherein the material that forms an elastic polymer membrane is selected from the groupconsisting of PDMS, Polyacrylamide (PA) gels, Polyethylene glycol (PEG) hydrogels, Collagen, and an extracellular matrix material (ECM).

[0078] Embodiment 57: The method of embodiment 56, wherein the material that forms an elastic polymer membrane includes PDMS.

[0079] Embodiment 58: A method of detecting mechanical forces of cells, the method comprising:

[0080] providing cells 114 disposed on the top surface 106a of an elastic polymer membrane 104 in a system according to any one of embodiments 30-41;

[0081] exposing the device 100 to illumination 112 substantially normal to the top surface 104a; and

[0082] detecting changes in illumination / diffraction patterns produced by light passing through the device as cells 114 alter the position of occluding elements 106a thereby providing a measurement of traction forces applied by the cells 114 to the top surface 104a of the elastic polymer membrane 104a and / or to the occluding element(s) 106a.

[0083] Embodiment 59: The method of embodiment wherein the detecting is by use of a detection system 206.

[0084] Embodiment 60: The method according to any one of embodiments 58-59, wherein the method further includes contacting the cells 114 with a fluorescent label / stain and detecting fluorescence fluorescent imaging of the cells 114 using a fluorescent imaging system 208.

[0085] Embodiment 61 : The method according to any one of embodiments 58-60, wherein the cells 114 include mammalian cells.

[0086] Embodiment 62: The method of embodiment 61, wherein the cells 114 include muscle cells.

[0087] Embodiment 63: The method according to any one of embodiments 61-62, wherein the incubator chamber 202 includes cell culture medium.Brief Description of the Drawings

[0088] FIG. 1 illustrates a perspective view of the device for Large- Area Mapping of Biomechanical Dynamics via Array of Diffractive Elements (LAMBDA) described herein.

[0089] FIG. 2A illustrates a side view of the device.

[0090] FIG. 2B illustrates a magnified view of one diffractive element from the device ofFIG. 2A.

[0091] FIG. 3 illustrates the working principal of the device. The single unit of a diffractive element includes a circular metal disk (in one embodiment) on the top surface of an elastic polymer material (e.g., PDMS) and a metal sheet with central circular aperture on the bottom surface of the elastic polymer material. Without any applied force, most of the illuminated light is blocked. Only a small portion of light diffracted from the edge can penetrate. When cells exert traction force on the PDMS membrane, the top metal will move accordingly while the bottom metal remains fixed. The relative movements of the top and bottom metal layers will create eclipse-like apertures with different sizes for each diffractive element while looking from top. Consequently, the transmitted light intensity7and the diffraction pattern resultant from the apertures are a direct indication of the cellular traction force applied on the PDMS membrane.

[0092] FIG. 4 schematically illustrates the LAMBDA system integrated with a stage-top incubator according to one embodiment.

[0093] FIG. 5 schematically illustrates another embodiment of the LAMBDA system integrated with a reflective-type fluorescence imaging system.

[0094] FIG. 6A illustrates various occluding element configurations (circle, circular ring, square, square ring).

[0095] FIG. 6B illustrates a transmission (left) and reflecting (right) embodiment of the diffractive element.

[0096] FIG. 7 A illustrates panels a-f. showing the time-course intensity change of the dynamic spiral wave propagation created by cardiac beatings of monolayer of neonatal rat ventricular myocytes (NRVMs). Field-of-view of each plot is 10.6 mm x 10.6 mm. The pixel with the maximum intensity from each disk (diffraction pattern) is selected as the representative pixel. The intensity of each pixel is normalized with its value over time. The center of the spiral wave is slightly offset from the center of the plot to the left top side and rotates counterclockwise.

[0097] FIG. 7B illustrates the stimulation schematic for the time-course intensity7change of the dynamic spiral wave propagation created by cardiac beatings of monolayer of NRVMs. The stimulating electrodes are positioned approximately 2 mm away from the active area on the right. A 1Hz, 50% duty7cycle square wave with a peak-to-peak voltage of 200 mV is utilized to pace the sample for 1 min.

[0098] FIG. 7C illustrates the time-course intensity7distributions of the sample before and after the electrical stimulation. Prior to stimulation, the spiral wave initiated slightly to theleft of the center and rotated counterclockwise. Following the electrical stimulation, the waveform started to propagate from right to left, corresponding to the location of the stimulating electrode on the right-hand side of the sample. These results strongly demonstrate the system’s capabilities to track large-area biomechanical dynamics in real time.

[0099] FIG. 7D illustrates the definition of dominant frequency calculation (top) and the dominant frequency maps (bottom) before and after electrical stimulation.

[0100] FIG. 8 illustrates panels a-f showing the time-course intensity change of the planar wave propagation after electrical stimulation. The electrodes are placed on the right-hand side of the area like illustrated in FIG. 7B, and they are about 2 mm away from the edge of the field-of-view. The planar wave propagates from right (stimulating site) to left.

[0101] FIG. 9 illustrates an activation map of a measured mechanical wave resulting from the cardiac beating of neonatal rat ventricular myocytes (NRVMs).

[0102] FIGS. 10A-10D compare the time-course intensities that indicate Ca2+ concentration and biomechanical dynamics from various locations (indicated by X and Y position).

[0103] FIG. 1 1 includes panels a-h illustrating the fabrication process flow showing: (a) patterning SU8 posts (b) patterning photoresist AZ5214 (c) stamping PDMS (d) removal of PDMS residue (e) removal of photoresist AZ5214 (f) isotropic metal deposition (g) stamping PDMS (h) final device.

[0104] FIG. 12 illustrates diffraction patterns with representative pixels marked by circles.

[0105] FIG. 13 A illustrates the time-course intensity of a representative pixel.

[0106] FIG. 13B illustrates the normalization of time-course intensity.

[0107] FIG. 13C illustrates the definition of activation time.

[0108] FIG. 14A illustrates the maps of activation time, each accompanied by a representative time-course intensity trace in the inset for that day. A defective region in the top-left comer of this device renders the diffraction patterns inaccessible, although the cells were present in that area. Day 3 and Day 4 present similar spatial patterns of wave propagation. While constant beatings were observed on Day 3, irregular beating was recorded on Day4. No clear beatings were observed on Day 5. On Day 6, a spiral wave pattern with a beating rate of 4.4 Hz appeared. On Day 7, a low-concentration phenylephrine was added to the sample as chemical stimulation. Just before the drug administration (time = -3 minutes), two clusters were observed at the top right and top bottom, beating with a rate of 1.29 Hz. By the end of the treatment, the beating frequency increased to 2.89 Hz, with the two clustersbecoming more connected. The drug was then removed, and fresh medium was replenished to the cells culture. After overnight incubation, the recording resumed on Day 8. At this time, the connected clusters separated, with a longer delay between their activation time. Lastly, on Day 9, only the cluster in the top-right comer remained constantly beating at 2.5Hz.

[0109] FIG. 14B shows the dominant frequency for each day, with a zoom-in figure (bottom) illustrating the change associated with the chemical stimulation performed on Day 7.

[0110] FIG. 15A illustrates time-course intensity distributions, revealing the biomechanical dynamics measured by the platform using diffractive elements. A spiral wave is clearly visible across the entire field of view-.

[0111] FIG. 15B shows time-course intensities indicating Ca2+ concentration (labelled as “F[Ca2+]) and biomechanical dynamics measured by the platform (curves labelled as “biomechanics”) from various locations. It is evident that the fluorescent signal and the biomechanical signal exhibit similar profiles, including certain elongated periods.Detailed Description of Illustrated Embodiments

[0112] Described herein is device 100 for a label-free optical mapping and methods of use therefore, e.g., for profiling real-time biomechanical dynamics (e.g., of cells 114) over large areas with high spatiotemporal resolution. It is realized by devices 100 w ith a massive number of optical diffractive elements 106 embedded periodically in an elastic polymer membrane 104 (referred to as an array of diffractive elements 106). The optical diffraction elements 106 convert the biomechanical properties of cells 114 seeded on top (or bottom) of the elastic polymer membrane 104 to optical diffraction patterns, which can be measured over a large area via one or more image sensors 110. In an illustrative, but non-limiting embodiment, an observation field of view up to 10.6 mm by 10.6 mm is achieved, which is 3 orders of magnitude improvement compared to traditional methods such as traction force microscopy or micropillar arrays. Meanwhile, high spatiotemporal resolution is maintained, allowing one to measure transient activities at the cellular level.

[0113] In addition to measuring the biomechanical properties alone, the innovative approach (designated large-area mapping of biomechanical dynamics via array of diffractive elements 106 (aka LAMBDA) holds promise for studying the complex interplay between biomechanical, biochemical, and bioelectrical properties in biological systems.

[0114] Large-Area Mapping of Biomechanical Dynamics via Array of Diffractive Elements (LAMBDA)

[0115] One illustrative, but non-limiting embodiment of the LAMBDA platform or system 200 is schematically illustrated in FIGS. 1 and 2A, 2B. In various embodiments the device 100 utilizes a number of optical diffractive elements 106 embedded in an elastic polymer membrane 104 (e.g., polydimethylsiloxane (PDMS)) to track the traction forces generated by the cells 114 seeded on the top thereof. In certain embodiments, the spacing of the diffractive elements 106 may be random, or haphazard, although preferably the spacing is periodic. The elastic polymer membrane 104 has a substantially planar top surface 104a and a substantially planar bottom surface 104b opposite the top surface. The LAMBDA system 200 utilizes a collimated light beam, emitted by a single- wavelength light source 116 (e.g.. light-emitting diode (LED)), illuminates the diffractive elements 106 and the transmitted diffraction patterns of each diffractive element 106 are detected on the one or more image sensors 110 below. When cells 114 apply traction forces on the PDMS membrane 104, the induced lateral displacements can be monitored by the changes of the transmitted intensities and diffraction patterns from each diffractive element 106 projected on the one or more image sensors 110.

[0116] The diffractive elements 106 are key components in the LAMBDA system 200. The diffractive elements 106 translate the lateral displacement induced by the cell-generated forces on the elastic polymer membrane 104 to discernible changes that can be measured by the one or more image sensors 1 10. As shown FIGS. 2A, 2B, the single unit of a diffractive element 106 includes an opaque occluding element 106a (e.g., a circular (or other shaped) metal disk) disposed on the top surface 104a of the elastic polymer membrane 104 and an opaque layer 106b with a central hole / aperture 106c (e.g.. a metal sheet with central circular aperture) disposed on the bottom surface 104b of the elastic polymer membrane 104. The occluding element 106a is typically aligned with hole 106c in the bottom surface 106b and in certain embodiments has the same shape and size as the hole / aperture 106c in the bottom surface 106b so that light impinging on the diffractive element 106 normal to the opaque occluding element 106a is substantially blocked by the occluding element 106a from passing through the hole / aperture 106c of the bottom surface 106b. In other words, the top opaque occluding element 106a and bottom surface 106b with hole 106c form a complementary shape in terms of the overall opacity of the diffractive element 106 but are offset vertically by the elastic polymer membrane 104 (e.g.. PDMS membrane).

[0117] In various embodiments, the elastic membrane 104 having the opaque occluding element(s) 106a is disposed on an optically transparent rigid substrate 108. Since the bottom surface 106b is disposed on the optically transparent substrate 108, movement of this portion of the diffractive element 106 is restrained. Without any applied force, most of the light shining normal to the top surface 104a of the elastic polymer membrane 104 is blocked from passing through the device 100. Only a small portion of light diffracted from the edge(s) can penetrate. When cells 114 are disposed, for example, on the top surface 104a of the elastic polymer membrane 104 and exert traction forces on the elastic polymer membrane 104, the top metal disks that form the opaque occluding elements 106a will move accordingly while the bottom metal islands of the opaque layer 106b remain fixed. The relative movements of the top occluding elements and bottom metal layers (106a and 106b, respectively) can create eclipse-like apertures with different sizes for each diffractive element 106 as illustrated in FIG. 3. The transmitted light intensity and the diffraction pattern are direct indicators of the cellular traction forces applied on the elastic polymer membrane 104.

[0118] There are several unique advantages of the LAMBDA system 200 compared to the existing technologies for measuring biomechanical properties, especially referring to traction force measurement. First, for conventional in traction force microscopy (TFM), force- induce displacements of beads or pillars are directly tracked. This often requires a high- magnification objective lens or large numerical aperture (N.A.) optics to accurately resolve the minute displacements of beads or pillars, thereby greatly limiting the observable region. By contrast, via the optical diffractive elements 106 described herein, the LAMBDA platform measures the changes of displacement-induced optical properties (transmitted intensity and diffraction patterns) as opposed to measuring the force-induced displacements directly. Therefore, the system does not carry the field-of-view (FOV) limitation imposed by the high N.A. optics. Consequently, the LAMBDA platform can capture fast and transient biomechanical dynamics propagating over a larger FOV with superior spatial resolution that is not achievable using traditional TFM. Secondly, no labeling chemicals such as fluorescent dyes are needed during measurements. Hence, the label-free nature of the LAMBDA platform can preserve native and physiological conditions of cells 114. enabling continuous and prolonged observation of biomechanical dynamics. Thirdly, the surface of the device 100 is relatively flat. Unlike elastic micropillars approaches, no protruded structures or gaps are present on the devices used by the LAMBDA system 200 to alter the focal adhesions of cells 114. Finally, the integration of the LAMBDA system 200 and other microscopes offersgreat potential to allow simultaneous monitoring of biomechanical dynamics along with other cellular properties such as molecular configuration, transmembrane voltage, or ion concentration.

[0119] The diffractive elements 106 used in the devices 100 described herein may be varied with respect to a number of different parameters including, but not limited to the opaque occluding element 106a shape and periodicity (density), elastic polymer membrane 104 thickness, and stiffness, illumination wavelength, and the like.

[0120] Occluding element shape

[0121] Thus, for example, in certain embodiments, the occluding elements 106a are circular and the hole 106c in the bottom surface 106b is also circular. In certain embodiments, the occluding element 106a and the hole 106c are the same shape so that, absent displacement of the occluding element(s) 106a, substantially all of the light falling on the diffractive element 106 (in a direction normal to the surface 104a) is blocked from passing through the structure, e.g., as illustrated in FIG. 6A (circle). Alternatively, in certain embodiments, the occluding element 106a has a smaller diameter than the hole 106c so that, absent displacement of the occluding element(s) 106a, an annulus of light can pass through the diffractive element 106 as illustrated in FIG. 6A, (circular ring). It will also be recognized that the occluding element(s) 106a and the hole(s) 106c can be any of a wide number of shapes. Such shapes include, but are not limited to regular polygons e.g., squares as illustrated in FIG. 6A (square and square ring), rectangles, triangles, and other regular polygons comprising 5 up to 100 or more sides. In certain embodiments the occluding element(s) 106a and the hole(s) 106c can be irregular polygons, ovals, and other shapes. It will be recognized however, that for analytic simplicity, in various embodiments circular occluding element(s) 106a and the hole(s) 106c may be preferred.

[0122] Occluding element size and periodicity (spatial density)

[0123] As in the case of traditional traction force microscopy, there exists a tradeoff between the occluding element 106a size / periodicity (density) and the spatial resolution of the device 100. Namely, a smaller occluding element 106a and higher density will render a better spatial resolution. In reality, however, the image sensor 110 typically has a finite pixel dimension, therefore, the occluding element 106a size / periodicity cannot be infinitesimal in order for the diffraction pattern to be resolvable. Given the typical pixel dimension of a modem image sensor (1 to 4 pm) and the average size of mammalian cells (e.g., cardiomyocytes is about 100 pm by 20 pm), in certain embodiments, the devices haveoccluding element 106a sizes and periodicity fall that range from about 5pm up to 15 pm (maximum dimension) and from about 20 pm up to about 40 pm spacing between diffractive elements. These ranges provide diffraction patterns that are able to be resolved by typical image sensors 110 while still maintaining spatial resolution at a cellular level.

[0124] Device orientation

[0125] It will also be recognized that while the diffractive elements 106 are described with the occluding element 106a on the top surface 104a of the elastic polymer membrane 104 and the opaque layer 106b with the hole 106c on the bottom surface 104b of the elastic polymer membrane 104 (see, e.g., FIG. 6B, left), in certain embodiments, this configuration can be reversed with the occluding element(s) 106a on the bottom surface 104b of the elastic polymer membrane 104 and the opaque layer 106b with the hole 106c on the top surface 104a of the elastic polymer membrane 104 as illustrated in FIG. 6B, right.

[0126] Elastic membrane thickness

[0127] In various embodiments the thickness of the elastic membrane 104 controls the offset distance between the top occluding element 106a and bottom 106b layers. This offset distance has impacts not only on the amount of light transmission through the elastic membrane layer 104 but also on the optical sensitivity7(change of the diffraction patterns with respect to the lateral displacements of the occluding element(s) 106a). Considering the extreme case that the offset distance drops to zero, the occluding element 106a will typically completely block light transmission through the diffractive element 106 if there is no lateral displacement. Once lateral displacements of the occluding element(s) 106a occur, the passage of light is permitted and there can be a steep increase in transmitted light intensity. Thus, where the elastic layer 104 is thin there is high optical sensitivity of the device. In the other extreme where there is a large case that there is a huge offset distance between the occluding element(s) 106a and the opaque layer 106b, the large distance in will permit high transmission of light, creating a bright background even without any lateral displacements of the occluding element(s) 106a. Hence, changes of diffraction patterns with lateral displacements of the occluding element(s) 106a only contributes a minute amount to the existing bright background.

[0128] On the other hand, mechanical sensitivity’ (change of the lateral displacement of occluding element(s) 106a with respect to the exerted cellular force) shows an opposite trend with respect to optical sensitivity. In general, the thicker the elastic membrane 104, the largerthe induced displacements of the occluding element(s) 106a under the same loading force, thereby providing higher mechanical sensitivity.

[0129] To sum up, a device 100 with a thin elastic polymer membrane 104 will have higher optical sensitivity but lower mechanical sensitivity, compared to a device 100 with a thick elastic polymer membrane 104. In practice, in certain embodiments the elastic polymer membrane 104 may have a thickness within the range from about 2 pm up to about 5 pm to provide a preferably tradeoff between optical sensitivity and mechanical sensitivity. It will be recognized, however, that the devices 100 need not be limited to this range of elastic membrane thicknesses.

[0130] Elastic membrane stiffness

[0131] In addition to the thickness of elastic polymer membrane 104, the stiffness of the elastic polymer membrane 104 also affects the amount of lateral displacement of occluding element(s) 106a produced by cells 14. Under the same loading force, the softer the elastic polymer membrane 104, the larger the resulting displacement of an occluding element 106a. However, for a biological system, cell-generated forces can vary significantly with changes in their micro-environment. It has been reported that cells 114 tend to exert larger forces when they are disposed on harder substrates. The relationship between the force and the substrate stiffness is complex and nonlinear. Therefore, in certain embodiments, elastic polymer membrane 104 stiffness may be chosen to mimic the physiological tissue environment of the cells 114.

[0132] The stiffness of a material is characterized by the Young’s modulus in the linear region of its stress-strain curve. In the present case different stiffness elastic polymer membranes 104 can readily be provided. For example, different stiffness of PDMS can be obtained, for example, by mixing Sylgard PDMS 184 and PDMS 527 with different ratios (see, e.g., Palchesko et al. (2012) PLoS ONE, 7(12): e51499, incorporated by reference herein). In experiments with neonatal rat ventricular myocytes (NRVM), PDMS with a Young's modulus of 50 kpa was used and provided by a mixture of PDMS 527 and 184 with a ratio of 10 to 1). Of course, in various embodiments, the Young's modulus of the elastic membrane 104 can readily be varied depending on the application.

[0133] Illumination Wavelength

[0134] Theoretically, the size of the diffraction patterns at a given observation plane is proportional to the illumination wavelength. In other words, the shorter the illumination light wavelength, the smaller the diffraction pattern. Nevertheless, in practice, the diffractionpatern can be digitalized by the finite pixel size of the image sensor 110. Therefore, the impact of wavelength on the diffraction is typically smoothed out. On the other hand, to avoid scatering and absorption of light from the biological sample, illumination wavelengths with a large penetration depth are favored. In certain embodiments this spectrum window can range from about 650 nm up to about 1350 nm, within which the penetration depth is typically maximized. Combining the above two points with the fact that the quantum efficiency of image sensor 110 can drop significantly for wavelengths above 1 um. red to near-infrared illumination (660 nm to 800 nm) was used in the experiments described herein. It will be recognized, however, that illumination wavelength need not be limited to the above ranges.

[0135] Illustrative devices for label-free optical mapping

[0136] In view of the foregoing, in certain embodiment a device 100 for label free optical mapping is provided. In certain embodiments the device 100 includes an optically transparent elastic polymer membrane 104 that includes a top surface 104a and a bottom surface 104b opposite the top surface 104a and wherein the elastic polymer 104 is disposed on an optically transparent rigid substrate 108. A plurality of diffractive elements 106 are disposed on the elastic polymer membrane 104, where each diffractive element 106 includes an opaque occluding element 106a disposed on the top surface 104a of the elastic polymer membrane 104 and an opaque layer 106b disposed on the bottom surface 104b of the elastic polymer membrane 104 wherein the opaque layer 106b includes a hole 106c the same shape as the corresponding occluding element 106a. In various embodiments the occluding element 106a is aligned with the hole 106c and so that light impinging on the diffractive element 106 normal to top surface 104a of the elastic polymer membrane 104 is substantially blocked by the occluding element 106a from passing through the hole 106c of the botom opaque layer 106b. In certain embodiments the elastic polymer membrane 104 is disposed on an optically transparent rigid substrate 108. In certain embodiments the hole 106c is the same size and shape as the occluding element 106. In certain embodiments the hole 106c is up to 10% smaller than the occluding element 106 and aligned with the occluding element 106 to allow a perimeter of light to pass through the diffractive element 106. Note that in a typical device there is an array of diffractive elements 106 disposed on the elastic polymer layer 104. Each diffractive element 106 is associated with an opaque occluding element 106a and corresponding hole 106c. In certain embodiments the botom layer 106b is a singlecontinuous layer, while in other embodiment the bottom layer 106b forms a discrete island for each diffractive element 106.

[0137] In certain embodiments the elastic polymer membrane 104 includes a material selected from the group consisting of PDMS, Polyacrylamide (PA) gels, Polyethylene glycol (PEG) hydrogels, Collagen and an extracellular matrix material (ECM) such as Matrigel, and others. In certain embodiments the elastic polymer membrane 104 is made from PDMS. In various embodiments the polymer membrane ranges in thickness from about 1 pm. or from about 2 pm up to about 8 pm, or up to about 6 pm, or up to about 5 pm, or up to about 4 pm. In certain embodiments the elastic polymer membrane is about 2 pm thick. In certain embodiments the elastic polymer membrane 104 has a surface area that ranges from about 0.1 cm2, or from about 0.2 cm2, or from about 0.3 cm2, or from about 0.4 cm2, or from about 0.5 cm2, up to about 200 cm2, or up to about 100 cm2, or up to about 100 cm2, or up to about 100 cm2, or up to about 60 cm2, or up to about 40 cm2, or up to about 30 cm2, or up to about 20 cm2, or up to about 10 cm2, or up to about 5 cm2. In certain embodiments the elastic polymer membrane 104 has a surface area that is about 1 cm2.

[0138] In certain embodiments the elastic polymer membrane 104 has a Young's modulus that ranges from about 0.2 kpa, or from about 0.5 kpa, or from about 1 kpa up to about 400 kpa, or up to about 300 kpa, or up to about 200 kpa. In certain embodiments the elastic polymer membrane 104 has a young's modulus that is about 50kpa. In certain embodiments the occluding element 106a and / or the bottom layer 106b includes a material selected from the group consisting of a metal, an opaque plastic, an opaque dielectric, a semiconductor such as silicon, or a composite layer comprising one or more of these materials. In certain embodiments the occluding element 106a and / or the bottom layer 106b includes a metal. In certain embodiments the occluding element 106a and / or the bottom layer 106b is a metal selected from the group consisting of aluminum, aluminum oxide, magnesium, magnesium oxide, palladium, copper, copper oxide, nickel, titanium, titanium dioxide, gold, silver, silver oxide, iron, iron oxide, cobalt, and alloys thereof. In certain embodiments the occluding element 106a and / or the bottom layer 106b includes aluminum. In various embodiments the occluding element 106a and the bottom layer 106b are sufficiently thick and opaque to completely block the passage of light. In certain embodiments the occluding element 106a and the bottom layer 106b ranges in thickness from about 50 nm, or from about 100 nm, or from about 150 nm up to about 5 pm, or up to about 4 pm, or up to about 3 pm, or up toabout 2 un, or up to about 1 pm. In certain embodiments the occluding element 106a and / or the bottom layer 106b have a thickness of about 200 nm.

[0139] In certain embodiments the occluding element(s) 106a have a maximum dimension (seen from the top) ranging from about 2 pm up to about 20 pm. In certain embodiments the diffractive elements 106a have an average spacing between elements ranging from about 5 pm, or from about 10 pm, or from about 15 pm, or from about 20 pm up to about 80 pm, or up to about 60 pm. or up to about 40 pm, or up to about 30 pm. or up to about 25 pm. In certain embodiments the occluding elements 106a in the elastic polymer membrane 104 are present at a density that ranges from about 20,000 elements / cm2, from about 30,000 elements / cm2or from about 40,000 elements / cm2up to about 4,000,000 elements / cm2, or up to about 3,000.000 elements / cm2, or up to about 2,000,000 elements / cm2, or up to about 1,000,000 elements / cm2, or up to about 500,000 elements / cm2, or up to about 300,000 elements / cm2. In certain embodiments the occluding elements 106a in the elastic polymer membrane 104 are present at a density of about 250,000 elements / cm2.

[0140] In certain embodiments the device 100 further includes one or more image sensors 110 configured to detect illumination passing through or reflected from the device 100. In certain embodiments the image sensor(s) 110 includes one or more CCD image sensors or cameras / camera elements. In certain embodiments the optical sensor(s) 110 are disposed against the optically transparent rigid substrate 108. In certain embodiments the image sensor 110 is disposed in an optical path from the optically transparent rigid substrate 108 and not in contact with the optically transparent rigid substrate 108 (e.g., a gap or space separates the two).

[0141] In certain embodiments the device 100 is disposed in a stage-top incubator chamber 202 and, optionally, may comprises cells 114 (e.g., mammalian cells) disposed on the top surface 104a of the elastic polymer membrane 104.

[0142] +Methods of fabricating the devices described herein are illustrated in Example 1.

[0143] Systems comprising the device for Large-Area Mapping of Biomechanical Dynamics via Array of Diffractive Elements (LAMBDA).

[0144] In certain embodiments, e.g., when in use, the LAMBDA device 100 described above is provided as a component of a system 200, e.g., as illustrated FIG. 4. In various embodiments the system 200 will include the device 100, a light source 116 disposed above (or below) the device 100 that provides illumination 112 substantially normal to the topsurface 104a of the elastic polymer membrane 104 (e.g., as best seen in FIG. 1). An image sensor 110 can be disposed below the optically transparent rigid substrate 108 or in a light path that has passed through the device 100 and the optically transparent rigid substrate 108. In certain embodiments the image sensor 110 can be a component of a detection system 206 as seen in FIG. 4. In certain embodiments the detection system 206 may include various optical elements (e.g., lenses, prisms, mirrors such as prism mirror 207 (90° prism mirror) to transmit the diffraction pattern to the image sensor 110. In certain embodiments the system 200 can include a computer 212 operably coupled to the image sensor 110 and / or control circuitry 210 to control the light source 116 and / or acquire image data obtained by the image sensor 110.

[0145] In certain embodiments the device 100 described above can be disposed in an incubation chamber 202 (e.g., a stage top incubator) as schematically illustrated in FIG. 4. In illustrative, but non-limiting embodiments, the incubation chamber 202 can be coupled to an incubation system 204, that may include, for example, a temperature controller 204a. a CO2 module 204b, a humidifier 204d, a CO2 source 204c, and the like.

[0146] FIG. 7A illustrates the dynamic spiral wave propagation created by cardiac beatings of monolayer of neonatal rat ventricular myocytes (NRVMs) while FIG. 8 presents the planar wave propagation from the same cell culture after electrical stimulation. The electrodes are placed on the right-hand side of the FIG. 8, and they are about 2 mm away from the edge of the field-of-view. The planar wave propagates from right (stimulating site) to left. The platform has captured this spiral pattern and subsequently observed cardioversion after applying electrical stimulation. The stimulation schematic is displayed in FIG. 7B. The stimulating electrodes are positioned approximately 2 mm away from the active area on the right. A 1Hz. 50% duty cycle square wave with a peak-to-peak voltage of 200 mV is utilized to pace the sample for 1 min. FIG. 7C demonstrates the time-course intensity distributions of the sample before and after the electrical stimulation. Prior to stimulation, the spiral wave initiated slightly to the left of the center and rotates counterclockwise. Following the electrical stimulation, the waveform starts to propagate from right to left, corresponding to location of the stimulating electrode on the right-hand side of the sample. These results strongly demonstrate the capability of the system 200 to track large-area biomechanical dynamics in real time.

[0147] Next, the dominant frequency of the constantly beating cells is calculated before and after electrical stimulation. This is accomplished by performing fast Fourier transform(FFT) of the time-course intensity traces from each diffractive element. The frequencycomponent with the largest intensity value is considered the dominant frequency for that element. The dominant frequency maps before and after electrical stimulation are given in FIG. 7D. The mode values (most frequent number) are 2.7 Hz and 2.3 Hz, respectively. The platform's high spatial resolution and large area features enable one to discern both the main beating frequency exhibited by the majority of cells and various heterogeneous behavior, which may express as slower or faster beats compared to the majority. This will be challenging to achieve for both traditional traction force microscopes or conventional optical mapping techniques using fluorescent dyes, in which the spatial resolution and large area cannot be attained simultaneously.

[0148] In certain embodiments, the LAMBDA platform or system 200 can be integrated with a fluorescent imaging system 208, e.g., as illustrated in FIG. 5, to conduct simultaneous mappings of the multiple properties. In embodiment illustrated in FIG. 5, a dichroic mirror 214, emission fdter 216, camera lens 218 and camera 220 are arranged and aligned along the reflective path to capture a fluorescent signal emitted by the cells 114 after staining. Meanwhile, biomechanical dynamics of the cells 114 can still be measured using the transmitted path without modification. FIG. 9 show s the activation map of the measured mechanical w ave resulting from the cardiac beating of NRVMs and FIGS. 10A-10D compares the time-course intensities that indicate the calcium ion concentration [Ca2+] and biomechanical dynamics from various locations. It is believed to be the first demonstration of detailed mechanical w ave propagation and the corresponding calcium ion transient.

[0149] Illustrative, but non-limiting methods of use of the devices 100 and systems 200 described herein are illustrated in Examples including Example 2.

[0150] EXAMPLES

[0151] The following examples are offered to illustrate, but not to limit the claimed invention.

[0152] Example 1 - Fabrication of the Devices

[0153] In certain embodiments the devices 100 described herein can be fabricated using photolithography, isotropic metal deposition and PDMS stamping. In one illustrative, but non-limiting embodiment, the optically transparent rigid substrates 108 are standard 1 mm- thick glass slides. The fabrication process flow- is schematically illustrated in given in FIG. 11 and described below-. Detailed parameters for each step can be found in the "Recipes" section, below.

[0154] (a): Patterning SU8 posts on the glass substrate.

[0155] In certain embodiments SU8 (epoxy-based negative photoresist) or other photoresist posts can serve as spacers and mechanical support for the subsequent PDMS stamping. The SU8 posts can be patterned using standard photolithography.

[0156] (b): Patterning photoresist AZ5214

[0157] In certain embodiments a positive photoresist (e.g.. AZ5214) can be used to mold the PDMS. It can be patterned using standard photolithography and aligned with the SU8 posts patterned as seen in in Step (a) (Figure Error! Reference source not found.).

[0158] (c): Stamping PDMS

[0159] Uncured PDMS (or other transparent elastic polymer) is poured on the patterned substrate and covered by a polyvinyl acetate (PVA)-coated glass. PVA is a water-soluble polymer and is used as a sacrificial layer during the stamping process. A constant pressure (23 kpa) is applied on PVA-coated glass through a PDMS buffer. The device is left in the room temperature for at least 48 hours until PDMS is fully cured. Then, the PVA-coated glass is removed by soaking the whole sample in deionized water.

[0160] (d): Removal of PDMS residue

[0161] After the first PDMS stamping, reactive ion etching (Oxford RIE) is utilized to clean and etch the PDMS residue until photoresist AZ5214 is completely exposed. The etching speed is about 400 nm per min and the etching time is about 2.5 minutes.

[0162] (e): Removal of photoresist AZ5214

[0163] Next, the device is soaked into acetone solution and put in the ultrasound bath for 5 seconds to strip the photoresist AZ5214. The PDMS disks, which are the main component to create displacements, are formed after this step.

[0164] (I): Isotropic metal deposition

[0165] Electron beam evaporation is utilized to implement isotropic metal deposition (SLOAN, SL 1800). Aluminum is chosen due to its high reflectivity in visible and near infrared spectra. According to the working principle of the diffractive element 106, however, any light-blocking material can be used.

[0166] (g): Stamping PDMS

[0167] Lastly, to ensure a flat surface, which is favored by most cells to develop focal adhesions physiologically, PDMS stamping process, the same as (c), is conducted again to fill PDMS to the air gaps in between PDMS disks. After removing the PVA-coated glass, the final device is shown in step (h) of FIG. 11.

[0168] Recipes for the fabrication process

[0169] Recipe for Patterning SU8

[0170] SU8 2002 (~ 2um thick)

[0171] Oxygen plasma treatment for the glass substrate, 30s (Technics fluorine RIE 800, 500 mTorr, 80 W).

[0172] Spin coating

[0173] Step 1 : rate 1000 rpm, ramp 500 rpm / s, 5s.

[0174] Step 2 : rate 3000 rpm, ramp 1000 rpm / s, 45s.

[0175] Soft bake 95°C, 1.5 mins.

[0176] Exposure 15 s (Lamp power 8mW), hard contact.

[0177] Post exposure bake 95°C. 1.5 mins.

[0178] Develop in SU8 developer, 1 min.

[0179] Hard bake 150°C, 10 mins.

[0180] Recipe for Patterning AZ5214 Photoresist

[0181] AZ5214 (~ 2um thick)

[0182] Vapor deposition of adhesive layer HMDS. 3 mins.

[0183] Spin coating.

[0184] Step 1 : rate 500 rpm, ramp 250 rpm / s, 5s.

[0185] Step 2 : rate 2000 rpm, ramp 1000 rpm / s, 45s.

[0186] Soft bake 100°C, 1 min 111°.

[0187] Exposure 13.5 s (Lamp power 8mW), hard contact.

[0188] Develop in diluted AZ 400K Developer (developer: water = 1:4), 25s.

[0189] Recipe for Making PVA-Coated Glasses

[0190] 4 %wt PVA solution is spined coat at 2000 RPM on glass substrate, baked at 65°C for 1 minute and then 130°C for 30 minutes.

[0191] Recipe for Removing PDMS Residual

[0192] SF6 = 80.0 seem, 02 = 20.0 seem, RF forward power = 270W, Process pressure = 0.24 Torr.

[0193] Recipe for Isotropic Metal Deposition

[0194] Metal: SiO2 / Ti / Al / Ti / SiO2; Thickness: lOnm / lOnm / lOOnm / lOnm / lOnm; Rate:1 A / s for SiO2 and Ti, 3 / s for Al.

[0195] Titanium and silicon dioxide are used to improve the adhesion in between glassmetal interface metal-PDMS interface.

[0196] Example 2 - Real-Time Visualization of Large-Area Cellular Biomechanical Dynamics using the LAMBDA Platform

[0197] To demonstrate the capabilities of the LAMBDA platform and system 200 for visualizing the large-area cellular biomechanical dynamics in real-time, monolayer tissue sheet composed of a few hundreds of thousands of neonatal rat ventricular myocytes (NRVMs) cells 114 were seeded on the devices 100. Various beating patterns were recorded. An observation field-of-view up to 10.6 mm by 10.6 mm was achieved. Meanwhile, high spatiotemporal resolution was maintained (with temporal resolution up to 130 frames per second and spatial resolution down to 20 pm). Hence, global beating behavior and local heterogeneity could readily be recognized at the same time. Moreover, several conditions such as spontaneous beating, electrical stimulation and chemical stimulation were included to illustrate the strengths for real-time monitoring of fast biomechanical dynamics.Additionally, the label-free nature of the platform allowed long-term observation with minimized perturbation of the physiological environment.

[0198] As cell cultures grow and mature over time, recording their biomechanical dynamics without disrupting their physiological activities can effectively eliminate uncertainties associated with external factors such as the impacts of fluorescence dyes on cells 114. This enables a more straightforward recognition of the causality7of biological events and pathological progression. One strength of the current platform or system 200 is the ability to conduct long-term observation of samples under native conditions by continually logging the biomechanical dynamics of the same cell culture daily for up to 7 days, which cannot be achieved by conventional optical mapping using fluorescent dyes. Limitations such as dye internalization, photobleaching and phototoxicity' typically restrict the maximum observation duration to only several hours

[0199] Here, the day when the NRVM cells were isolated and seeded on the device 100 is denoted as Day 0. The cells 114 usually begin beating within 24 to 48 hours after seeding. Measurements started on Day 3 and were repeated daily until Day 9. On Day 7, a chemical stimulation was conducted using a low concentration of phenylephrine and recorded the corresponding response. Phenylephrine, being a kind of a-1 adrenergic receptor agonist, frequently induces hypertrophic responses in cardiomyocytes. FIG. 14A displays the maps of activation time, each accompanied by a representative time-course intensity trace for each day. There is a defective region in the top left comer of this device 100, rendering the data (diffraction patterns) inaccessible. However, the cells 114 are present in that area. FIG. 14Bpresents the dominant frequency for each day with a zoom-in figure illustrating the change associated with the chemical stimulation performed on Day 7.

[0200] As can be seen in FIG. 14A, on Day 3, regular beatings with a rate of 0.81 Hz were observed. The onset of the beating was from the top-left and propagated to the bottom-right comer. Then, on Day 4, the beating pattern followed the same spatial arrangement, traveling from the top-left to the bottom-right. However, the beating of NRVM is not consistent in culture, with only two beats occurring over the entire 16.8-second recording. Although the beating activity was not steady, the captured two beatings had similar time difference as those on the previous day (1.25s). Interestingly, the activation time map shows denser points compared to that of Day 3. This may indicate that more cells 114 were engaged in the beating activities, resulting in large traction forces being applied on the device 100 to activate more diffractive elements 106. Next, no clear beating of the cells 1 14 was recorded on Day 5. Subsequently, on Day 6, a spiral pattern appeared with a beating rate of 4.4 Hz. Afterward, chemical stimulation was performed on Day 7. Specifically , a low-concentration (1 pM) phenylephrine (PE) was added to the culture medium and treated for 153 minutes. Just before the drug administration (time = -3 minutes), two clusters were observed at the top-center and bottom-center, beating with a rate of 1 .29 Hz. Phenylephrine is an alpha agonist and know n to increase the frequency of beating cardiac muscle cells in culture. By the end of the treatment, the beating frequency increased to 2.89 Hz, with the two clusters becoming more connected. The drug was then removed, and fresh medium was replenished to the cell culture. After overnight incubation, the recording resumed on Day 8. At this time, the connected clusters separated, with a longer delay between their activation time. The majority of cells beat at 0.51 Hz. Lastly, on Day 9, only the cluster in the top-center remained constantly beating at 2.5Hz. The beating was initiated from the bottom of the cluster and traveled to the top, opposite to the directions shown in the previous few days. Additionally, it is worth noting that there were tw o consecutive peaks associated with each beating.

[0201] In summary, long-term observations of large-area biomechanical dynamics are achieved with the span extended to a week, with minimal disruption to the physiological activities of cells 114. It is crucial to emphasize that the designed experiment is not aimed at drawing any biological conclusions from a single sample, but rather to demonstrate the platform's capabilities to dynamically monitor cells 114 under native conditions over an extended period. By continuously observing biomechanical dynamics and minimizingdisturbances to cell cultures, the platform holds great potential for applications in studying disease development and evaluating the effectiveness of drugs.

[0202] Finally, the LAMBDA system 200 was integrated with a fluorescent imaging system 208 that included a florescence microscope to conduct simultaneous mapping of the calcium concentration and biomechanical transient. As a proof-of-concept, simultaneous measurements of calcium ion (Ca2>) concentration and biomechanical dynamics were performed. The high-affinity calcium ion dye. Rhod-2, was utilized for fluorescent imaging.

[0203] FIG. 15A illustrates the time-course intensity distributions, revealing the biomechanical dynamics measured by the platform or system 200 using diffractive elements 106. A spiral wave is clearly visible across the entire field of view. In FIG. 15B, the timecourse intensities indicating Ca2+concentration (curves labelled as "F|Ca21]) and biomechanical dynamics from various locations are compared. It is evident that the fluorescent signal and the biomechanical signal exhibit similar profiles, including certain elongated periods. This observation verifies that calcium concentrations and biomechanical dynamics are closely related in the physiological activities of muscle contractions.

[0204] It should be noted that with the cunent fluorescent imaging system 200. changes in the fluorescent signal that reflects Ca2+ concentration across the entire field of view could not be obtained. Only a few pixels exhibit discernible changes in fluorescent intensity, and these pixels are sparsely scattered, making it difficult to recognize any spatial pattern. The primary limitations stem from the low signal nature of optical mapping using fluorescent dyes. Specifically, the signal relies on the '‘change” in fluorescent intensity to track change in ion concentration or transmembrane voltage. However, typically, the fractional change of these fluorescent dyes is not significant (less than 30% for most dyes). Moreover, the high-speed feature of dynamical cellular activities imposes an ultimate restriction on the exposure time of the image sensor 110 (maximum exposure time = I / frame rate), resulting in a low signal due to the short exposure time. To address these challenges, conventional optical mapping often relies on specialized equipment to collect fluorescent signals for large-area measurements during short exposure periods. For example, the cameras adopted in optical mapping often have extremely large pixel sizes (ranging from a few tens of micrometers to a few hundred micrometers per pixel) but small pixel numbers (range from 256x256 to 16x16). The large pixel size requirement also limits the spatial resolution of conventional optical mapping approaches.

[0205] Although the dynamics of Ca2tconcentration cannot be profiled with fine resolution over a large area in the current setup, the experiment represents the first demonstration of detailed mechanical wave propagation and the corresponding calcium ion transient. It also underscores the platform’s ability to provide high spatial resolution for recording the biomechanical dynamics. It should be appreciated that the sy stem 200 may be modified or upgraded to incorporate a high-quality camera specialized for optical mapping, thus enabling the acquisition of changes in fluorescent intensity across the entire field of view. This advancement will allow for simultaneous mappings of ion concentration or transmembrane voltage alongside the biomechanical dynamics measured by the platform. Consequently, this technology can provide rich information about cellular activities, serving as an invaluable tool to study the complex mechanisms underlying the interrelationships between various bio-properties.

[0206] The results demonstrate the potential of combining the LAMBDA system with conventional optical mapping to further study the coupling and interplay between biomechanical, biochemical, and bioelectrical properties.

[0207] Experimental setup

[0208] Stage Top Incubator

[0209] To keep the seeded cells 114 in a native environment for long-term, continuous monitoring of their behavior and minimize interference while they grow and mature a homemade incubation system 204 was integrated into the system 200. The incubation system 204 includes a stage-top incubator with incubation chamber 202 that provided a constant temperature environment and supplied the desired gas mixture optimized for cell growth. In the experiments, constant temperature at 37°C and humid gas mixture (95% air, 5% CO2) was adopted if otherwise specified.

[0210] Illumination, Image sensor and Control Circuit

[0211] In various embodiments the intensity and homogeneity of illumination are important for large-area measurements. A monochromatic LED chip (Chanzon, 3W, 450mA) was used as the light source 116. The LED chip was soldered onto a heat sink and the diverging beam from the LED chip was collimated by a plano-convex lens (focal length = 75 - 100mm) to provide a uniform illumination. A 90° prism mirror was used to turn the illumination direction from horizontal to vertical. Deep red LED (wavelength = 660 nm) w as selected for the experiments to minimize light scattering in the tissue if not otherwise specified.

[0212] To offset the image plane for easy configuration of devices 100 and the image sensor 110. a 90° prism mirror was placed below the stage-top incubator to project the transmitted diffraction patterns to the camera lens (0.727x magnification) and the image sensor 110 (Emergent Vision Technologies, HB25000-SB-M). The data acquisition was done automatically by incorporating switches and an Arduino circuit board as a control circuit.

[0213] Cell Seeding Procedure

[0214] To prepare the device 100 for cell seeding, it undergoes several treatment steps. First the device 100 is first rinsed with 75% isopropyl solution (IP A) followed by deionized water, and then dried with nitrogen. Subsequently, PDMS well with the desired size is bonded to the device 100 after oxygen plasma treatment. The PDMS confines the culture medium during experiments. After bonding, the device 100 is dried in a vacuum chamber with calcium chloride (as desiccant) overnight. Prior to seeding cells, the device 100 surface is coated with a dopamine (Alfa Aesar Chemicals) solution (0.01%, w / v in IM Tris-HCl buffer, Boston BioProducts, Inc.) at 4°C for an hour. Then, the device is washed twice with phosphate buffered saline (PBS) solution (Coming Inc.) and deionized water, respectively. Then, a 35-minutes ultraviolet (UV) sterilization is conducted. Once sterilized, Geltrex (83 pL / mL, Thermo Fisher Scientific Inc.) is coated on the device for 1 hour immediately before seeding cells. Neonatal rat ventricular cardiomyocytes (NRVMs) (i.e., cells 114) were seeded with a density 0.2 - 0.25 millions / cm2to form a confluent monolayer tissue sheet over a 4 cm2device area (active area ~1 cm2). To allow the observation of cells 1 14 via phase contrast microscope, a small transparent window was usually made at the edge of device. Typically , the cells 114 started beating 24 to 48 hours after seeding. 5% Fetal Bovine Serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) is used as the culture medium for NRVMs. Fresh medium was changed every other day.

[0215] Loading protocol of fluorescent dye

[0216] High-affinity calcium ion dye (Rhod-2) was utilized for fluorescent imaging. The ImM stock solution of Rhod-2 is prepared by mixing 44.5 pL dimethyl sulfoxide (DMSO. Thermo Fisher Scientific Inc) with 50 pg Rhod-2 AM (Invitrogen). Following this, 44.5 pL Pluronic F-127 (20% w / v solution in DMSO, Thermo Fisher Scientific Inc) is added to the stock solution for assisting the dispersion of the nonpolar AM ester. For fluorescent imaging, 5 pL of the ImM stock solution is diluted in ImL of buffer solution (DPBS, calcium, magnesium, Gibco™, 14-040-133) and incubated with the cells 114 seeded on the device100. The final concentration of Rhod-2 AM is 5 pM. After incubating for 1 hour, the buffer solution containing the dye is removed, and the sample is rinsed with DPBS twice. The cell culture is incubated for an additional 30 minutes before fluorescent imaging is performed.

[0217] Measurement Setup for Biomechanical Dynamics and Fluorescent Imaging

[0218] To keep seeded cells 114 in their native environment for long-term, continuous monitoring of their behaviors during growth and maturation, an incubation system 204 (e.g., stage top incubator) is integrated into the platform. This incubator ensures a constant temperature environment and supplies the desired gas mixture optimized for cell growth. In these experiments, a constant temperature of 37°C and a humid gas mixture (95% air, 5% CO2) is adopted unless otherwise specified. The chamber of the stage top incubator comprises a heatable mounting frame with a 47.5mm x 21mm observation window (Zeiss, heatable mounting frame KH-L S) and a plastic cover with slidable opening and gas inlet (Zeiss, CO2 cover micromanipulation K). The temperature control module and the CO2 module are connected to a desktop and can be controlled remotely.

[0219] A deep red LED (wavelength = 660 nm) is selected for the experiments to minimize light scattering in the biological tissue by default. To offset the image plane for easy configuration of devices and the image sensor, a 90° prism mirror 207 is placed below the stage top incubator 202 to project the transmitted diffraction patterns to the camera lens 218 (0.727x magnification) and the image sensor 110 (Emergent Vision Technologies, HB25000-SB-M). Data acquisition is automated using switches and an Arduino circuit board as the control circuit. In experiments involving fluorescent images, the wavelength of LED (520-525nm) is chosen to match the excitation spectrum of the dye. A separated CMOS image sensor 110 (IDS, UI-3080CP-M-GL, Rev.2) is utilized to record fluorescent signals. To synchronize the two cameras during data acquisition, they are fed using the same trigger signal. The dichroic mirror 214 (Thorlabs MD568) and emission filter 216 (Thorlabs FBH580-010) are used in accordance with the fluorescent imaging setup. See FIG. 5.

[0220] Representative Pixels of Diffractive Elements

[0221] As described above, each diffractive element 106 will generate a diffraction pattern depending on the applied cellular traction force and will change over time according to the beating behaviors of cells 114. To clearly visualize the biomechanical dynamics, the representative pixel of a diffractive element 106 was designed to be the pixel that undergoes the maximum intensity change over time. FIG. 12 presents an example of 3x3 diffraction patterns with their representative pixels marked by circles while FIG. 13A shows an exampleof the time-course intensity' of a representative pixel at certain location. Since the pixel intensity is a function of both time and spatial location, it is denoted as I(t; x. y) for easy explanation hereafter.

[0222] To distinguish the signals generated by cellular activities from the fluctuated noise, the diffractive element 106 yvas considered to be “activated” only yvhen its representative pixel has intensity change larger than a certain threshold (AI>Ithres). The intensity threshold yvas adjusted according to the noise levels from different measurement conditions. The noise level is often related to the stability of illuminating light 112, exposure time and electronic noise of the image sensor 110. For an activated diffractive element 106, the intensity of its representative pixel yvas normalized to he between zero and one (FIG. 13B) to extract the “activation time (AT)” and “activation duration (AD).

[0223] The activation time is a figure-of-merit to characterize the timing yvhen the beating yvave arrives at a certain position. In conventional optical mapping, it is often defined as the point with the steepest change of fluorescent intensity and can be obtained by finding the maximum value of the first derivative (dl / dtmax). Here, to enhance the accuracy from the discrete data points, the rising part of the normalized intensity yvas fitted by a logistic curve and the midpoint of the logistic function (to) is considered as the activation time (FIG. 13C). Next, the activation duration is how long the beating lasts at a certain location. Mimicking the common definition of activation potential duration (APD), the activation duration yvas defined to be the interval between the time yvhere the intensities are half of its maximum value (Normalized intensity = 0.5). It is denoted as AD50 in FIG. 13B.

[0224] It should be noted that activation time yvas employed during the upstroke phase to demonstrate the platform’s capability' in studying the propagation of mechanical waves. Alternatively, users or researchers can explore other crucial time points, such as the doyvnstroke time and repolarization midpoint, to investigate various physiological stages during cardiac beats.

[0225] Finally, in addition to the time-course intensity , activation time and activation duration, beating frequency is another important characteristic. It can be obtained by calculating the frequency spectrum of the time-course intensity by fast Founer transform (FFT).

[0226] Intensity Sum and Diffraction Pattern

[0227] The representative pixels discussed above allow one to quickly retrieve some important properties of biomechanical dynamics including beating pattern, beating frequencyand activation time. On the other hand, the diffraction pattern, along with the intensity sum of all the pixels in a diffraction pattern, contains rich information for extracting cellular traction force quantitively.

[0228] While embodiments of the present invention have been show n and described, various modifications may be made without departing from the scope of the present invention. The invention, therefore, should not be limited, except to the following claims, and their equivalents.

Claims

What is claimed is:

1. A device for label free optical mapping comprising: a transparent elastic polymer membrane comprising a top surface and a bottom surface opposite the top surface wherein the elastic polymer membrane is disposed on an optically transparent substrate; a plurality of diffractive elements disposed on the elastic polymer membrane, each diffractive element comprising: an opaque occluding element disposed on the top surface of the elastic polymer membrane; and an opaque layer disposed on the bottom surface of the elastic polymer membrane wherein the opaque layer comprises a hole having the same shape as the occluding element; and wherein the occluding element is aligned with the hole such that light impinging on the diffractive element normal to the top surface of the elastic polymer membrane is substantially blocked by the occluding element from passing through the hole of the bottom opaque layer.

2. The device of claim 1 , wherein the hole is the same size and shape as the occluding element.

3. The device of claim 1, wherein the hole is up to 10% smaller than the opaque occluding element and aligned with the opaque occluding element to allow a perimeter of light to pass through.

4. The device according to any one of claims 1-3, wherein the bottom surface comprises a single continuous layer.

5. The device according to any one of claims 1-3, wherein the bottom surface comprises a discrete island for each diffractive element.

6. The device according to any one of claims 1-3, wherein the elastic polymer membrane comprises a material selected from the group consisting of PDMS, apolyacrylamide gel, polyethylene glycol (PEG) hydrogel, collagen, an extracellular matrix material (ECM), and collagen / ECM combinations.

7. The device according to any one of claims 1-3, wherein the elastic polymer membrane ranges in thickness from about 2 pm up to about 5 pm.

8. The device according to any one of claims 1-3, wherein the elastic polymer membrane has a surface area that ranges from about 0.2 cm2up to about 100 cm2.

9. The device according to any one of claims 1-3, wherein the elastic polymer membrane has a Young's modulus that ranges from about 1 kpa up to about 1 MPa.

10. The device according to any one of claims 1-3, wherein the opaque occluding element and / or the bottom surface comprises a material selected from the group consisting of a metal, an opaque plastic, an opaque dielectric, a semiconductor, or a composite layer of these materials.

11. The device of claim 10, wherein the opaque occluding element and / or the bottom surface comprises a metal selected from the group consisting of aluminum, aluminum oxide, magnesium, magnesium oxide, palladium, copper, copper oxide, nickel, titanium, titanium dioxide, gold, silver, silver oxide, iron, iron oxide, cobalt, and alloys thereof.

12. The device according to any one of claims 1-3, wherein the opaque occluding element ranges in thickness from about 50 nm up to about 5 pm.

13. The device according to any one of claims 1-3, wherein the occluding element(s) have a maximum dimension ranging from about 2 pm up to about 205 pm.

14. The device according to any one of claims 1-3, wherein the diffractive elements have an average spacing between diffractive elements ranging from about 20 pm up to about 40 pm.

15. The device according to any one of claims 1-3, wherein the diffractive elements in the elastic polymer membrane are present at a density that ranges from about 40,000 elements / cm2up to about 4,000,000 elements / cm216. The device according to any one of claims 1-3, wherein the device further comprises an image sensor configured to detect illumination passing through the device or reflected from the device.

17. The device of claim 16, wherein the image sensor comprises one or more CCD image sensors.

18. The device of claim 16, wherein the image sensor is disposed against the optically transparent rigid substrate.

19. The device of claim 16, wherein the image sensor is disposed in an optical path from the optically transparent rigid substrate.

20. The device according to any one of claims 1-3, wherein the device is disposed in a stage-top incubator chamber.21 . The device according to any one of claims 1 -3, wherein cells are disposed on the top surface of the elastic polymer membrane.

22. A system for label free optical mapping, the system comprising: a device according to any one of claims 1-3; a light source configured to produce illumination normal to the top surface of the elastic polymer membrane; and one or more image sensors configured to detect illumination passing through the plurality of diffractive elements.

23. The system of claim 22, wherein the light source produces monochromatic illumination.

24. The system of claim 23, wherein the light source comprises a laser or a light emitting diode (LED).

25. The system of claim 22, wherein the one or more image sensors is a component of a detection system comprising one or more of a mirror, a lens, control circuitry', and a computer.

26. The system of claim 22, wherein the system further comprises an incubation chamber in which the device is disposed.

27. The system of claim 26, wherein the incubation chamber is operably coupled to an incubation system.

28. The system of claim 27, wherein the incubation system comprises one or more of a CO2 module, a humidifier, a CO2 source, a temperature regulator, and a culture medium.

29. The system of claim 22, wherein the system further comprises a fluorescence microscopy system.

30. The system of claim 29, wherein the fluorescence microscopy system comprises a lens or lens system and an imaging device.

31. The system of claim 22, wherein cells are disposed on the top surface of the elastic polymer membrane.

32. The system of claim 31, wherein the cells comprise mammalian cells.

33. The system of claim 31, wherein the cells comprise muscle cells.

34. A method of fabricating a device according to any one of claims 1-3, the method comprising: a) patterning photoresist posts on a substrate using a first photoresist;b) paterning a second photoresist to provide a mold comprising a plurality of pillars for molding an elastic polymer membrane; c) covering the substrate with a material that forms the elastic polymer membrane; d) removing residue of the elastic polymer membrane; e) removing the second photoresist; f) depositing layers of opaque material on the substrate to form opaque occluding elements and the opaque layer; and g) applying the material that forms the elastic polymer membrane again to fill air gaps between the opaque occluding elements.

35. The method of claim 34, wherein the first photoresist is a negative photoresist and the second photoresist is a positive photoresist.

36. The method of claim 34, wherein steps c) and g) comprise a stamping process.

37. The method of claim 34, wherein the opaque material comprises a metal selected from the group consisting of aluminum, aluminum oxide, magnesium, magnesium oxide, palladium, copper, copper oxide, nickel, titanium, titanium dioxide, gold, silver, silver oxide, iron, iron oxide, cobalt, and alloys thereof.

38. The method of claim 34, wherein the material that forms the elastic polymer membrane is selected from the group consisting of PDMS, polyacrylamide (PA) gels, polyethylene glycol (PEG) hydrogels, collagen, and an extracellular matrix material (ECM).

39. A method of detecting mechanical forces of cells, the method compnsing: providing cells disposed on the top surface of the elastic polymer membrane in a system according to claim 22; exposing the device to illumination substantially normal to the top surface; and detecting changes in illumination / diffraction paterns with the one or more image sensors produced by light passing through the device as cells alter the position of occluding elements thereby providing a measurement of traction forces applied by the cells to the top surface of the elastic polymer membrane and / or to the occluding element(s).

40. The method of claim 39, further comprising contacting the cells with a fluorescent label / stain and detecting fluorescence fluorescent imaging of the cells using a fluorescent imaging system.

Images