Fluid exchange chamber for light sheet imaging and scattering

Abstract

A chamber for a sample imaging system, in which the chamber is defined by an enclosure, a plurality of optically transparent walls forming the enclosure and configured to retain an aqueous sample, and one or more inner semipermeable membranes disposed in relation to comers of the chamber. The one of more membranes are configured to separate the aqueous sample from the optically transparent walls of the chamber and define channels that are further configured to control the environment within the formed enclosure.

Description

FLUID EXCHANGE CHAMBER FOR LIGHT SHEET IMAGING AND SCATTERINGCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to USSN 63 / 622,159, entitled: FLUID EXCHANGE CHAMBER FOR LIGHT SHEET IMAGING AND SCATTERING, filed January 18, 2024, pursuant to relevant portions of 35 USC §119 and 35 USC §120. This noted application is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This application relates generally to the field of sample imaging and sample imaging systems, and more specifically to a novel fluid exchange chamber for purposes of light sheet microscopy, spectroscopy and related aspects of sample imaging.BACKGROUND

[0003] Biomolecular condensation, driven by liquid-liquid phase separation (LLPS) or coacervation has been observed in protein solutions since the 1950s. Recent observations of LLPS have been observed in bacteria, plants, and mammalian systems and linked to a variety of cellular processes. LLPS appears to be a universal phenomenon of cellular self-organization. When liquidlike droplets separate they can alter the local chemistry to enhance or limit biochemical reactions. The ability of condensed droplets to alter the environment is even more pronounced if enzymes or small molecules separate into or are excluded from the droplets. LLPS condensates in cells have been implicated as chemical reaction centers, acting as “membraneless organelles.” Such phase- separated domains self-assemble, acting as organizing centers that colocalize high concentrations of enzymes, polymers and scaffolds. Condensed domains have been implicated in myriads of biological functions including protein homeostasis, DNA replication, RNA transcription, circadian-mediated processes, and muscle organization.

[0004] Since 2009, when it was observed that round, liquid droplets of GFP-labeled p- granule proteins were forming in living cells and could be recapitulated in vitro, there has been a resurgence of interest in protein condensates; specifically the realization that proteins can phase separate into liquid-like droplets that can transiently form and dissolve again to perform specificfunctions in cells. Prior to these observations, condensation of proteins was mostly associated with disease states (prions, occlusions, aggregates) or used for protein purification strategies. A greater understanding has therefore been made of the importance of these structures, and the need for new technologies to study these systems in a non-perturbative manner.

[0005] Accordingly, a major conceptual advance in cell biology over the last decade has been the ability to observe a diversity of intracellular, membraneless droplets involved in important cellular processes such as gene regulation, RNA processing, and degradation. The ability of proteins, RNA, DNA, and other molecules to separate within the aqueous environment in cells and in the test tube is not only important for normal biological functions, but can also go wrong and lead to dysfunction and disease. A major challenge to elucidating the phase diagram for biological systems is the need for chemostatic and temperature control of an in vitro environment, since the condensation transition is highly sensitive to the pH, ions, temperature, and other small molecules.

[0006] Currently, technologies for chemostatic chambers for microscopy utilize microfluidics, which inherently have a very high surface area to volume ratio. As a result, a considerable amount of an aqueous sample remains adhered to the glass wall of the sample chamber. Furthermore, the glass surfaces of the sample chamber themselves can nucleate changes in phase to cause the samples under study to phase separate at inopportune times. Larger sample chambers are used for spectroscopy, but these larger chambers are not capable of chemostatic control or visualization using objective lenses or microscopy. Some spectrophotometers will allow sample flow, but this flow is constantly ongoing, which means different molecules are being examined throughout an experiment.

[0007] By way of example and in terms of overall need, the cytoplasm is complex, dynamic environment teeming with biological enzymes performing chemical processes far from equilibrium, and, therefore not well described by any known understanding of equilibrium physics in general, and more specifically, of protein phase transitions. It is postulated that the energy landscape within cells is controlled by the background enzyme reactions (metabolism) that function as an active bath to modify biological processes. In this regard, quantitative models governing the non-equilibrium nature of cells are presently lacking. Therefore, and to directlyexplore how background activity can alter biological processes, an in vitro reconstitution of a model system of enzymes, for example, urease, and a protein, such as ubiqulin2, can be created, which condenses via liquid-liquid phase separation (LLPS). Biological condensation can be the “silver bullet” for exploring the effects of the energy landscape for at least the following reasons. First, condensation processes are highly sensitive to the environment, yet all experiments in vitro are currently performed in the absence of background activity. Second and as previously noted, protein condensation results in membraneless organelles, which are compartments that organize and perform essential functions in cells.

[0008] There is, however, a major experimental hurdle in performing experiments of liquid-liquid phase separation (LLPS) in active baths. The experimental platform is required to control the chemical environments and image the bulk properties of the condensate system away from surfaces. Current platforms designed for this purpose or similar purposes are either: (i) UV- Vis spectrophotometry systems with low surface area to volume ratios, but without direct imaging or chemical control capabilities; or (ii) microfluidic devices that are compatible with imaging and chemical control, but which have high surface area to volume ratios, which leads to the afore mentioned and undesirable nucleation, growth, and stabilization of condensates at the glass surfaces. Condensation at or near the glass surfaces of the sample chamber will confound the ability to precisely quantify how enzyme activity affects liquid-liquid phase separation.

[0009] Accordingly, there is a prevailing need in the field to provide a sample (chemostatic) chamber that can effectively control the chemical environment with an imaging system that allows for rapid and thorough examination of the phase diagram of a biomolecular condensate with a single sample. The sample chamber would permit the same molecules of an aqueous sample to be studied over time within the chemostatic chamber, as the environment is held constant. There is another similar need to provide a sample imaging platform having a sample chamber in which the chemical environment of aqueous samples in the chamber can be effectively controlled through constant diffusive exchange of small molecules, for example, to feed the enzyme reactant substrates and also remove any product molecules that could potentially poison the sample imaging system.BRIEF SUMMARY

[0010] Therefore, and according to at least one aspect of the present invention, there is provided a sample imaging platform comprising a bespoke sample chamber to permit direct visualization of biochemical and biophysical processes, while simultaneously enabling parameters such as temperature, ionic strength, and the activity of non-interacting enzymes in the sample solution. According to at least one embodiment, the herein described system / platform permits examinations and investigations to determine how background enzymes that mimic the nonequilibrium, active nature of the cell, alters the phase separation of biological condensates.

[0011] According to at least one aspect, there is provided a sample chamber for an imaging system, the chamber comprising an enclosure; a plurality of optically transparent walls forming the enclosure which is configured to retain an aqueous sample; and one or more inner semipermeable membranes disposed in relation to corners of the sample chamber. The one or more inner semipermeable membranes are configured to separate the aqueous sample from the optically transparent walls and further configured to control the environment within the formed enclosure, wherein the one or more membranes create flow channels.

[0012] In at least one version, a total of four (4) inner semipermeable membranes are disposed, each of the membranes being attached at 45 degree angles relative to respective comers of the sample chamber, the latter being defined by a rectilinear configuration. In at least one embodiment, the inner semipermeable membranes are made from a suitable hydrogel, such as PEGDA.

[0013] The optically transparent walls of the sample chamber according to at least one embodiment are made from glass, though other suitable optically transparent materials can be utilized. According to at least one version, the imaging system used in conjunction with the sample chamber is a light sheet microscopy system, although the herein described sample chamber can alternatively be used for other applications, including but not limited to spectroscopy or x-ray light scattering systems.

[0014] According to another aspect of the present invention, there is provided an imaging system comprising a sample chamber having an interior enclosure formed by a plurality of optically transparent walls at opposing sides of the interior enclosure, a first objective lens disposed in relation to a first optically transparent wall to enable light to enter the sample chamber, and a second objective lens disposed in relation to an second optically transparent wall of the sample chamber that is orthogonal to the first optically transparent wall of the chamber. The first objective lens is disposed along an illumination axis and the second objective lens is disposed along a detection axis and in which one or more inner semipermeable membranes are disposed in relation to the optically transparent walls of the sample chamber, the membranes separating the walls of the chamber from an aqueous sample disposed in the sample enclosure. According to one or more versions, either of the first and second objective lens can be selectively used for illumination or detection with the other being used selectively for detection or illumination.

[0015] According to at least one embodiment, the one or more inner semipermeable membranes form one or more channels to permit flow of a buffer solution. In a preferred version, the semipermeable membranes are disposed at comers of the sample chamber and at 45 degree angles such that the membranes do not obscure either the illumination axis or the detection axis of the imaging system. In one or more versions, four (4) inner semipermeable membranes are disposed at each corner of the sample chamber, the defined buffer channels extending between respective top and bottom surfaces of the chamber. Inlet and outlet tubes can extend into and out of each defined channel for permitting a buffer or other solution to be added and removed wherein various gradients can be created in regard to a sample being assessed within the sample chamber.

[0016] The imaging system according to at least one version is a light sheet microscopy system (LSMS), although the sample chamber can be configured for other suitable imaging uses involving light scattering or spectroscopy, among others.

[0017] According to yet another aspect of the present invention, there is provided a method for imaging of an aqueous sample, the method comprising the steps of providing the aqueous sample in a sample chamber, the chamber having a plurality of optically transparent walls defining an interior enclosure and one or more inner semipermeable membranes in which the aqueoussample is separated from the walls of the sample chamber; and adding one or more agents within one or more channels that are formed between the at least one semipermeable membrane and the optically transparent walls of the chamber. The semipermeable membrane(s) are further configured to permit passage of the one of more agents, but preventing the sample from passing into the formed channel(s).

[0018] In at least one version, the one or more agents can comprise at least one of urea and salt (or other material) in order to create various gradients in the sample chamber through one or more channels created by the semipermeable membranes and the walls of the sample chamber. In at least one version, four (4) inner semipermeable membranes are disposed in comers of the sample chamber and at 45 degrees thereto. In at least one embodiment, the inner semipermeable membranes can be fabricated from a hydrogel, such as PEGDA, or other suitable semipermeable material.

[0019] In at least one version of the herein described method, the contained sample is illuminated along an illumination axis and the sample is observed along a detection axis orthogonal to the illumination axis and in which the semipermeable membranes are disposed not to impede or block the illumination and detection axes. In at least one version, illumination and detection can be selectively toggled along either of the orthogonal axes to facilitate imaging of a contained sample.

[0020] Advantageously, the herein described sample chamber can be used for controlling the environment of aqueous samples during imaging, spectroscopy and various other experiments. For example, and according to at least one application, the herein described sample chamber can be used for fluorescence imaging using light sheet microscopy, though it will be readily understood that the novel sample chamber described herein can alternatively be used in connection with other types of measurements.

[0021] Moreover, the novel imaging system that includes the sample chamber will provide unprecedented views, while at the same time providing environmental regulation. According to atleast one embodiment, the herein described sample chamber enables elucidation of how organisms control their internal structures.

[0022] The herein described system advantageously permits the control, exchange, and gradients of small molecules that can control the phase of the samples. Exchange can induce the phase changes. Gradients can induce the phases in only one region of the chamber. Using this methodology and structure allows a single continuous procedure to reveal the entire condensation phase diagram of a molecule in one sweep of the scattering light. Moreover and if different concentrations of small molecules are used in the exchange (buffer) channels of the herein described sample chamber, small molecule gradients will be created, such that a range of concentrations can be evaluated simultaneously. As such, the herein described sample chamber addresses a number of the noted deficiencies of current approaches to assess the phase diagram of biomolecular condensates quickly and easily. That is and while UV-Vis spectrophotometry can reveal information pertaining to the concentration and temperature dependence of condensation, this technique is incapable of revealing the properties of the condensate, e.g., liquid-like or solid aggregate, and also cannot control the chemical environment. In addition, traditional microscopy approaches can measure the material properties of the condensate and control the chemical environment to some extent, but the imaging is done at or near the surface of the cover slip, which can produce undesired phase separation. The herein described sample chamber (also synonymously referred to throughout as a “chemocontrol chamber” or “chemostatic chamber”) used in conjunction with a suitable imaging system combines the best attributes of spectrophotometry and imaging into a single platform.

[0023] Additionally, the novel combination imaging and spectroscopy platform that includes the herein described chamber permits both spectrophotometric and imaging measurements throughout the chamber to know how a contained sample scatters light and the shape and density of the scattering objects.

[0024] These and other features and advantages will be readily apparent from the following Detailed Description, which should be read in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will now be more fully understood and appreciated from reading the following Detailed Description in conjunction with the accompanying drawings, in which:

[0026] FIG. 1(a) is a top schematic view of a macroscopic sample chamber and imaging system, the latter of which is partially shown and in accordance with an embodiment;

[0027] FIG. 1(b) is a side schematic view of the macroscopic sample chamber and partially shown imaging system of FIG. 1(a);

[0028] FIG. 2(a) depicts an imaged top view of a horizontal gradient of two small molecules in the sample chamber of FIGS. 1(a) and 1(b);

[0029] FIG. 2(b) depicts an imaged top view of a vertical gradient of the two small molecules in the sample chamber of FIGS. 1(a) and 1(b);

[0030] FIG. 2(c) depicts an imaged top view of simultaneous gradients for each of the two small molecules in the sample chamber of FIGS. 1(a) and 1(b);

[0031] FIGS. 3(a) and 3(b) illustrate bulk-phase diagrams of a sample disposed in a sample chamber made in accordance with an embodiment;

[0032] FIG. 4(a) schematically depicts configurations of alternative designs of sample chambers made in accordance with various embodiments;

[0033] FIG. 4(b) graphically depicts estimated equilibration times to 90 percent of a source concentration for square configured sample chambers, based upon different cross sections;

[0034] FIG. 4(c) depicts surface to volume ratios for the sample chambers of FIG. 4(b);

[0035] FIG. 4(d) depicts a sample chamber configuration in accordance with another alternative embodiment;

[0036] FIG. 5 is a schematic view of an imaging system for use with a sample chamber in accordance with an embodiment;

[0037] FIG. 6 is a top perspective view of a sample chamber made in accordance with another embodiment; and

[0038] FIG. 7 is a top perspective view of a sample chamber made in accordance with yet another embodiment.DETAILED DESCRIPTION

[0039] The following relates to an imaging platform that includes a chemocontrol sample chamber and related imaging system in accordance with one or more embodiments. It will readily apparent that other variations or modifications can be made to the herein described embodiments. In addition, a number of terms are used throughout this discussion in order to provide an adequate frame of reference for the accompanying drawings. These terms, which include “top,” “bottom,” “inner,” “outer,” “above,” “below,” “first,” “second” and the like are not intended to overly limit the inventive concepts discussed herein, except where so specifically indicated.

[0040] First and with reference to FIGS. 1(a) and 1(b), there is schematically shown an imaging platform 100 that includes a macroscopic fluid exchange chamber 140 made in accordance with an exemplary embodiment. More specifically, top and side elevational views are shown, respectively, of the sample chamber 140 with aspects of an imaging system that are disposed on orthogonal sides of the sample chamber 140 for purposes of illumination and detection, as will be later described in greater detail.

[0041] More specifically and according to this embodiment, the platform 100 is a light sheet microscopy system that incorporates the sample chamber 100. As noted, details relating to examplary imaging systems will be made in a later portion of this description. The sample chamber140 is defined by a rectilinear configuration, as shown with further reference to FIGS. 1(a) and 1(b), including a plurality of walls that combine to form an interior enclosure 144. The herein described chamber 140 is defined by a rectilinear configuration and more specifically a square shape according to this specific embodiment. It will be understood, however, that other alternative shapes / configurations, including various sized and / or triangular designs, can be utilized, such as those shown by way of example in FIGS. 4(a) and 4(d). In FIG. 1(a), a small portion of the interior enclosure 144 is shown in magnified form directly beneath the sample chamber 140, including a pictorial view of a sample 146 that is disposed therein as well as small molecules 147.

[0042] More specifically, the sample chamber 140 includes four (4) side walls 145, 147, 149 and 151, a top wall or surface 146, and a bottom or base surface 148. In this specific embodiment, each of the side walls 145, 147, 149, 151 and the top wall 146 are optically transparent with the base surface 148 being optionally transparent. According to another version, only the side walls disposed along a defined illumination axis of the imaging system are optically transparent. Furthermore, and according to this specific embodiment, the optically transparent walls are made from glass, although it will be understood that other optically transparent materials, including various transparent polymers / plastics that include but are not limited to polycarbonate, polyvinylchloride (PVC), polystyrene, polyethylene terephthalate (PET), acrylics, and polypropylene can be utilized. The optically transparent walls of the sample chamber 140 are preferably thin, having a thickness of about 170 micrometers, with the overall size of the interior enclosure 144 being about 10 mm in size, according to this specific embodiment. The foregoing parameters can be suitably varied. For example, FIG. 4(a) illustrates a number of alternative sample chambers 140A, MOB and 140, respectively, each commonly defined by a square configuration, but different wall sizes of 1 mm, 3 mm and 10 mm walls, respectively. As noted, other variations are possible. The size of the sample chambers 140 can be tailored or scaled accordingly.

[0043] Still referring to FIGS. 1(a) and 1 (b), a plurality of inner semiperm eable membranes 152 are further provided within the confines of the herein described sample chamber 140. According to this specific embodiment, the semipermeable membranes 152 are made from a polyethylene-glycol-diacrylate (PEGDA) hydrogel system, which is separately polymerized forfabrication purposes. It will be understood that other porous materials can alternatively be utilized in lieu of PEGDA hydrogel or in combination therewith, such as but not limited to polyacrylymide and agarose, which are also hydrogels. According to at least one embodiment, there are also cellulose membrane versions used for small molecule exchange (dialysis tubing), these latter membranes being made from cellulose acetate.

[0044] According to this specific embodiment, respective end surfaces of the inner semipermeable membranes 152 are disposed at the comers of the sample chamber 140, and extend at approximately 45 degrees in relation to the illumination and detection axes 166, 168 of the sample imaging system. A spacing is formed between each of the inner semipermeable membranes 152 and the chamber walls to which the membranes 152 are attached, defining an elongated channel 156 extending between the top surface 146 and the bottom surface 148 of the sample chamber 140. In this specific embodiment, four (4) flow channels 156 are formed, each having a substantially triangular shape as viewed from the top of the sample chamber 140. A series of inlet and outlet tubes 160, 162 are connected to the top and bottom of each of the defined flow channels 156. Variations of this configuration are possible. For example, and though a total of four (4) inner semipermeable membranes 152 are provided according to this specific embodiment, it will be understood that the overall number of membranes 152 and channels 156 can be varied depending, for example, on the application. In addition, and though the chamber according to this specific embodiment is defined by a rectilinear (square) configuration, the sample chamber could also be formed into other shapes (circular, rhombic, polygonal) such as the sample chamber 140C, FIG. 4(d) having a triangular configuration, and including only two flow channels 156 at opposing comers based on the positioning of a pair of semipermeable membranes 152.

[0045] With reference to FIG.4(b), estimated equilibration times to 90 percent of the source concentrations for square sample chambers with sides ranging in length from 1 mm to 10 mm are depicted with the calculated surface area to volume ratios for the same sample chambers being depicted in FIG. 4(c) The equilibration times were determined by modeling the diffusion equation with a source at the edges having the semipermeable membrane walls. The time that it takes for the center of the chamber, initially at zero concentration of a small molecule with D ~ 0.001mm2 / s, to reach 90 percent of the maximum concentration scales with the size of the chambersquared (proportional to the area). The surface area to volume ratio decreases with increasing side edge. A chamber with a side edge of 3 mm takes about 30 minutes to equilibrate and has a surface area to volume ratio of approximately 1 mm-1. This chamber also uses a fairly small volume of sample (less than 20 microliters). For applications requiring smaller sample volumes, the geometry shown in FIG. 4(d) having the triangular cross section with semipermeable membranes at the opposing corners can be used. This latter design permits a single concentration gradient and requires less than 10 microliters of sample and about 42 minutes for equilibration, although the surface area to volume ratio (~2 mm'1) is nearly double to that of the square shaped sample chamber. It will be understood that any suitable design can be utilized, provided that the semipermeable membranes do not impede illumination or detection by the imaging system, while also effectively separating the walls of the sample chamber from the contained sample.

[0046] As further discussed herein, the resulting sample chamber 140 having the disposed inner semipermeable membranes 152 enables effective control of the environment within the defined enclosure 144 of the chamber. For example, and according to this specific embodiment, the influx of salt and urea as agents can be controlled relative to the contained aqueous sample within the defined enclosure 144 of the sample chamber 140, such as urease, to drive protein (such as ubiqulin2) condensation, while keeping temperature constant.

[0047] According to this specific embodiment and still referring to FIGS. 1(a) and 1(b), the herein described sample imaging system is a light sheet microscopy system, in which the optically accessible sample chamber 140 includes orthogonally placed illumination and detection paths. More specifically and according to this embodiment, the illumination path extending along a defined illumination axis 166 includes a first objective lens 170 specifically adjacent to the side wall 145 of the chamber 140. A light source (not shown), such as a visible light laser, typically having an emission wavelength of about 405 nm, 478 nm, 488 nm, 532 nm, 561 nm, 640 nm or other suitable fluorescence wavelength for the molecules of the contained sample, enables light to be directed through the first objective lens 170, as well as through the optically transparent side wall 145 of the sample chamber 140 along the illumination axis 166 with the directed light passing through the interior enclosure 144 and the contained aqueous sample. Light is then caused to exit the sample chamber 140 through the opposing optically transparent wall 149 to a focusing lens179 also arranged along the illumination path (axis 166), the lens 179 then directing the received light to a photodetector 180, which can be used for scattering and spectrophotometric measurements.

[0048] The detection path of the herein described imaging system 100 includes a second objective lens 174, which is disposed in relation to side wall 147 of the sample chamber 140 for purposes of viewing the illuminated aqueous sample along a detection or optical axis 168. According to this specific embodiment, the two objective lenses 170, 174 being placed orthogonally around the chamber 140 will allow for optically sectioned images of ubiquilin2 condensates 205, FIGS. 4(a) and 4(b), within the bulk of the aqueous sample. For this specific example, far red light (spectrally separated from the wavelengths used for imaging) can be used to detect changes in absorption / scattering.

[0049] According to at least one embodiment, buffer will flow from inlets 160 along flow paths / directions 190, 194 with specific salt, urea and temperature to control both the chemical environment, as well as the temperature within the sample chamber 140. According to at least one version, additional temperature control can be applied using a resistive or similar heater (not shown) connected to a metal sheath (not shown) that fits around a portion of the sample with apertures for viewing. The buffers can also be heated or cooled prior to flowing through the channels of the sample chamber to assist to equilibrate temperature. Temperature changes diffuse faster than ions, and it is estimated to take about 12 seconds in order to equilibrate the temperature of a 20 microliter chamber by 25 degrees. The concentrations of urea and salt in the channels 156, as well as temperature, can produce defined gradients. For example, and as depicted in in FIG. 3(a), one side 149 of the sample chamber 140 can be flowed with buffer having high urea concentration added to the flow channels 156, and at the opposite side 144 of the sample chamber 140 a quantity of buffer having low urea concentration can be made to flow through the channels 156, resulting in gradients of urea (or other small molecules) as shown with materials passing through the defined semipermeable membranes 152 into the interior enclosure 144 of the chamber 140 to feed the enzymes. At the same time, and as shown in FIG. 3(b), buffer with low salt concentration can be added to the channels 156 disposed at the top 146 of the chamber 140 and buffer with high salt concentration can be added to the channels 156 disposed at the bottom 148of the sample chamber 140 resulting in the formation of a gradient in salt concentration across the chamber in an orthogonal direction from that of the urea gradient. The depictions in FIGS. 3(a) and 3(b) are merely examples, wherein other conditions to the sample environment can be effected.

[0050] The strength of the gradient (i.e., the difference between low and high concentrations) and the small molecules used will change the location of the phase transition within the chamber 140. The gradients established will be fixed using constant concentrations in each of the flow channels 156. The different gradients can be established simultaneously if, for example, the channel 156 between walls 146 and 149 has high urea and low salt, the channel 156 between walls 149 and 148 has high urea and high salt, the channel 156 between walls 148 and 147 has low urea and high salt, and the channel between walls 147 and 146 has low urea and low salt. Similar gradients are shown generically in FIGS. 2(a) and 2(b). In addition, this pattern will simultaneously create gradients in urea and salt running in perpendicular directions to induce the phase transition by two different small molecules at the same time, such as depicted in FIG. 2(c), by way of example.

[0051] FIG. 5 illustrates an imaging platform in accordance with another embodiment. For discussion purposes, this platform includes a sample chamber 140 similar to that of FIGS. 1(a) and 1(b), but having side walls (3 mm), and an imaging system 500. More specifically and according to this embodiment, the imaging system 500 is a light sheet fluorescent microscope that permits incident light to be transmitted to the chamber for purposes of excitation and imaging of a sample from orthogonal sides of the sample chamber 140, creating a dual-view microscope that is capable of imaging condensate formation across two chemical gradients nearly simultaneously. The herein described dual view light sheet microscope 500 includes a light source 504, such as a laser, whose output is coupled to a pair of optical fibers 508, 512, in which each optical fiber extends along a pair of orthogonal optical arms; namely a first microscope arm 515 and a second microscope arm 517. According to this embodiment, the first microscope arm 515 extends from the first optical fiber 508 distally through an electrically tunable lens 516 and a first pair of relay lenses 520 to a pair of galvo mirrors 524 and a second pair of relay lenses 528 to a beamsplitter 532, such as a dichroic mirror. The beamsplitter 532 is optically coupled to an objective lens 536 that is disposed proximate to one lateral sidewall of the sample chamber 140.

[0052] The second microscope arm 517 similarly links the output of the second optical fiber 512 through an electrically tunable lens 540 and a first relay lens pair 544 to a pair of galvo mirrors 548 and a second relay lens pair 552 to a beamsplitter 556, in this instance, a dichroic mirror. The beamsplitter 556 is optically coupled to an objective lens 560 disposed in relation to an orthogonal side wall of the sample chamber 140. Light can be transmitted to the sample chamber 140 for purposes of excitation / illumination by the light source 504 selectively over either or both of the first or second microscope arms 515, 517. In addition, image sensors, such as cameras 564, 568, are disposed to receive light from the sample chamber 140 for purposes of imaging the sample along respective orthogonal optical paths. More specifically and according to this embodiment, the first microscope arm 515 defines a first imaging path via the objective lens 536, extending proximally through the beamsplitter 532 and a focusing lens 572 that is aligned with the first camera 564, while the second microscope arm 517 defines a similar orthogonal second imaging path proximally extending through the objective lens 560, beamsplitter 556 and a focusing lens 580 to the second camera 568. According to this embodiment, the microscope 500 can easily alternate which microscope arm 515, 517 is being used for illumination and which microscope arm 515, 517 is being used for detection, enabling the acquisition of orthogonal planes through the sample and detection of simultaneous gradients, such as those illustrated in FIGS. 2(a) - 3(b), whether sequentially or simultaneously. In at least one version, the imaging system is configured to provide magnification (e.g., 4x, lOx, 20x) sufficient to capture a field of view that encompasses nearly all of the gradient, but the imaging system can be tailored in accordance with known means to accommodate higher magnification and high numerical aperture (NA) lenses. For example and according to at least one version, a 20x / 1.0 NA objective can be used to achieve an optical resolution of about 300 nm.

[0053] In operation, the herein described imaging system permits imaging of condensate formation across two chemical gradients nearly simultaneously. With both arms 515, 517 of the system 500 able to provide illumination and detection, the herein described system is able to switch between the two orthogonal views in less than about 30 milliseconds. A sheet of excitation light is created by sweeping the focused region of a Gaussian beam through the sample using the two galvo mirrors and the electrically tunable lens of either arm 515, 517 of the imaging system 500.This same system can be used to control the optical elements and the power of the excitation light source (e.g., laser) such that the excitation beam dwells on a specific region of a droplet or an entire droplet for an extended time period for photobleaching. The tightest focus region of the light sheet of excitation light will be swept across the sample using one of the electrically tunable lenses 516, 540 depending on which microscope arm 515, 517 is performing the excitation of the sample. The tightest focus will then move along the optical axis of the microscope (whether left to right or right to left on the image sensor (camera)). This sweeping of the excitation focus is synchronized with the rolling shutter readout of the appropriate imaging sensor (camera 564 or camera 568).

[0054] As previously noted, the foregoing example is that of a light microscopy system. However, the concepts discussed including the semipermeable membranes that separate the glass walls of the chamber from the sample and further provide environmental control can be similarly extended to other sample imaging platforms, including spectroscopy and X-ray scattering, among others.

[0055] FIGS. 6 and 7 depict perspective views of sample chambers made in accordance with other embodiments. FIG. 6 depicts a macroscopic sample chamber 640 defined by a square open-ended frame 644 that is fabricated from a suitable durable plastic. For example, the frame 644 can be fabricated using a 3-D printer, at least for testing and / or prototyping. In this depicted version, a plurality of transparent (e.g., glass) observation walls 648 and semi-permeable membranes 652 installed to interior walls of the chamber 640 in each corner thereof, in which each side of the sample chamber 640 is approximately 25 mm in length, although as noted this latter parameter can be suitably adjusted, as previously discussed for example, with reference to the graphs of FIGS. 4(b) and 4(c). In this specific example, the membranes are made from a cellulose material intended for small molecule exchange. Variations, as previously noted, are possible.

[0056] FIG. 7 depicts a smaller sample chamber 740 that is similar in shape to that of the chamber 640 and defined by an open-ended aluminum frame 744, but in which each side of the chamber 740 is approximately 10 mm in length. Similarly, transparent (e.g., glass) observation walls and semipermeable membranes (not shown in this view), such as those previously discussed, can be adhered or otherwise attached to the chamber frame 744.

[0057] While the invention has been described in terms of particular variations and illustrative figures, those of ordinary skill in the art will recognize that the invention is not limited to the variations or figures described. In addition, where methods and steps described above indicate certain events occurring in certain order, those of ordinary skill in the art will recognize that the ordering of certain steps may be modified and that such modifications are in accordance with the variations of the invention. Additionally, certain of the steps may be performed concurrently in a parallel process when possible, as well as performed sequentially as described above. Therefore, to the extent there are variations of the invention, which are within the spirit of the disclosure or equivalent to the inventions found in the claims, it is the intent that this patent will cover those variations as well.

[0058] To the extent that the claims recite the phrase “at least one of’ in reference to a plurality of elements, this is intended to mean at least one or more of the listed elements, and is not limited to at least one of each element. For example, “at least one of an element A, element B, and element C,” is intended to indicate element A alone, or element B alone, or element C alone, or any combination thereof. “At least one of element A, element B, and element C” is not intended to be limited to at least one of an element A, at least one of an element B, and at least one of an element C.

[0059] This Detailed Description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

[0060] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,”and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprise” (and any form of comprise, such as “comprises” and “comprising”), “have” (and any form of have, such as “has” and “having”), “include” (and any form of include, such as “includes” and “including”), and “contain” (and any form of contain, such as “contains” and “containing”) are open-ended linking verbs. As a result, a method or device that “comprises,” “has,” “includes,” or “contains” one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more steps or elements. Likewise, a step of a method or an element of a device that “comprises,” “has,” “includes,” or “contains” one or more features possesses those one or more features, but is not limited to possessing only those one or more features. Furthermore, a device or structure that is configured in a certain way is configured in at least that way, but may also be configured in ways that are not listed.

[0061] The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below, if any, are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed. The description set forth herein has been presented for purposes of illustration and description, but is not intended to be exhaustive or limited to the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the disclosure. The embodiment was chosen and described in order to best explain the principles of one or more aspects set forth herein and the practical application, and to enable others of ordinary skill in the art to understand one or more aspects as described herein for various embodiments with various modifications as are suited to the particular use contemplated and in accordance with the following appended claims. Additional embodiments include any one of the embodiments described above and described in any and all exhibits and other materials submitted herewith, where one or more of its components, functionalities or structures is interchanged with, replaced by or augmented by one or more of the components, functionalities or structures of a different embodiment described above and as set forth in the following appended claims.PARTS LIST FOR FIGS. 1 (a) - 7100 imaging platform140 macroscopic sample chamber140 A sample chamberMOB sample chamber140C sample chamber144 interior enclosure145 optically transparent side wall146 top surface147 optically transparent side wall148 bottom or base surface149 side wall151 side wall152 inner semipermeable membranes156 buffer / flow channels160 inlet tubes162 outlet tubes166 illumination axis168 detection (optical) axis170 first objective lens174 second objective lens179 focusing lens180 photodetectorflow di recti on / path flow direction / path condensates imaging system light source optical fiber optical fiber first microscope arm electrically tunable lens second microscope arm first relay lens pair pair of galvo mirrors second relay lens pair beamsplitter objective lens electrically tunable lens first relay lens pair pair of galvo mirrors second relay lens pair beamsplitter objective lens first image sensor second image sensor572 focusing lens580 focusing lens640 sample chamber644 chamber frame648 glass observation walls652 semipermeable membranes740 sample chamber744 chamber frame

[0062] It will be understood that the description related to specific embodiments of a sample chamber and imaging system. As such, it will be further understood that there are variations and modifications that will be readily apparent for practicing the invention and as described and covered in accordance with the following appended claims.

Claims

CLAIMS1. A sample chamber for an imaging system, the chamber comprising; an enclosure; a plurality of optically transparent walls forming the enclosure which is sized and configured to retain an aqueous sample; and one or more inner semipermeable membranes disposed in relation to corners of the sample chamber, the one or more membranes being configured to separate the aqueous sample from the optically transparent walls and further configured to control the environment within the formed enclosure.

2. The sample chamber according to claim 1, in which four (4) inner semipermeable membranes are disposed, each of the membranes being attached at 45 degree angles relative to respective corners of the sample chamber.

3. The sample chamber according to claim 1, wherein the optically transparent walls are made from glass.

4. The sample chamber according to claim 1, wherein the inner semipermeable membranes are made from PEGDA hydrogel.

5. The sample chamber according to claim 1, wherein the imaging system is a light sheet microscopy system.

6. The sample chamber according to claim 1, wherein the imaging system is a spectroscopy system.

7. The sample chamber according to claim 1, wherein the imaging system is an x-ray scattering system.

8. An imaging system comprising:a sample chamber having an interior enclosure formed by a plurality of optically transparent walls at opposing sides of the sample enclosure; a first objective lens disposed in relation to a first optically transparent wall to enable light to enter the sample chamber; a second objective lens disposed in relation to an second optically transparent wall of the sample chamber that is orthogonal to the first optically transparent wall of the chamber, wherein the first objective lens is disposed along an illumination axis and the second objective lens is disposed along a detection axis and in which one or more inner semipermeable membranes are disposed in relation to the optically transparent walls of the sample chamber, the membranes separating the walls of the chamber from an aqueous sample disposed in the sample enclosure.

9. The imaging system according to claim 8, wherein the one or more semipermeable membranes form a channel to permit flow of a buffer solution.

10. The imaging system according to claim 9, wherein the one or more semipermeable membranes are disposed at comers of the sample chamber and at 45 degree angles such that the membranes do not obscure either the illumination axis or the detection axis of the imaging system.

11. The imaging system according to claim 10, wherein the imaging system is a light sheet microscopy system.

12. The imaging system according to claim 10, in which four semipermeable membranes are disposed at each corner of the sample chamber, the defined channels extending between a top surface and bottom surface of the sample chamber.

13. The imaging system according to claim 12, including inlet and outlet tubes extending into and out of each defined channel.

14. The imaging system according to claim 11, wherein the light sheet microscopy system comprises a light source linked to a pair of optical fibers, wherein each of the optical fibers are configured to direct light to orthogonal side walls of the sample chamber.

15. A method for imaging of an aqueous sample, the method comprising: providing the aqueous sample in a sample chamber, the chamber having a plurality of optically transparent walls defining an enclosure and one or more inner semipermeable membranes in which the aqueous sample is separated from the walls of the chamber; and adding one or more agents within a channel formed between the at least one semipermeable membrane and the optically transparent walls of the chamber, the semipermeable membrane further configured to permit passage of the one of more agents but preventing the sample from passing into the channel.

16. The method according to claim 15, in which the agents comprise at least one of urea and salt.

17. The method according to claim 15, wherein four (4) inner semipermeable membranes are disposed in the sample chamber, each membrane being disposed at respective comers of the sample chamber and at 45 degrees thereto.

18. The method according to claim 15, wherein the inner semipermeable membranes are fabricated from a PEGDA hydrogel.

19. The method according to claim 17, further comprising illuminating the sample along an illumination axis and observing the sample along a detection axis orthogonal to the illumination axis and in which the semipermeable membranes are disposed not to impede or block the illumination and detection axes.

20. The method according to claim 19, wherein illumination and detection can be toggled selectively along either of the illumination and detection axes.

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