Patent Application
20240003905
Temperature is a fundamental environmental parameter that is important in cellular activity regulations. The classical temperature control techniques employ heating sources to conduct the heat transfer, which can be time-consuming and nonuniform over the target surface. In recent decades, plasmon resonances in nanostructures, especially metallic nanoparticles, have been proved to be efficient in regulating localized heat rapidly and feasibly. These nanometer-sized heaters are capable of harvesting light due to the internal decay of hot carriers, facilitating many practical applications, such as photothermal therapy, neuron activation, phase separation, gas sensing, and heterogeneous catalysis. However, localized heating achieved by metallic nanoparticles still suffers from poor space precision of heating due to the random distribution of metallic nanoparticles, resulting in bulk heating on the sample. Moreover, the nanocavities created by the tightly positioned metal nanoparticles may also generate excessive heat, leading to overheating on the target.
Therefore, there is a need for methods, and related aspects, for rapid temperature regulation and uniform temperature distribution over detection fields in various analytical applications.
This present disclosure provides rapid temperature modulation processes using plasmonic scattering microscopy. The temperature modulation strategies of the present disclosure have many applications, including activities associated with cellular membrane temperature changes as part of drug screening processes and the like. These and other aspects will be apparent upon a complete review of the present disclosure, including the accompanying figures.
According to various embodiments, a method of modulating temperature in a detection field is presented. The method includes introducing an incident light toward a second surface of a substrate to induce a plasmonic wave at least proximal to a first surface of the substrate such that a temperature in a selected heating space within the detection field is substantially uniformly changed. The first surface of the substrate is coated with a metallic layer. In addition, the selected heating space comprises a Z-dimension that extends above the metallic layer about 110 nm or less, thereby modulating the temperature in the detection field.
According to various embodiments, a system for modulating temperature in a detection field is presented. The system includes a substrate receiving area configured to receive a substrate that comprises first and second surfaces, wherein the second surface is coated with a metallic layer that is configured to create surface plasmon resonance when incident light is introduced toward the second surface at a suitable incident angle via the first surface of the substrate, and wherein the metallic layer comprises at least a first set of analyte binding moieties; a light source configured to introduce an incident light toward the substrate receiving area; a detector configured to collect light scattered by at least one analyte disposed on the metallic layer when the substrate is received in the substrate receiving area and the incident light is introduced from the light source; and a controller that comprises, or is capable of accessing, computer readable media comprising non-transitory computer-executable instructions which, when executed by at least one electronic processor, perform at least: disposing a fluidic sample that comprises the analyte on the second surface of the substrate such that at least a portion of the analyte binds to at least a portion of the first set of analyte binding moieties to produce one or more surface-bound analytes when the substrate is received in the substrate receiving area; introducing the incident light from the light source at the suitable incident angle toward the second surface of the substrate when the substrate is received in the substrate receiving area; introducing the incident light toward the second surface of the substrate such that an area defined by X- and Y-dimensions of a selected heating space within the detection field disposed at least proximal to the second surface of the substrate is within a range of about 1 to about 1000 μm2; adjusting a power density of the incident light such that a temperature in the selected heating space within the detection field is substantially uniformly changed to a selected temperature; and, detecting light scattered by the surface-bound analytes over a duration to produce an analyte imaging data set to thereby at least detect the surface-bound analytes using the detector when the substrate is received in the substrate receiving area. In some embodiments, a fluidic device comprises the substrate. In some embodiments, the fluidic material is substantially free of plasmonic metallic nanoparticles when the fluidic sample that comprises the analyte is disposed on the second surface of the substrate.
According to various embodiments, a computer readable media is presented. The computer readable media comprises non-transitory computer-executable instructions which, when executed by at least one electronic processor, perform at least: disposing a fluidic sample that comprises an analyte on a second surface of a substrate such that at least a portion of the analyte binds to at least a portion of a first set of analyte binding moieties to produce one or more surface-bound analytes when the substrate is received in a substrate receiving area; introducing incident light from a light source at a suitable incident angle toward the second surface of the substrate to create surface plasmon resonance when the substrate is received in the substrate receiving area; introducing the incident light toward the second surface of the substrate such that an area defined by X- and Y-dimensions of a selected heating space within a detection field disposed at least proximal to the second surface of the substrate is within a range of about 1 to about 1000 μm2; adjusting a power density of the incident light such that a temperature in the selected heating space within the detection field is substantially uniformly changed to a selected temperature; and, detecting light scattered by the surface-bound analytes over a duration to produce an analyte imaging data set to thereby at least detect the surface-bound analytes using the detector when the substrate is received in the substrate receiving area.
Various optional features of the above embodiments include the following. A temperature within the detection field that is outside of the selected heating space is substantially unchanged. The method includes flowing a fluidic material over the first surface of the substrate in the selected heating space, which fluidic material is substantially free of plasmonic metallic nanoparticles. The Z-dimension extends above the metallic layer about 100 nm. The selected heating space comprises X- and Y-dimensions and the method comprises introducing the incident light toward the second surface of the substrate such that an area defined by the X- and Y-dimensions of the selected heating space is within a range of about 1 to about 1000 μm2. The method includes changing a focus level of the incident light to adjust the area defined by the X- and Y-dimensions of the selected heating space within the range of about 1 to about 1000 μm2. The metallic layer comprises gold (Au). The selected heating space comprises at least one analyte and the method comprises detecting light scattered by the analyte to produce an analyte imaging data set. The analyte comprises one or more biomolecules. One or more cells comprise the biomolecules. The biomolecules comprise transient receptor potential vanilloid 1 (TRPV1) ion channels. The analyte comprises one or more fluorescent labels and wherein the method further comprises detecting fluorescent light emitted from the analyte. The method includes adjusting a power density of the incident light such that the temperature in the selected heating space within the detection field is substantially uniformly changed to a selected temperature. The power density of the incident light is no more than about 3 kW/cm2. The selected temperature is in a range of about 33° C. to about 80° C. The incident light comprises is 660 nm p-polarized light.
In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth throughout the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In describing and claiming the methods, systems, and devices, the following terminology, and grammatical variants thereof, will be used in accordance with the definitions set forth below.
About: As used herein, “about” or “approximately” or “substantially” as applied to one or more values or elements of interest, refers to a value or element that is similar to a stated reference value or element. In certain embodiments, the term “about” or “approximately” or “substantially” refers to a range of values or elements that falls within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value or element unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value or element).
Binding: As used herein, the term “binding”, typically refers to a non-covalent association between or among two or more entities.
Detect: As used herein, “detect,” “detecting,” or “detection” refers to an act of determining the existence or presence of one or more analytes in a given sample.
Binding Moiety: As used herein, “binding moiety” refers to a molecule or compound that is capable of binding to an analyte, for example, via a protein or other biomolecule displayed on a surface of a cell.
In some embodiments: As used herein, the term “in some embodiments” refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.
Moiety: As used herein, “moiety” in the context of chemical compounds or structures refers to one of the portions into which the compound or structure is or can be divided (e.g., a functional group, a substituent group, or the like).
Sample: As used herein, “sample” or “fluidic sample” refers to a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g., a polypeptide), which is assayed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells, cell components, or non-cellular fractions.
Subject: As used herein, the term “subject” means any member of the animal kingdom. In some embodiments, “subject” refers to humans. In some embodiments, “subject” refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a ferret, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, a subject may be a transgenic animal, genetically-engineered animal, and/or a clone. In some embodiments, the subject is an adult, an adolescent or an infant. In some embodiments, terms “individual” or “patient” are used and are intended to be interchangeable with “subject.”
Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
System: As used herein, “system” in the context of analytical instrumentation refers a group of objects and/or devices that form a network for performing a desired objective.
Plasmonic absorption of light can create significant local heat and has become a promising tool for rapid temperature regulation. Current plasmonic heating usually relies on specially designed nanomaterials randomly distributed in the space and hardly provides uniform temperature regulation in a wide field. In some embodiments, the technology provided in the present disclosure is a rapid temperature regulation strategy on a plain gold-coated glass slip using plasmonic scattering microscopy, which can be referred to as wide-field plasmonic thermal microscopy (W-PTM). W-PTM has nondestructive local temperature-regulating and concurrent fluorescence imaging capability, and can be a powerful tool to study cellular activities associated with cellular membrane temperature changes.
In some embodiments, W-PTM can provide a temperature regulation range of 33-80° C. at nanometer scale. In some embodiments, W-PTM provides imaging capability, thus allowing statistical analysis of phase-transitioned polymeric nanoparticles, including for drug screening and discovery applications. In some embodiments, W-PTM can be used for noninvasive and local regulation of the transient receptor potential vanilloid 1 (TRPV1) ion channels in the living cells, which can be monitored by simultaneous fluorescence imaging of calcium influx. In some embodiments, W-PTM does not record the propagating plasmonic waves with long decaying length along the surface into the images, thus providing high spatial resolution and Gaussian-distributed point spread function for automatic image processing with, for example, conventional open-source software. In some embodiments, W-PTM enables easy monitoring of thermal dynamics in the time domain by tracking the formation of polymeric aggregation. In some embodiments, W-PTM does not record the strong reflection, thus allowing the incident intensity up to about 3 kW/cm2 so that a wide temperature regulation range can be achieved from room temperature to ˜80° C. In some embodiments, W-PTM records the light from the top of the gold surface, making it possible to integrate with fluorescence detection approaches. In some embodiments, the experimental set-up of W-PTM includes a flow channel thickness of about ˜100 μm in which a p-polarized 660 nm laser is directed to a gold coated (Au) chip to reach SPR.
In some embodiments, the systems and methods described herein include implementations of near field optical imaging in which the near field is created by surface plasmon resonance (SPR) or total internal reflection (TIF). Rather than detection of reflected light, however, scattered light from the sample molecules and sensor surface is detected. Light scattered by a molecule in free space scales with the 6th power of the molecular diameter. For this reason, the scattered light intensity diminishes quickly with the molecular size, making it difficult to image single molecules. To overcome this issue, a sensor surface with a selected roughness is used, such that the sensor surface scatters light with a magnitude comparable with that of the scattered light from the target single molecules. There are different ways to define surface roughness, and one of which is given by
where yi is the height at position i, and n is the number of positions. Using this definition, the surface roughness of a gold surface is −1.5 nm.
In some implementations, a roughness of the sensor surface is in a range of about 1 nm to about 100 nm. The interference of light scattered from the protein and sensor surface produces an image contrast that scales with the 3 rd power of the molecular diameter. This slows down the decay in image contrast with the molecular size, which favors imaging of small objects (e.g., single cells, single protein molecules, etc.).
Rough features and impurities on the sensor surface, and features associated with imperfect optics, all contribute to image contrast, which can mask weak images of single cells or molecules. As described herein, a differential-integral imaging processing algorithm is used to subtract out background features that contribute to image contrast above from each frame of the time sequence images and integrate the differential images to recover the binding and unbinding of single cells on the sensor surface. A drift or motion correction algorithm is introduced to track the drift or motion pattern of one or more features on the sensor surface and correct the drift or motion from each image frame, thereby reducing the impact of drift in position of the sensor surface or the optics or mechanical vibrations of the environment. Binding kinetics are assessed by counting the individual cells on the sensor surface. This digital counting approach allows a precise measurement of binding kinetics. In addition, this approach obviates the need to measure the shift in the surface plasmon resonance angle (determined not only the number of the cells that bind to the sensor surface, but also by the size of the cells) either directly or indirectly.
In some implementations, the objective of the system in
System 100 can include controller 128. Controller 128 can be configured to control one or more components of system 100 (e.g., cameras 108, 114, SLD 111), to control fluid flow to and away from system 100, and to process data or images collected one or more components of system 100 (e.g., cameras 108, 114). In some cases, controller 128 can be used to correct for mechanical drift in system 100.
In one example, gold-coated glass slides were prepared by evaporating 2 nm thick chromium on BK-7 glass slides, followed by 47 nm gold. Before loading into the vacuum chamber for chromium and gold evaporation, the BK-7 glass slides were cleaned by acetone and by deionized water thoroughly. The gold surfaces were examined by Atomic Force Microscopy (AFM), showing islands of variable sizes.
The SPR imaging system has several unique features. First, the evanescent field intensity is localized within −100 nm from the SPR sensor surface (e.g., gold-coated glass slide), making it immune to interference of molecules and impurities in the bulk solution, thus particularly suitable for studying surface binding. Second, there is a large enhancement (20-30 times) in the field near the sensor surface, which is responsible for the high sensitivity of SPR. Finally, the resonance condition of SPR depends on the refractive index near the sensor surface, such that surface charging, small molecules or ions, and biochemical reactions that do not scatter light strongly can also be measured with the same setup from the simultaneously recorded traditional SPR images.
Referring to
I˜|E
v
+E
e
+E
r|2, (2)
where Ep is the plasmonic wave excited by the incident light, Es describes the scattering of the plasmonic wave by an exosome on the sensor surface, and Er is the reflection of the incident wave from the backside of the gold surface. The SPR image contrast is determined by the interference between the planar plasmonic wave and the spherical scattered plasmonic wave, given by 2|Ep∥Es|cos(θ), where Q is the phase difference between the two waves, which produces a spot at the location of the exosome with a parabolic tail. Es is proportional to the optical polarizability of the exosome, which scales with the mass of the exosome or d3, where d is the diameter.
Er in Eq. 2 produces a large background in the SPR image, which masks the weak scattered wave (Es) from a single exosome. To overcome this difficulty, plasmonic waves scattered by the exosome are imaged with a second objective placed on top of the sample, in addition to recording the traditional SPR images from the bottom. This avoids the collection of the strong reflection and also eliminates the parabolic tail, providing a high contrast image of the exosome. At first glance, the image contrast should scale according to |Es|2˜d6. This would lead to a rapid drop in the image contrast with decreasing d, making it challenging to detect small exosomes. However, the gold surface is not atomically flat. Atomic Force Microscopy (AFM) has revealed nm-scaled gold islands, which scatter the surface plasmonic waves and produce a background (Eb) also collected by the top objective. Consequently, the plasmonic image is given by
I˜|E
b
+E
z|2=|Eb|2+2|Eb∥Es|cos(β)+|Es|2, (3)
where β is the phase difference between light scattered by the exosome and by the gold surface. The interference term, 2|Eb∥Es|cos(β), in Eq.3 produces image contrast that scales with d 3, or the mass of the exosome. To differentiate this plasmonic imaging method from the traditional SPR imaging, it is referred to as PSM.
To obtain a high contrast PSM image, |Eb|2 is removed from Eq. 3, which is achieved with the following imaging processing flow. Starting from the raw images captured with a high frame rate, the image frames are averaged (e.g., over 50 ms) to remove pixel and other random noise in the images. Differential images are then obtained by subtracting a previous frame from each frame, or I(N)−I(N−1), where I(N) and I(N−1) are the Nth and (N−1)th image frames. The subtraction removes background features and captures the binding of an exosome to the surface on Nth image frame. To view all the exosomes on the surface on Nth frame, the differential images are integrated from 1 to N. Due to thermal and mechanical drift of the optical system, a drift correction mechanism is introduced to ensure effective removal of the background.
To illustrate,
In some embodiments, a temperature within the detection field that is outside of the selected heating space is substantially unchanged. In some embodiments, method 200 includes flowing a fluidic material over the first surface of the substrate in the selected heating space, which fluidic material is substantially free of plasmonic metallic nanoparticles. In some embodiments, the Z-dimension extends above the metallic layer about 100 nm. In some embodiments, the selected heating space comprises X- and Y-dimensions and method 200 comprises introducing the incident light toward the second surface of the substrate such that an area defined by the X- and Y-dimensions of the selected heating space is within a range of about 1 to about 1000 μm2. In some embodiments, method 200 includes changing a focus level of the incident light to adjust the area defined by the X- and Y-dimensions of the selected heating space within the range of about 1 to about 1000 μm2. In some embodiments, the metallic layer comprises gold (Au). In some embodiments, the selected heating space comprises at least one analyte and the method comprises detecting light scattered by the analyte to produce an analyte imaging data set. In some embodiments, the analyte comprises one or more biomolecules. In some embodiments, one or more cells comprise the biomolecules. In some embodiments, the biomolecules comprise transient receptor potential vanilloid 1 (TRPV1) ion channels. In some embodiments, the analyte comprises one or more fluorescent labels and wherein the method further comprises detecting fluorescent light emitted from the analyte. In some embodiments, method 200 includes adjusting a power density of the incident light such that the temperature in the selected heating space within the detection field is substantially uniformly changed to a selected temperature. In some embodiments, the power density of the incident light is no more than about 3 kW/cm2. In some embodiments, the selected temperature is in a range of about 33° C. to about 80° C. In some embodiments, the incident light comprises is 660 nm p-polarized light.
The present disclosure also provides various systems and computer program products or machine readable media. In some aspects, for example, the methods described herein are optionally performed or facilitated at least in part using systems, distributed computing hardware and applications (e.g., cloud computing services), electronic communication networks, communication interfaces, computer program products, machine readable media, electronic storage media, software (e.g., machine-executable code or logic instructions) and/or the like. To illustrate,
As understood by those of ordinary skill in the art, memory 306 of the server 302 optionally includes volatile and/or nonvolatile memory including, for example, RAM, ROM, and magnetic or optical disks, among others. It is also understood by those of ordinary skill in the art that although illustrated as a single server, the illustrated configuration of server 302 is given only by way of example and that other types of servers or computers configured according to various other methodologies or architectures can also be used. Server 302 shown schematically in
As further understood by those of ordinary skill in the art, exemplary program product or machine readable medium 308 is optionally in the form of microcode, programs, cloud computing format, routines, and/or symbolic languages that provide one or more sets of ordered operations that control the functioning of the hardware and direct its operation. Program product 308, according to an exemplary aspect, also need not reside in its entirety in volatile memory, but can be selectively loaded, as necessary, according to various methodologies as known and understood by those of ordinary skill in the art.
As further understood by those of ordinary skill in the art, the term “computer-readable medium” or “machine-readable medium” refers to any medium that participates in providing instructions to a processor for execution. To illustrate, the term “computer-readable medium” or “machine-readable medium” encompasses distribution media, cloud computing formats, intermediate storage media, execution memory of a computer, and any other medium or device capable of storing program product 308 implementing the functionality or processes of various aspects of the present disclosure, for example, for reading by a computer. A “computer-readable medium” or “machine-readable medium” may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks. Volatile media includes dynamic memory, such as the main memory of a given system. Transmission media includes coaxial cables, copper wire and fiber optics, including the wires that comprise a bus. Transmission media can also take the form of acoustic or light waves, such as those generated during radio wave and infrared data communications, among others. Exemplary forms of computer-readable media include a floppy disk, a flexible disk, hard disk, magnetic tape, a flash drive, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave, or any other medium from which a computer can read.
Program product 308 is optionally copied from the computer-readable medium to a hard disk or a similar intermediate storage medium. When program product 308, or portions thereof, are to be run, it is optionally loaded from their distribution medium, their intermediate storage medium, or the like into the execution memory of one or more computers, configuring the computer(s) to act in accordance with the functionality or method of various aspects disclosed herein. All such operations are well known to those of ordinary skill in the art of, for example, computer systems.
In some aspects, program product 308 includes non-transitory computer-executable instructions which, when executed by electronic processor 304, perform at least: disposing a fluidic sample that comprises an analyte on a second surface of a substrate such that at least a portion of the analyte binds to at least a portion of a first set of analyte binding moieties to produce one or more surface-bound analytes when the substrate is received in a substrate receiving area; introducing incident light from a light source at a suitable incident angle toward the second surface of the substrate to create surface plasmon resonance when the substrate is received in the substrate receiving area; introducing the incident light toward the second surface of the substrate such that an area defined by X- and Y-dimensions of a selected heating space within a detection field disposed at least proximal to the second surface of the substrate is within a range of about 1 to about 1000 μm2; adjusting a power density of the incident light such that a temperature in the selected heating space within the detection field is substantially uniformly changed to a selected temperature; and, detecting light scattered by the surface-bound analytes over a duration to produce an analyte imaging data set to thereby at least detect the surface-bound analytes using the detector when the substrate is received in the substrate receiving area.
Typically, imaging is obtained using device or subassembly 318. As shown, device or subassembly 318 includes a gold coated glass slide. Incident light is introduced via a second surface of the slide and light scattered from analyte molecules disposed on the first surface is detected using the cameras.
Introduction
This example shows wide-field plasmonic thermal microscopy (W-PTM), which provides rapid temperature regulation and uniform temperature distribution over its detection field (
The W-PTM was developed based on the plasmonic scattering microscopy (PSM), a novel wide-field surface plasmon resonance (SPR) imaging approach. In addition to the wide-field imaging ability, which is beyond the capabilities of localized SPR detection with metallic nanoparticles, the W-PTM also shares the advantages of PSM over the traditional SPR systems: 1) the W-PTM does not record the propagating plasmonic waves with long decaying length along the surface into the images, thus providing high spatial resolution and Gaussian-distributed point spread function for automatic image processing with conventional open-source software such as ImageJ. As a result, W-PTM enables easy monitoring of thermal dynamics in the time domain by tracking the formation of polymeric aggregation (i.e., phase transition) particles. 2) W-PTM does not record the strong reflection, thus allowing the incident intensity up to 3 kW/cm2 so that a wide temperature regulation range can be achieved from room temperature to ˜80° C. 3) W-PTM records the light from the top of the gold surface, making it possible to integrate with fluorescence detection approaches, whose signals are massively dissipated through the gold surface in traditional SPR systems.
To investigate the local heating behavior on W-PTM, polymers with low critical solution temperature (LCST) were chosen due to their phase transition and non-photobleaching features. Moreover, a wide range of responsive temperatures (i.e., LCST) can be easily achieved by applying the salt effect to a specific polymer. Temperature-dependent dynamic light scattering (DLS) results showed that 8 groups of LCST polymer precursor with LCST ranging from 33-80° C. could be achieved by dissolving hydroxypropyl cellulose (HPC) or poly(ethylene oxide)(PEO, Mw=100,000) in Na2HPO4 or NaCl aqueous solution with various salt concentrations. As shown in
In order to further confirm the feasibility of LCST polymer in sensing the local temperature of W-PTM, we tested the phase transition processes of four LCST polymer precursors (LCST=33, 45, 62, 72° C., referred to as 33° C. LCST, 45° C. LCST, 62° C. LCST, 72° C. LCST, respectively) on three independent Au chips, respectively. By measuring ensemble image intensity as a function of excitation power density, we found that the phase transitions of these LCST polymers are consistent among these chips. Interestingly, HPC-based (LCST=33, 45° C.,
Next, we calibrated the surface temperature of the gold chip using these LCST polymers. We gradually increased the W-PTM excitation power density in a step-by-step manner while monitoring the phase transition of a specific LCST polymer. The W-PTM intensity becomes very sensitive to the excitation power when the local temperature reaches the phase transition temperature, defined by the W-PTM intensity change exceeding five times the fluctuation of the background signal. And its equilibrium temperature was defined as the phase transition temperature of the specific LCST polymer.
To validate the above findings, low-density lipoprotein (LDL), a natural heat-sensitive protein with a transition temperature at ˜78° C., was used as a reference. We determined the phase transition power density and the corresponding phase transition time of LDL to be 2.1 kW/cm2 and 16 s, respectively, which is comparable to that of 80° C. LCST (2.5 kW/cm2 and 19 s). Both LDL and 80° C. LCST showed phase transition behavior when excited at the same power density (3 kW/cm2), but the ensemble W-PTM intensity change of LDL is close to an exponential increase, while that of 80° C. LCST is closer to linear increase. These dynamics can be explained by the slight lower phase transition temperature of LDL making burst nucleation of LDL at this power density, while the 80° C. LCST is just reaching the phase transition temperature and polymerizing at a slower rate. Nevertheless, this result confirms that local temperature calibration using LCST polymer is reliable and accurate.
Taking advantage of the single-particle analysis capability of W-PTM, an image processing algorithm was employed to analyze the size change of the generated particles during the phase transition. We first subtract a previous frame from each frame to remove background features, resulting in the differential images (
We expect W-PTM to be applied to biological micro-heaters, owing to the advantages of non-invasiveness, label-free feature, accurate temperature control, and light-triggered fast switching feature. As a proof of concept, we attempted to exploit the local thermal effect of W-PTM to selectively activate transient receptor potential vanilloid 1 (TRPV1) ion channels. The TRPV1 ion channel is a calcium-permeable non-selective cation channel that can be activated by capsaicin, heat (>42° C.), pH (<5.9), voltage, and other stimuli. After activated, the action potential can be triggered to promote downstream intracellular signal transduction processes. Thus, elucidating how TRPV1 responds to heat or drugs is vital to understanding diseases that affect every major organ system of the body. However, the activation of TRPV1 is often limited to a few ensemble triggering methods, such as halogen lamps, focused lasers, and drug flows, making it difficult to achieve selective activation.
To investigate the response of TRPV1 ion channels to the localized heat of the W-PTM, we employed the calcium imaging by integrating a set of fluorescence (FL) imaging pathways along with W-PTM (
Compared with the traditional micro-heating methods, such as plasmonic metallic nanoparticles heating, W-PTM provides a more precise and controllable micro-region heating. Owing to the evanescent properties of surface plasmonic waves on W-PTM, the Z-dimension of heating space is limited to ˜100 nm nearby the gold surface, while the XY-dimension can be controlled by the laser focus ranging from ˜1 to hundreds pmt. Moreover, the local temperature on the W-PTM is controllable within 33-80° C. with no overheating effects. Meanwhile, W-PTM can also analyze thermal transition kinetic processes at the single-molecule or single-particle scale, such as the phase transition process of LCST polymers. In addition to quantify the particle size of these generated nanoparticles, W-PTM can also provide 1 ms temporal resolution for rapid real time counting of the particles, better than ensemble measuring methods like DLS.
Until now, the heat activation of TRPV1 is often limited to ensemble triggering methods that lack of selectivity at cell level. In this example, we applied W-PTM in selective heating of the TRPV1 transfected cells, while calcium fluorescence imaging was used as a reference. We demonstrated that W-PTM can selectively activate the cells in the field of view (as few as several cells) in an excitation power-dependent manner without affecting surrounding cells. Our previous study demonstrated that PSM, the parent technique of W-PTM, can image cell deformation at the single focal adhesion level. Therefore, in future studies we could monitor the dynamic cell deformations to characterize the thermally controlled ion channel states. We anticipate that W-PTM will become a powerful tool for studying TRPV1 and other thermal responsive cellular activities, and accelerating drug screening throughput in the future.
While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the methods, systems, and/or computer readable media or other aspects thereof can be used in various combinations. All patents, patent applications, websites, other publications or documents, and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/354,890 filed Jun. 23, 2022, the disclosure of which is incorporated herein by reference.
This invention was made with government support under R01 GM107165 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63354890 | Jun 2022 | US |
