This work was supported by the National Science Foundation (NSF CHE-1847830).

1. Making polyvinyl alcohol (PVA) solution
<ol>
	<li>Measure water and PVA polymer pellets (see Table of Materials) to create a 10 mL solution at a 20% PVA to water ratio by weight.</li>
	<li>Heat the water in the glassware over a hot plate set to 100 &#176;C.</li>
	<li>Place the PVA polymer pellets into the heated water. Insert a magnetic stir bar.</li>
	<li>Reduce the heat to 80 &#176;C and stir until the PVA fully dissolves.</li>
	<li>Cover the top of the glassware to prevent contamination.</li>
	<li>Once fully dissolved, place the 20% PVA solution into an appropriate storage container for storage at room temperature.</li>
</ol>
2. Preparing PVA-coated silicon (Si) wafers
<ol>
	<li>Cut a silicon (Si) wafer (see Table of Materials) into ~10 x 10 mm2 squares.</li>
	<li>Clean the Si substrate using isopropyl alcohol and let it dry.</li>
	<li>Place the clean Si wafer on the chuck of the spin coater (see Table of Materials).
	<ol>
		<li>Drop cast approximately 10 &#181;L of PVA solution onto the center of the Si substrate. Try to avoid bubble formation.</li>
		<li>Coat the Si substrate with a uniform PVA film by spin coating for 30 s at 1,500 rotations per minute (rpm). 
		NOTE: The specified volume of liquid and spin coating parameters create a uniform layer of PVA across the surface of the substrate with sufficient thickness to prevent rapid drying between the spin coating and placing the PS beads onto the PVA surface in the next step.</li>
	</ol>
	</li>
	<li>Remove the sample from the spin coater and place it into a clean sample container to prevent contamination before transferring the PS beads.</li>
</ol>
3. Placing the PS beads onto the PVA-coated surface
<ol>
	<li>Clean a Si substrate using isopropyl alcohol and let it dry.</li>
	<li>Using a pipette, place 1 &#181;L of PS beads suspended in water onto the center of the substrate.</li>
	<li>Let the water evaporate by placing the sample in a storage compartment containing bentonite clay desiccant. 
	NOTE: This step preserves the sample by reducing exposure to humidity.</li>
	<li>Place the PVA-coated substrate (step 2.4) and the substrate with the dried PS beads (step 3.3) under an optical microscope. Depending on their size, a single bead will be visible using simple binoculars or will require higher optical magnification.</li>
	<li>Gently loosen the beads using ultrafine tweezers (see Table of Materials). Use a fine hair paintbrush to collect a few loose beads and lightly tap the hairs of the paintbrush over the freshly PVA-coated wafer. Multiple sweeps should allow the beads to accumulate within the hairs of the brush. Tap the top of the paintbrush hairs to disturb the PS bead powder to release beads onto the tacky PVA surface. 
	NOTE: It is important that the paintbrush is of high quality and clean to avoid releasing fibers and contaminants onto the PVA film&#39;s surface. Moving quickly during this step is essential so that the PVA does not completely dry.</li>
	<li>Repeat this step until it is confirmed by optical microscopy inspection that individual PS beads adhere to the PVA surface.</li>
	<li>Store the sample in a clean container. Allow the sample to dry fully. 
	NOTE: The sample should be allowed to cool and dry completely before further analysis attempts are performed. AFM height measurements or surface profiler measurements can assess the thickness of successive PVA films.</li>
</ol>
4. Loading the sample for AFM characterization
NOTE: The protocol described is based on standard operating procedures of a nanoIR2 (see Table of Materials) platform, but should be adapted according to the AFM model used for the measurement.
<ol>
	<li>Mount the PVA and PS bead sample onto the AFM stage using a metallic AFM disk and adhesive tabs, so that the sample is firmly attached to the sample holder.</li>
	<li>Mount a nanoIR probe (e.g., FORTGG) onto the AFM probe holder. 
	&#8203;NOTE: The AFM cantilever is 225 &#181;m long, 27 &#181;m wide, and 2.7 &#181;m thick, with a tip radius of less than 10 nm. The cantilever is coated with 45 nm thick gold film on both sides to limit its response to the top-side IR illumination of the sample (see Table of Materials). For nanoIR spectroscopy measurements, preferably use a cantilever that has been stored in a polydimethylsiloxane-free environment prior to use.</li>
	<li>Align the read-out laser at the free end of the cantilever beam by turning the laser alignment knobs (x and y adjustments of the laser position and vertical adjustment of the detector position).
	<ol>
		<li>Maximize the SUM signal of the detector.</li>
		<li>Adjust the position of the detector by turning the deflection knob so that the laser is aligned with the center of the position-sensitive detector of the AFM read-out system, corresponding to a vertical deflection signal of ~0 V.</li>
	</ol>
	</li>
	<li>Click on the Load icon in the AFM &#34;Probe&#34; control panel.
	<ol>
		<li>Follow the prompts within the wizard screen. Use the focus arrows to determine the focal plane of the nanoIR cantilever. Use the XY-displacement controls to position the cantilever in the center of the screen (aligned with the white cross).</li>
		<li>Next, click on the focus arrows to find the focal plane of the surface of the sample.</li>
		<li>Use the optical view of the system and the XY-displacement controls to position the cantilever tip above the bead of interest and click on Next.</li>
		<li>On the engage screen, set the &#34;standoff&#34; to 50 &#181;m and click on Approach Only.</li>
	</ol>
	</li>
	<li>Initiate the Engage procedure to approach the tip for imaging.</li>
</ol>
5. Creating topographical and nanoIR images of the multipolymer sample
<ol>
	<li>Acquire topography images in standard &#34;Contact Mode&#34;. Once the position of the cantilever with respect to the PS bead is set, initiate the approach by clicking on the engage icon in the AFM &#34;Probe&#34; control panel. An engage setpoint of a deflection differential of 0.2 V is used for the entire study here, corresponding to a force of ~100 nN. 
	In the AFM &#34;Scan&#34; control panel, set the scan rate to 0.8 Hz, scan size (height and width), and the number of points per line and number of lines per image to use for imaging (512 x 512 was used here). Click on Scan to acquire the topography image. 
	NOTE: Calibration of the cantilever18 is done by determining the deflection sensitivity (in nm/V) from the slope of the deflection-distance curve obtained with the cantilever interacting with a sapphire substrate (Supplementary Figure 1A). The cantilever stiffness is determined from thermal tuning19 (Supplementary Figure 1B). The resonance of the cantilever is fitted using a Lorentzian function. The cantilever stiffness (in N/m) is determined using the equipartition theorem <img alt="Equation 1" src="/files/ftp_upload/65357/65357eq01.jpg" style="margin: auto;" />, where KB is the Boltzmann constant, T is the temperature (T = 295K), and P is the area of the power spectrum of the thermal fluctuations of the cantilever determined by integrating the Lorentzian fit of the thermal tuning data20.</li>
	<li>For nanoIR measurements, position the AFM tip on the feature of interest identified from the topography image.
	<ol>
		<li>Select the tuning fork icon in the nanoIR control panel to determine the contact resonance frequencies of the cantilever. Set an illumination wavenumber that will excite photothermal expansion in the material. Set a range of laser pulse frequency to sweep and set the duty cycle of the nanoIR laser. Select Acquire within the &#34;Laser Pulse Tune Window&#34;.</li>
		<li>Select the second contact resonance of the tip-sample system for nanoIR measurements by positioning the marker bar (green vertical line) at the peak of the second contact resonance. 
		NOTE: The selection of the contact resonance mode can vary depending on the type of cantilever and sample.</li>
		<li>Click on Optimize to align the center of the IR laser focal region with the position of the cantilever tip. Alignment is done at a selected IR illumination wavenumber, corresponding to an absorption band of the material probed. The cantilever is positioned at the center of the laser footprint (Supplementary Figure 2). 
		NOTE: Alignment can vary for different wavenumbers depending on the laser model.</li>
		<li>Acquire a background of the IR laser illumination. This consists of measuring the output of the IR quantum cascade laser (QCL) in the wavelength range of emission at the pulse frequency selected (Supplementary Figure 3). This is important for background correction of the nanoIR spectra.</li>
		<li>Acquire the nanoIR spectrum by selecting the wavenumber range (here, Start and Stop are set to 1,530 cm-1 and 1,800 cm-1, respectively), the step size (2 cm-1), and the number of averages used for the measurement. Perform background correction of the spectra displayed by dividing the photothermal amplitude measured by the attenuated background, which consists of multiplying the background collected in step 5.2.4 by the percentage of power selected for the measurement.</li>
	</ol>
	</li>
	<li>For nanoIR imaging, select the region of interest for imaging.
	<ol>
		<li>Enable phase-locked loop (PLL) auto-tune in the &#34;Laser Pulse Tune Window&#34; (accessed by the Tuning fork icon).</li>
		<li>Adjust the minimum and maximum frequency to create a sweep range centered at the second resonance mode in the general control panel.</li>
		<li>Zero the phase by clicking on zero in the PLL control panel and then click on OK in the &#34;Laser Pulse Tune window&#34;.</li>
		<li>Select IR Imaging Enabled by putting a checkmark in the box within the nanoIR control panel.</li>
		<li>In the &#34;Imaging View&#34; control panel, choose Height (Imaging View 1), Amplitude 2 (Imaging View 2), and Phase 2 (Imaging View 3) to acquire the topographical and chemical images of the sample. Set the acquisition direction to Trace (or Retrace). A line fit Line is often required to observe the topography image of the sample being acquired. Capture fit should be set to None. 
		NOTE: Scanning preferences, such as scan direction captured or color palette used, can be adjusted as needed.</li>
		<li>In the AFM &#34;Scan&#34; control panel, select the Scan icon.</li>
		<li>To save the image, select the Now or End of frame icon in the &#34;Capture&#34; control panel.</li>
	</ol>
	</li>
	<li>To export the data, right-click on the image or spectrum file names within the data lists. Select Export and choose the format of the file to export. Save the file in the desired computer folder location.</li>
</ol>

Exploring Multiphase Polymeric Systems by Nanoscale Infrared Spectroscopy

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The authors have nothing to disclose.

AFM combined with nanoIR spectroscopy can provide nanoscale chemical information using a cantilever in contact mode and a pulsed tunable IR light source. Model systems, such as embedding an absorber with finite dimensions in the volume of a polymeric material, are important to improve the understanding of image formation mechanisms and to determine the performance of the tool. In the case of the PS/PVA configuration presented here, optimization was carried out to obtain a stable PS bead positioned above or below the surf...

Atomic force microscopy (AFM) has become essential to image and characterize the morphology of a wide variety of samples with nanoscale resolution1,2,3. By measuring the deflection of an AFM cantilever resulting from the interaction of the sharp tip with the sample surface, nanoscale functional imaging protocols for local stiffness measurements and tip-sample adhesion have been developed4,5. For soft condensed matter and polymer analysis, AFM measurements exploring the nanomechanical and nanochemical properties of local domains are highly sought after6,7,8. Before the emergence of nanoscale infrared (nanoIR) spectroscopy, AFM tips were chemically modified to assess the presence of different domains from the AFM force curve and deduct the nature of the tip-sample interaction. For instance, this approach was used to unveil the transformation of microdomains of poly(tert-butyl acrylate) at the surface of cyclohexane-treated polystyrene-block-poly(tert-butyl acrylate)block copolymer thin films at the sub 50 nm level9.
The combination of infrared (IR) light with AFM has had a significant impact on the field of polymer science6. Conventional IR spectroscopy is a widely used technique for studying the chemical structure of polymeric materials10,11, but it fails to provide information on individual phases and interphase behavior, as the regions are too small compared to the size of the IR beam used to probe the sample. The problem holds with IR microspectroscopy, as it is restricted by the optical diffraction limit6. Such measurements average the contributions of the entire region excited by the IR light; the signals resulting from the presence of nanoscale phases inside the probed region either exhibit complex fingerprints that should be deconvoluted during post-processing or are lost due to a signal level below the detectable level. Hence, it is essential to develop tools capable of nanoscale spatial resolution and high IR sensitivity to explore nanoscale chemical features in complex media.
Schemes to achieve nanoIR spectroscopy have been developed, first using a metallic AFM tip as a nanoantenna12,13, and more recently exploiting the AFM cantilever&#39;s ability to monitor changes in the photothermal expansion incurred during IR illumination of the sample12,14,15. The latter uses a pulsed, tunable IR light source tuned to an absorption band of the material probed, which causes the sample to absorb radiation and undergo photothermal expansion. This approach is well-suited for organic and polymeric materials. The pulsed excitation makes the effect detectable by the AFM cantilever in contact with the sample surface in the form of an oscillation. The amplitude of one of the contact resonances of the system observed in the frequency spectrum is then monitored as a function of illumination wavelength, which constitutes the nanoIR absorption spectrum of the material beneath the AFM tip15. The spatial resolution of nanoIR imaging and spectroscopy is limited by various effects of the photothermal expansion of the material. It has been evaluated that photothermal nanoIR spectroscopy using contact mode AFM can acquire the vibrational absorption spectra properties of materials with sub 50 nm scale spatial resolution14, with recent work demonstrating the detection of monomers and dimers of &#945;-synuclein16,17. However, quantitative studies of the performance of nanoIR measurements on heterogeneous polymeric materials assembled in various configurations, such as the case of absorbers of finite dimensions embedded in the volume of various polymeric films, remain limited.
This article aims to create a polymeric assembly with an embedded feature of a known dimension to evaluate the sensitivity of photothermal expansion and spatial resolution of nanoIR during surface analysis. The protocol covers the preparation of a polyvinyl alcohol (PVA) polymer thin film on a silicon substrate and the placement of a three-dimensional polystyrene (PS) bead onto or embedded in the PVA film, which constitutes the formation of the model system. NanoIR imaging and spectroscopy measurements are described in the context of evaluating the signals generated by the same PS bead positioned on or beneath the PVA film. The influence of the bead position on the nanoIR signals is evaluated. Methods to assess the spatial footprint of the bead in the nanoIR map are discussed, and the effects of several parameters are considered.

<table><tbody><tr><td>10|0 2200 Golden Taklon Round</td><td>Zem</td><td></td><td></td></tr><tr><td>5357-8NM Tweezers</td><td>Pelco</td><td></td><td></td></tr><tr><td>Adhesive Tabs</td><td>Ted Pella</td><td>16079</td><td></td></tr><tr><td>AFM metal specimen disks</td><td>Ted Pella</td><td>16208</td><td></td></tr><tr><td>Binocular</td><td>AmScope</td><td></td><td></td></tr><tr><td>Cantilever for nanoIR measurements</td><td>AppNano</td><td></td><td>FORTGG</td></tr><tr><td>Cell culture dishes</td><td>Greiner bio-one GmbH</td><td></td><td></td></tr><tr><td>Desiccator</td><td></td><td></td><td></td></tr><tr><td>Floating optical table</td><td>Newport</td><td>RS 4000</td><td></td></tr><tr><td>Hotplate</td><td>VWR</td><td></td><td></td></tr><tr><td>Isopropanol&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Kimwipes</td><td>KIMTECH</td><td></td><td></td></tr><tr><td>Magnetic stir bar</td><td></td><td></td><td></td></tr><tr><td>Microparticles based on polystyrene size: 5 &amp;micro;m</td><td>SIGMA-ALDRICH</td><td>79633</td><td></td></tr><tr><td>nanoIR2 microscope</td><td>Bruker</td><td></td><td>Contact mode NanoIR2</td></tr><tr><td>Nitrogen Tank</td><td>Airgas</td><td></td><td></td></tr><tr><td>Petri dishes</td><td>Greiner bio-one GmbH</td><td></td><td></td></tr><tr><td>Polyvinyl Alcohol</td><td>SIGMA-ALDRICH</td><td>363170</td><td>this polymer was only 87%-89% hydrolyzed, which explains the presence of residual C=O at 1730 cm&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Quantum Cascade Laser</td><td>Daylight Solutions</td><td></td><td>1550-1800 cm&lt;sup&gt;-1&lt;/sup&gt; range</td></tr><tr><td>Silicon wafer</td><td>MEMC St. Peters</td><td>#901319343000</td><td></td></tr><tr><td>Spin coater</td><td>Oscilla</td><td></td><td></td></tr></tbody></table>

advances in nanoscale infrared spectroscopy to explore multiphase polymeric systems

Multiphase polymeric systems encompass local domains with dimensions that can vary from a few tens of nanometers to several micrometers. Their composition is commonly assessed using infrared spectroscopy, which provides an average fingerprint of the various materials contained in the volume probed. However, this approach does not offer any details on the arrangement of the phases in the material. Interfacial regions between two polymeric phases, often in the nanoscale range, are also challenging to access. Photothermal nanoscale infrared spectroscopy monitors the local response of materials excited by infrared light with the sensitive probe of an atomic force microscope (AFM). While the technique is suitable for interrogating small features, such as individual proteins on pristine gold surfaces, the characterization of three-dimensional multicomponent materials is more elusive. This is due to a relatively large volume of material undergoing photothermal expansion, defined by the laser focalization onto the sample and by the thermal properties of the polymeric constituents, compared to the nanoscale region probed by the AFM tip. Using a polystyrene (PS) bead and a polyvinyl alcohol (PVA) film, we evaluate the spatial footprint of photothermal nanoscale infrared spectroscopy for surface analysis as a function of the position of PS in the PVA film. The effect of the feature position on the nanoscale infrared images is investigated, and spectra are acquired. Some perspectives on the future advances in the field of photothermal nanoscale infrared spectroscopy are provided, considering the characterization of complex systems with embedded polymeric structures.

PS ((C8H8)n) beads were deposited on a clean Si substrate (Figure 1A) and on PVA ((CH2CHOH)n) (Figure 1B,C). Due to the poor adhesion of the bead on Si, nanoIR imaging in contact mode could not be acquired for this sample. Instead, using the optical view of the sample on nanoIR, the gold-coated AFM probe was engaged on top of the PS bead in contact mode, with an estimated force of about 100 ...

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Author Spotlight: Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems  

This protocol describes the application of atomic force microscopy and nanoscale infrared spectroscopy to evaluate the performance of photothermal nanoscale infrared spectroscopy in the characterization of three-dimensional multi-polymeric samples.

Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems

Multiphase polymeric systems encompass local domains with dimensions that can vary from a few tens of nanometers to several micrometers. Their composition ...

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Research

JoVE Journal

Engineering

739 Views.  University of Central Florida. This protocol describes the application of atomic force microscopy and nanoscale infrared spectroscopy to evaluate the performance of photothermal nanoscale infrared spectroscopy in the characterization of three-dimensional multi-polymeric samples. 

Video: Author Spotlight: Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems  

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers. A typical inkjet ink is a colloidal dispersion containing approximately ten components including solvent, the nano to micron scale particles which will comprise the printed layer, polymeric dispersants to stabilize the particles, and polymers to tune layer strength, surface tension and viscosity. To rationally and efficiently formulate such an ink, it is crucial to know how the components interact. Specifically, which polymers bond to the particle surfaces and how are they attached? Answering this question requires an experimental procedure that discriminates between polymer adsorbed on the particles and free polymer. Further, the method must provide details about how the functional groups of the polymer interact with the particle. In this protocol, we show how to employ centrifugation to separate particles with adsorbed polymer from the rest of the ink, prepare the separated samples for spectroscopic measurement, and use Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) for accurate determination of dispersant/particle bonding mechanisms. A significant advantage of this methodology is that it provides high level mechanistic detail using only simple, commonly available laboratory equipment. This makes crucial data available to almost any formulation laboratory. The method is most useful for inks composed of metal, ceramic, and metal oxide particles in the range of 100 nm or greater. Because of the density and particle size of these inks, they are readily separable with centrifugation. Further, the spectroscopic signatures of such particles are easy to distinguish from absorbed polymer. The primary limitation of this technique is that the spectroscopy is performed ex-situ on the separated and dried particles as opposed to the particles in dispersion. However, results from attenuated total reflectance spectra of the wet separated particles provide evidence for the validity of the DRIFTS measurement.

Formulation of stable, functional inks is critical to expanding the applications of additive manufacturing. In turn, knowledge of the mechanisms of dispersant/particle bonding is required for effective ink formulation. Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) is presented as a simple, inexpensive way to gain insight into these mechanisms.

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers.  A typical inkjet ink is a colloidal ...

Chemistry

Orientational Transition in a Liquid Crystal Triggered by the Thermodynamic Growth of Interfacial Wetting Sheets

In liquid crystal (LC) physical chemistry, molecules near the surface play a great role in controlling bulk orientation. Thus far, mainly to achieve desired molecular orientation states in LC displays, the "static" surface property of LCs, so-called surface anchoring, has been intensively studied. As a rule of thumb, once the initial orientation of LCs is "locked" by specific surface treatments, such as rubbing or treatment with a specific alignment layer, it hardly changes with temperature. Here, we present a system exhibiting an orientational transition upon temperature variation, which conflicts with the consensus. Right on the transition, the bulk LC molecules experience the orientational rotation, with 90&#176; between the planar (P) orientation at high temperatures and the vertical (V) orientation at low temperatures in the first-order transitional manner. We have tracked thermodynamic surface anchoring behavior by means of polarizing optical microscopy (POM), dielectric spectroscopy (DS), high-resolution differential scanning calorimetry (HR-DSC), and grazing incidence X-ray diffraction (GI-XRD) and reached a plausible physical explanation: that the transition is triggered by a growth of surface wetting sheets, which impose the V orientation locally against the P orientation in the bulk. This landscape would provide a general link explaining how the equilibrium bulk orientation is affected by surface-localized orientation in many LC systems. In our characterization, POM and DS are advantageous by offering information on the spatial distribution of the orientation of LC molecules. HR-DSC gives information about the precise thermodynamic information on transitions, which cannot be addressed by conventional DSC instruments due to limited resolution. GI-XRD provides information on surface-specific molecular orientation and short-range orderings. The goal of this paper is to present a protocol for preparing a sample that exhibits the transition and to demonstrate how the thermodynamic structural variation, both in the bulk and on surfaces, can be analyzed through the abovementioned methods.

Here, we present a protocol to trigger an orientational transition of a liquid crystal in response to temperature. Methodologies are described for preparing a sample in order to observe the transition and the detailed transitional evolution.

In liquid crystal (LC) physical chemistry, molecules near the surface play a great role in controlling bulk orientation. Thus far, mainly to achieve ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various surfaces and interfaces. This non-invasive optical technique can provide the local molecular-level information with monolayer or submonolayer sensitivity. We here are providing experimental methodology on how to selectively detect the buried interface for both macromolecules and biomacromolecules. With this in mind, interfacial secondary structures of silk fibroin and water structures around model short-chain oligonucleotide duplex are discussed. The former shows a chain-chain overlap or spatial confinement effect and the latter shows a protection function against the Ca2+ ions resulting from the chiral spine superstructure of water.

Being comprehensively utilized, sum frequency generation (SFG) vibrational spectroscopy can help to reveal chain conformational order and secondary structural change happening at polymer and biomacromolecule interfaces.

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various ...

Biochemistry

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

Infrared Degenerate Four-wave Mixing with Upconversion Detection for Quantitative Gas Sensing

We present a protocol for performing gas spectroscopy using infrared degenerated four-wave mixing (IR-DFWM), for the quantitative detection of gas species in the ppm-to-single-percent range. The main purpose of the method is the spatially resolved detection of low-concentration species, which have no transitions in the visible or near-IR spectral range that could be used for detection. IR-DFWM is a nonintrusive method, which is a great advantage in combustion research, as inserting a probe into a flame can change it drastically. The IR-DFWM is combined with upconversion detection. This detection scheme uses sum-frequency generation to move the IR-DFWM signal from the mid-IR to the near-IR region, to take advantage of the superior noise characteristics of silicon-based detectors. This process also rejects most of the thermal background radiation. The focus of the protocol presented here is on the proper alignment of the IR-DFWM optics and on how to align an intracavity upconversion detection system.

Here, we present a protocol to perform sensitive, spatially resolved gas spectroscopy in the mid-infrared region, using degenerate four-wave mixing combined with upconversion detection.

We present a protocol for performing gas spectroscopy using infrared degenerated four-wave mixing (IR-DFWM), for the quantitative detection of gas species ...

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &#960;-conjugate Systems

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near infrared (near-IR) transitions, because it can overcome the low sensitivity of spontaneous Raman spectrometers in the near-IR region. Here, we describe technical details of a femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer that we have recently developed. A description of signal generation and optimization, measurement, data acquisition, and calibration and correction of recorded data is provided as well. We present an application of our spectrometer to analyze the excited-state dynamics of &#946;-carotene in toluene solution. A C=C stretch band of &#946;-carotene in the second lowest excited singlet (S2) state and the lowest excited singlet (S1) state is clearly observed in the recorded time-resolved stimulated Raman spectra. The femtosecond time-resolved near-IR stimulated Raman spectrometer is applicable to the structural dynamics of &#960;-conjugate systems from simple molecules to complex materials.

Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of &#946;-carotene in toluene is shown as a representative application.

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near ...

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated biochemical information. FT-IR imaging of tissue is based on the principle that different regions of the mid-infrared are absorbed by different chemical bonds (e.g., C=O, C-H, N-H) within cells or tissue that can then be related to the presence and composition of biomolecules (e.g., lipids, DNA, glycogen, protein, collagen). In an FT-IR image, every pixel within the image comprises an entire Infrared (IR) spectrum that can give information on the biochemical status of the cells that can then be exploited for cell-type or disease-type classification. In this paper, we show: how to obtain IR images from human tissues using an FT-IR system, how to modify existing instrumentation to allow for high-definition imaging capabilities, and how to visualize FT-IR images. We then present some applications of FT-IR for pathology using the liver and kidney as examples. FT-IR imaging holds exciting applications in providing a novel route to obtain biochemical information from cells and tissue in an entirely label-free non-perturbing route towards giving new insight into biomolecular changes as part of disease processes. Additionally, this biochemical information can potentially allow for objective and automated analysis of certain aspects of disease diagnosis.

High-definition Fourier Transform Infrared FT-IR Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

Fourier Transform Infrared (FT-IR) spectroscopic imaging is a fast and label-free approach to obtain biochemical data sets of cells and tissues. Here, we demonstrate how to obtain high-definition FT-IR images of tissue sections towards improving disease diagnosis.

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated ...

Medicine

Using a VSFG Microscope for Multimodal, Rapid, Hyperspectral Chemical Analysis

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 &#181;m. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 &#181;m level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.

Author Spotlight: Unveiling the Potential of VSFG Microscopy in Studying Mesoscopically Heterogeneous Self-Assembled Structures 

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a ...