This project was funded by the National Science Foundation BRIGE Award (1221019).

1. Setting up the System
<ol>
	<li>Use cyanoacrylate epoxy to adhere a 9 mm diameter, 1 mm thick red latex rubber cylinder to the front face of one 10 MHz ultrasonic transducer and use cyanoacrylate epoxy to adhere a 9 mm diameter, 1 mm thick red latex rubber cylinder to a 6 mm thick acrylic block which is then epoxied the same way to the reference ultrasonic transducer to act as an acoustic spacer.</li>
	<li>Set up an optical train that has a beam expander being hit by the laser first. Then place a manually adjustable aperture second. Finally use a polarizing beam splitter cube as the third element and place the ultrasonic transducer not in the EFPA prism holder and the transducer in the EFPA prism holder at each output of the non-polarizing beam splitter. 
	Note: The polarizing beam splitter cube is used to ensure a pure, single polarization for excitation as this is critical to the proper functionality of all EFPA techniques.</li>
	<li>Expand the outgoing laser beam using lenses to create a beam expander of at least 3X from the q-switched ND:YAG laser. 
	Note: The beam is intentionally oversized compared to the latex rubber absorber on the sample transducer in order to ensure proper sensor function despite laser light walk off due to refraction through the prism at varying angles of incidence.</li>
	<li>Align the optical train and EFPA prism holder such that the flat side of the mount nearest the prism will be set to a 0&#176; angle using a digital level. This ensures a correct starting point for the angular spectrum data that will be collected during the experiments.</li>
	<li>Connect and power on external devices such as the oscilloscope, stepper motor driver to the computer, the ultrasonic transducers, and XY stage motors. Physically connect the transducer not in the EFPA prism mount to Ch0 and physically connect the transducer in the EFPA prism mount to Ch1 via 50 ohm BNC cables. The software is preprogrammed to recognize acoustic signals from these specific channels.</li>
</ol>
2. EFPA System Initialization and Optical Alignment
<ol>
	<li>Manually adjust the adjustable aperture to block the beam to a 1 mm diameter.</li>
	<li>Start the programming software (e.g., LabVIEW), set the angle to 70&#176; by pressing the green &#34;move&#34; button to move the mount to the angle necessary for 70&#176; excitation at the prism/sample interface.</li>
	<li>Using appropriate laser safety glasses (OD 7+ at 532 nm), look into the prism from the side perpendicular to the laser beam and manually move the stage in the X and Y axes using the hand wheels until the 1 mm laser spot is fluorescently visible on the rubber latex. Ensure that the beam is centered on the latex.</li>
	<li>Expand the manually adjustable aperture to its maximum opening and look at the running front panel of the program to ensure both the laser energy measurement photoacoustic signal from the EFPA prism mount (red line) and the photoacoustic signal from the laser energy measurement side (white line) are visible and are approximately the same amplitude.</li>
	<li>Stop the program by pressing the &#34;STOP&#34; button. 
	Note: If the button is not pressed the prism will have to be manually reset before continuing the testing. Once the initialization protocol has been completed, TIRPAS, PAS/TIRPAS refractometry, or OTPAS can be performed.</li>
</ol>
3. TIRPAS Technique
<ol>
	<li>Place the prism in the plastic prism mount adapter as shown in Figure 3. Next, place 2.5 &#181;l of immersion oil index matched to the type of prism used, onto the center of the prism and sandwich the oil by placing a substrate on top of the oil layer.</li>
	<li>Place 25 &#181;l of sample on the latex rubber connected to the transducer in the EFPA transducer mount as shown in Figure 3 so that it coats the entire surface without bubble formation. The sample can be any optically absorbing material such as a solution of dye, a biological fluid, or an analyte suspended in a solution. No preparation of the sample is needed. Compress the prism mount and tighten the mount together with mounting screws to a set torque of 16.75 g/mm for each screw.</li>
	<li>Select the &#34;Setup&#34; tab and select &#34;Setup&#34; on the drop-down menu.</li>
	<li>Run the program entitled OTPAS thin film analyzer_USB-5133.vi (Supplementary File).</li>
	<li>View the acoustic signal generated by the sample as shown in Figure 4. 
	Note: The angle of incidence can be changed to control the optical penetration depth of the evanescent field to observe thinner or thicker optical sections of the sample.</li>
</ol>
4. PAS/TIRPAS Refractometry
<ol>
	<li>Place the prism in the plastic prism mount adapter as shown in Figure 3. Next, place 2.5 &#181;l of immersion oil index matched to the type of prism used, onto the center of the prism and sandwich the oil by placing a substrate on top of the oil layer.</li>
	<li>Place 25 &#181;l of sample onto the rubber piece connected to the transducer in the EFPA transducer mount as shown in Figure 3. Compress the prism mount and tighten the mount together with mounting screws to a set torque of 16.75 g/mm for each screw.</li>
	<li>Select the &#34;Angular Spectrum&#34; tab and select &#34;Angular Spectrum&#34; on the drop down menu. Next, input the appropriate parameters into the program as shown in Table 1.</li>
	<li>Run the program and wait until the angular spectrum has been completed and the program has ended.</li>
	<li>Right click on the angular spectrum graph and select &#34;Export &#8594; Export data to excel&#34; to save the data and open the .csv file.
	<ol>
		<li>Open this data in a graphing program (e.g., KaleidaGraph), and perform a numerical derivative on it clicking on &#34;Macros&#34; and selecting &#34;Derivative&#34;. Input the appropriate columns to take the derivative on and press &#34;Okay&#34; and the numerical derivative will be computed.</li>
		<li>Graph the numerical derivative vs. angle and select &#34;Curve fit&#34;. Select the &#34;Smooth&#34; option5,18,98 and select the checkbox of the data under &#34;Curve fit selections&#34; to fit to smooth noise from the data. Select the down arrow under &#34;View&#34; and select &#34;Copy curve fit to data window&#34; to extract the curve fit data to another column.</li>
	</ol>
	</li>
	<li>Manually search through the curve fit to find the local minimum and its corresponding angle of incidence that indicates a transition from the PAS to TIRPAS regimes. That minimum corresponds to the measured critical angle, as shown in Figure 5. Using the equation nsample = nprismsin&#952;c, calculate the bulk refractive index of the unknown sample at the wavelength used for laser interrogation. Typical results are shown in Table 1.</li>
</ol>
5. OTPAS
<ol>
	<li>Place 2.5 &#181;l of immersion oil (index matched to type of glass used) on the center of the prism.</li>
	<li>Place the film or substrate to be tested film side up (away from the prism) and ensure that no bubbles are formed during placement. 
	Note: If bubbles form, remove sample film or substrate and reattempt application.</li>
	<li>Place 25 &#181;l of immersion oil on the latex rubber so that the immersion oil coats the entire surface without bubble formation.</li>
	<li>Compress the substrate/film layers as shown in Figure 3. Tighten the mounting screws to a set torque of 16.75 g/mm that must be identical for each screw. 
	Note: Torque wrench in protocol is in oz.-in., therefore 16.75 g/mm ~ 15 oz.-in.</li>
	<li>Select the &#34;Angular Spectrum&#34; tab and select &#34;Angular Spectrum&#34; on the drop down menu. Next, input the appropriate parameters into the program as shown in Table 3.</li>
	<li>Run the program and wait until the angular spectrum has been completed and the program has ended.</li>
	<li>Rerun the test by performing steps 5.1-5.6 using the substrate or film (whichever wasn&#8217;t done previously) as shown in Figure 6.</li>
	<li>Select &#8220;Curve Fitting&#8221; in the drop down box and select the &#8220;Curve fitting&#8221; tab. Next, input the appropriate parameters as shown in Table 5. Select the film scan under &#8220;Sample&#8221;. Select the substrate scan under &#8220;Substrate&#8221;.</li>
	<li>Input the refractive index, polarization, and other options for the scans previously run in steps 5.1-5.6 as shown in Table 4.</li>
	<li>Run the program by selecting &#34;Curve Fitting&#34; in the drop down box and selecting the &#34;Curve Fitting&#34; tab. Observe the refractive index and thickness under &#34;Film RI&#34; and &#34;Thickness of film&#34; shown at the top right of the program&#39;s graphical user interface. Typical data is shown in Figure 7.</li>
	<li>Use the &#34;Batch fit&#34; option to fit many scans at once by inputting the number of scans to batch fit and selecting a .csv file to output the data to and repeat step 5.10. 
	Note: Once the program is run it will fit each set of data and output all refractive index, thickness, and residual values to the .csv. In order for this to work, the scans must be in a numerical list such as scan_001.csv, scan 002.csv, etc.</li>
</ol>

The authors declare that they have no competing financial interests.

The use of devices for the characterization of materials at the nanoscale will undoubtedly continue far into the future in order to produce new materials and combinations of materials to solve difficult and costly societal problems. Many methods such as ellipsometry, atomic force microscopy (AFM), and traditional photoacoustics are currently used to evaluate material properties at the nanoscale despite their inherent limitations due to few alternatives. EFPA shows promise as a consolidated set of techniques that can push beyond the typical limitations of the photoacoustic effect to provide a single instrument to characterize materials at the nanoscale.
The protocol described herein and its associated sub-techniques have the capability to estimate both bulk and thin film refractive indices of a material, film thickness, and have detection capabilities for a variety of biosensing applications. OTPAS specifically has the advantage that it can estimate the thickness and refractive index of nanometer scale films despite the fact that the films themselves do not contain inherently absorbing pigmentation. EFPA as a consolidated set of techniques has a few advantages over techniques such as ellipsometry, atomic force microscopy, and the traditional photoacoustic effect. The first advantage is that EFPA is comparatively inexpensive to set up. A typical EFPA setup can estimate refractive index from both the bulk and surface, can estimate thickness, and can probe absorbing materials and will cost around $5,000 to set up. In contrast, AFM setups cost around $50,000 and cannot estimate optical properties. Secondly, EFPA can estimate these properties within a few minutes without complex interaction such as in ellipsometry where input parameter error can cause differences in results. Finally, EFPA, when compared to the traditional photoacoustic effect, has axial resolution 100X smaller as it can estimate thickness of 200 nm films6 whereas the traditional photoacoustic effect is limited to 7,500 nanometers when using similar lasers and setup82. Conveniently, almost every material has independent and differing surface and bulk refractive index properties as well as absorptive properties, which collectively make for functional differences in their use in optical research systems.
Critical steps within the protocol come down to three primary considerations. First, optical coupling of the sample, whether that be a liquid or a film, is crucial to obtain reliable and repeatable results. Air bubbles within the immersion oil or sample will lead to inaccurate characterization of the thickness, refractive index, and in some cases a complete lack of sensitivity of the transducer to the excited sample owing to the inability of ultrasound to adequately pass through a liquid/air interface due to vastly differing acoustic impedance values. Second, with regard to OTPAS, a sample that is homogeneously thick in the area irradiated by the laser beam in order to provide consistent thickness values during scanning since as of yet EFPA cannot determine thickness differences within the area of excitation. Finally, again with regard to OTPAS, the sample film must be transparent to the wavelength being used. This is a fundamental assumption made in the mathematical equations that describe how OTPAS finds thickness and refractive index values. If absorption is substantial enough that more than 1% of the incident light is absorbed by the film, characterization will not match the real thickness and refractive index values of the film.
Modifications to the EFPA technique can be made with relative ease and other types of components can be substituted in for more effective scans or functionality. The only major issues that have to be addressed when building an EFPA system are the following. First, it is crucial to have a method of automatically rotating a prism mount and acquiring acoustic signals at small (0.05 degree) increments. Second, having a nanosecond pulse width or shorter laser system to excite samples using the photoacoustic effect. Third, having a graphical user interface or program to acquire the data to do mathematical curve fitting and numerical derivatives for estimating thickness and bulk/thin film refractive index.
There are three fundamental limitations to EFPA. The first is that in order to find the bulk refractive index, thin film refractive index, thickness, or detect materials using TIRPAS, the sample must have a refractive index that is less than that of the prism or substrate it is in contact with so that total internal reflection can occur which is the basis of all three of these techniques. The second limitation is that EFPA currently requires physical contact in order to estimate thickness, refractive index, and for detection of properties. The final limitation is that OTPAS, a sub-technique of EFPA, currently requires optically transparent materials to estimate thickness and refractive index of the thin film of interest.
This type of consolidated instrument could find many applications in characterization environments including research cores and industrial environments. Future work regarding EFPA techniques is focused on the improvement of the refractive index accuracy through improvements in instrumentation and translational/rotational stages. Additional advancements will focus around the improvement of signal-to-noise ratio for the detection of smaller quantities of absorbing materials, so as to characterize weakly absorbing materials, as are found most commonly in biological systems.

Advancements in the understanding of optical materials1,3,4,6,7,10,13-16 have provided new insights into the creation of thin film materials for a host of optical devices, including antireflection coatings on lenses, high extinction ratio optical filters, and highly absorbing slab waveguides17. These advancements would not be possible without the use of many characterization techniques, such as ellipsometry4,6,18, contact angle measurement, atomic force microscopy7,11,19, and scanning/transmission electron microscopy, which assist in the iterative improvement of these technologies by providing direct measures or indirect estimates of fundamental optical material properties. Said properties, such as the refractive index, govern how the materials interact with incident photons, which directly affects their function and their use in optical applications. However, each of these techniques has limitations relating to resolution, sample preparation, cost, and complexity, and each generates only a subset of the data needed to fully characterize the material. That being said, a new set of techniques, known as evanescent field-based photoacoustics (EFPA)5,6,15,18,20-49 as shown in Figure 1, has the potential to estimate material properties at the nanoscale in a consolidated set of experiments. EFPA encompasses the sub-techniques of total internal reflection photoacoustic spectroscopy (TIRPAS)23,25,26,33-35,43-45 , photoacoustic spectroscopy/total internal reflection photoacoustic spectroscopy refractometry (PAS/TIRPAS refractometry)18, and optical tunneling photoacoustic spectroscopy (OTPAS)6, and has been used to estimate bulk and thin film refractive index, film thicknesses, as well as to detect absorbing materials at a prism/sample or substrate/sample interface.
In order to understand the EFPA mechanism, one must first understand the concept of photoacoustic spectroscopy (PAS), which refers to the generation of ultrasonic pressure waves by the rapid thermoelastic expansion of a chromophore, following the absorption of an ultra-short (&#60; &#181;sec) pulse of light (Figure 1). Theoretical and mathematical framework for the photoacoustic effect discussed in this paper can be obtained here50-59. The resulting change in pressure can be detected by an ultrasonic microphone or transducer. The photoacoustic effect, originally discovered in 1880 with the invention of Alexander Graham Bell&#39;s photophone, was &#34;rediscovered&#34; in the early 1970s due to advancements in laser and microphone technology, and eventually put into practical use to fill niche applications from biomedical imaging to thin film analysis to non-destructive testing of materials.1,53-57,59-82 This effect can be mathematically described with one-dimensional wave equations, wherein the wave is a simple acoustic source whose pressure (p) varies in both position (x) and time (t):
<img alt="Equation1" src="/files/ftp_upload/54192/54192equation1.jpg" />
with solutions for simple acoustic sources of the form64
<img alt="Equation2" src="/files/ftp_upload/54192/54192equation2.jpg" />
where p is pressure, &#915;=&#945;vs2/Cp where &#945; is the volume thermal expansion coefficient, vs is the speed of sound in the medium, and Cp is the heat capacity at constant pressure, H0 is the radiant exposure of the laser beam, c is the speed of sound in the excited medium, x is length, and t is time. The magnitude of the resulting acoustic wave relies directly upon the optical absorption coefficient of the material, &#181;a, which is the inverse of the optical penetration depth, &#948;, which is in turn a measure of the distance the light travels until it decays to 1/e of its initial optical intensity. While Equation (1) is a general equation for a one dimensional plane wave source, typical absorbers will emit a spherical acoustic wave in three dimensions. Beyond the mathematical description, applications of the photoacoustic effect54 span many imaging modalities such as microscopy, tomography, and even molecular imaging owing to the photoacoustic effect having high sensitivity due to the large optical absorption due to the naturally present chromophore hemoglobin. Other applications of the photoacoustic effect even include the estimation of various thin film properties15,16,20,21,24,26-32,36-39,41,42,56,83,84. However, PAS does have certain limitations: (1) its extensive optical penetration depth eliminates the ability to probe near-field optical properties at surfaces (2) its efficiency of capturing the emitted acoustic energy is low due to spherical propagation of the majority of the energy away from the detector (3) samples must include chromophores in the wavelength regime under consideration.
When combined with evanescent field-based techniques, however, many of these limitations can be ameliorated. The evanescent field occurs when a beam of light undergoes total internal reflection (TIR), as described by Snell&#39;s Law, which effect also allows fiber optic waveguides to guide light large distances (km) for computation and telecommunication applications. In practical applications, the evanescent field is used in a variety of characterization and imaging technologies, including attenuated total reflectance spectroscopy (ATR). Imaging is achieved with high contrast due to the confinement of the light to within the first few hundred nanometers into the sample of interest. The evanescent field takes the form of an exponentially decaying field that extends into the external medium to an optical penetration depth that is typically on the order of the wavelength being used (usually ~500 nm or less) as shown in equations 3 and 4.
<img alt="Equation3" src="/files/ftp_upload/54192/54192equation3.jpg" />
where I is the light intensity in % at a location z from the prism/sample interface, I0 is the initial light intensity in % at the interface, z is distance in nanometers, and &#948;p is the optical penetration depth as shown in equation 4. With such a small optical penetration depth, the evanescent field is able to interact with the environment very close to the interface of the two materials, and well below the optical and acoustic diffraction limits. The optical properties of materials or particles within this range may perturb the field or otherwise alter its generation, which interaction can be detected by a variety of methods3,5,6,10,15,17,18,21,23,25-27,29-47,84-95.
When evanescent techniques are combined with PAS, the photoacoustic waveforms produced can be used to characterize materials or particles interacting with the evanescent field, creating the evanescent-field based photoacoustics (EFPA) family of techniques, as shown in Figure 1. This family includes, but is not limited to, total internal reflection photoacoustic spectroscopy (TIRPAS), optical tunneling photoacoustic spectroscopy (OTPAS), and surface plasmon resonance photoacoustic spectroscopy (SPRPAS). The interested reader should refer to the following references for derivations of the equations used for TIRPAS5,6,18,23,25,26,33-35,43-47, PAS/TIRPAS refractometry18, and OTPAS6. In each case, the photoacoustic effect is generated through a different excitation mechanism than simple transmittance through a prism; for instance, in TIRPAS, the light is evanescently coupled through a prism/substrate/sample interface into the chromophores (which could include the sample material itself, or guest molecules within the sample), whereas in SPRPAS, the primary mode of excitation is instead through the absorption of a surface plasmon, which is a secondary E-M wave created when the energy of the evanescent field is transferred into the electron cloud of a metal layer deposited on the prism surface. This family of techniques was originally invented in the early 1980s by Hinoue et al., and improved upon by T. Inagaki et al. with the invention of SPRPAS, but saw very little development due to technical limitations of the light sources and available detection equipment. More recently, previous investigations have shown that increased sensitivity and utility are possible with modern polyvinylidene fluoride (PVDF) ultrasonic detectors and q-switched neodymium-doped yttrium aluminum garnet (Nd:YAG) lasers. Specifically, nanosecond-pulsed Nd:YAG lasers result in a 106 fold increase in the peak power, which enables EFPA techniques to become useful tools for evaluating the optical properties of a variety of materials and interfaces5,6,15,18,21-29,31-47,84,96. Additionally, previous work has further shown the capability of such techniques to determine structural information about materials at an interface, which was previously never achievable with traditional photoacoustic spectroscopy (PAS) technologies due to their relatively large penetration depth53,55,57,59,61,62,69,73,75,80,81.
This capability is shown in the protocols that follow under the OTPAS technique; however, on a more fundamental level the three techniques each rely on a different definitive equation, which determines the capabilities of the technology. For example, in TIRPAS, the optical penetration depth of the evanescent field, &#948;&#39;p, primarily drives the resulting acoustic signal intensity to an absorbing sample, and is described by:
<img alt="Equation4" src="/files/ftp_upload/54192/54192equation4.jpg" />
where &#955;1 is the wavelength of light traveling through the prism medium and is defined by the relation &#955;1 = &#955;/n1 wherein n1 is the refractive index of the prism material. Additionally, &#952; refers to the angle of excitation, and n21 refers to the ratio of the refractive indices of each medium and is defined by n21 = n2/n1, wherein n2 is the refractive index of the sample material. The larger the optical penetration depth, the more material is being irradiated. For the photoacoustic effect, the greater the optical penetration depth, the more material is being excited that can produce acoustic waves leading to a larger acoustic signal.
Unlike TIRPAS however, in PAS/TIRPAS refractometry the primary equation is Snell&#39;s law:
<img alt="Equation5" src="/files/ftp_upload/54192/54192equation5.jpg" />
where n1 is the refractive index of the prism, &#952;1 is the angle of incidence at the prism/sample interface, n2 is the refractive index of the sample, and &#952;2 is the angle of the light that is refracted through the second medium. The sensitivity of estimating the refractive index of a material is primarily driven by the accuracy of the estimation of &#952;1. In total internal reflection, which is achieved when &#952;1 is beyond the critical angle which generates an evanescent field, sin &#952;2 = 1 and therefore, Equation 5 reduces to n2 = n1sin&#952;1. (Note: &#952;1= &#952;critical) Knowing the angle at which the numerical derivative (dP/d&#952; where P is the peak to peak voltage of the photoacoustic signal and &#952; is the angle of incidence of the light with the sample) of the photoacoustic signal has a local minima allows for the estimation of &#952;1 that allows the user to solve for n2 and thus estimate the bulk refractive index of a sample as shown in Figure 1.
Finally, in OTPAS, the following equation relates optical transmission in % to photoacoustic peak to peak voltage by:
<img alt="Equation6" src="/files/ftp_upload/54192/54192equation6.jpg" />
where T is the percent optical transmission, p is the peak-to-peak voltage generated by the angular spectrum of a substrate with a film on it, p0 is the peak-to-peak voltage generated by the angular spectrum of a substrate, &#946; is the coupling constant based upon the refractive index of prism and the immersion oil, &#945; is the attenuation factor, and is a factor that includes thickness and refractive index of the sample film within the evanescent field. The sensitivity of this technique to thickness and refractive index is driven by the accuracy of estimating the peak to peak acoustic signal intensities, p and p0 at each angle of incidence in the angular spectrum. It has been shown that &#946; can be directly calculated based upon the refractive indices of the prism and the immersion oil; consequently, it is a straightforward task to calculate the optical transmission at each angle of incidence and to then extract an estimate for the refractive index and thickness of the film through statistical curve fitting analysis. The interested reader should refer to Goldschmidt et al. for more information.5,6
The EFPA system is a photoacoustic based system capable of estimating the thickness, thin film refractive index, bulk refractive index, and generating acoustic signals through optical absorption for detection. The system is comprised of a laser, an optical train to guide the light to the prism/sample and to the laser energy measurement side. The laser energy measurement side is used to normalize the photoacoustic signal to the incident laser energy as shown in Figure 2. The EFPA system is driven by a stepper motor driver to rotate the prism/sample for the angular spectra in PAS/TIRPAS refractometry and OTPAS. The system acquires data through a digital acquisition card and provides a user interface and automated stage control through an in house program.

<table><tbody><tr><td>100 mm plano convex lens</td><td>Thorlabs</td><td>LA1509</td><td>Plano convex lens for beam expander</td></tr><tr><td>-30 mm plano concave lens</td><td>Thorlabs</td><td>LC2679</td><td>Plano concave lens for beam expander</td></tr><tr><td>10 MHz Ultrasonic transducers</td><td>Harisonic</td><td>I31006T</td><td>Ultrasonic sensors used for photoacoustic detection, both for laser energy measurement and for OTPAS mount</td></tr><tr><td>Immersion oil Type A</td><td>Cargille</td><td>16482</td><td>Index of 1.519 to match that of NBK-7 substrates</td></tr><tr><td>Natural latex rubber sheet (red)</td><td>McMastercarr</td><td>86085K11</td><td>Index ~1.519 to match that of Type A immersion liquid to act as an optical absorber to measure optical tunneling and laser energy</td></tr><tr><td>Laser goggles</td><td>VERE</td><td>53</td><td>Used to protect eyes from laser light</td></tr><tr><td>Cage mounted non polarizing beam splitter cube</td><td>Thorlabs</td><td>CM1-BS013</td><td>Split laser light so that one half can be measured and one half can be used for excitation</td></tr><tr><td>Cage mounted polarizing beam splitter cube</td><td>Thorlabs</td><td>CM1-PBS251</td><td>Ensure light is polarized before being used for optical tunneling experiments</td></tr><tr><td>Graduated ring activated iris diaphragm</td><td>Thorlabs</td><td>SM1D12C</td><td>Cut beam down to a smaller size for alignment</td></tr><tr><td>Data acquisition card</td><td>National Instruments</td><td>USB-5133</td><td>USB oscilloscope to acquire data</td></tr><tr><td>Stepper motor driver</td><td>National Instruments</td><td>MID-7604</td><td>Stepper motor driver to drive stepper motors for angular spectra</td></tr><tr><td>Sherline XY stage (14&amp;rdquo;)</td><td>Sherline</td><td>5600-CNC/5610-CNC</td><td>Sherline XY stage</td></tr><tr><td>4-jaw self centering chuck</td><td>Sherline</td><td>1076/1034</td><td>Sherline rotational attachment</td></tr><tr><td>Right angle attachment</td><td>Sherline</td><td>3701</td><td>Right angle attachment to attach rotational mount</td></tr><tr><td>CNC rotary table</td><td>Sherline</td><td>8730</td><td>Rotary table for holding OTPAS prism/sample</td></tr><tr><td>Surelite I-20 laser system</td><td>Continuum</td><td>I-20</td><td>Q-switched Nd:YAG laser for exciting samples</td></tr><tr><td>NBK-7 prism</td><td>Thorlabs</td><td>PS911</td><td>Right angle prism for EFPA</td></tr><tr><td>Adjustable torque wrench</td><td>Tohnichi</td><td>RTD40Z</td><td>Adjustable torque wrench to equally tighten down the EFPA mount for each technique to 16.75 g/mm</td></tr><tr><td>Digital level</td><td>Micromark</td><td>84519</td><td>Digital level to ensure EFPA prism holder starts at 0 degree.</td></tr></tbody></table>

evanescent field based photoacoustics: optical property evaluation at surfaces

Here, we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. Optical property evaluation of thin films and the surfaces of bulk materials is an important step in understanding new optical material systems and their applications. The method presented can estimate thickness, refractive index, and use absorptive properties of materials for detection. This metrology system uses evanescent field-based photoacoustics (EFPA), a field of research based upon the interaction of an evanescent field with the photoacoustic effect. This interaction and its resulting family of techniques allow the technique to probe optical properties within a few hundred nanometers of the sample surface. This optical near field allows for the highly accurate estimation of material properties on the same scale as the field itself such as refractive index and film thickness. With the use of EFPA and its sub techniques such as total internal reflection photoacoustic spectroscopy (TIRPAS) and optical tunneling photoacoustic spectroscopy (OTPAS), it is possible to evaluate a material at the nanoscale in a consolidated instrument without the need for many instruments and experiments that may be cost prohibitive.

Results have been shown for TIRPAS, PAS/TIRPAS refractometry, and OTPAS that are the subtechniques within the EFPA platform. Figure 4 shows a representative TIRPAS acoustic wave generated from an absorbing sample. The bipolar nature of the acoustic wave is characteristic of the TIRPAS technique and indicates that TIRPAS is occurring. This bipolar waveform occurs due to the acoustic reflection at the interface between the sample and the glass substrate due to a large difference in acoustic impedance. For PAS/TIRPAS refractometry Figure 5 and Table 1 was obtained. Figure 5 shows the angular spectrum and numerical derivative obtained for a sample undergoing testing to estimate the bulk refractive index. Table 1 shows the results of using the PAS/TIRPAS refractometry to estimate the bulk refractive index of a Water/PEG/Direct red dye mixture as compared to the bulk refractive index estimate using a standard handheld refractometer. Finally, OTPAS results are shown in Figure 7 and Table 2. Figure 7 shows two figures of the angular scans that are taken during OTPAS. Table 2 shows a comparison between OTPAS and spectroscopic ellipsometry of the same thin film samples.
<img alt="Figure 1" src="/files/ftp_upload/54192/54192fig1.jpg" /> 
Figure 1. Subtechnologies of EFPA. EFPA is currently comprised of three distinct sub-technologies. These technologies are TIRPAS, PAS/TIRPAS refractometry, and OTPAS. Each technique can evaluate materials to derive or determine different properties. TIRPAS detects materials based upon their optical absorption for biosensing purposes, PAS/TIRPAS refractometry evaluates bulk refractive index, and OTPAS evaluates thin film refractive index and thickness. In TIRPAS, the light beyond the critical angle &#952;c creates an evanescent field that can generate an acoustic wave upon interacting with an optical absorber. In PAS/TIRPAS refractometry, both TIRPAS and PAS waveforms are obtained from both evanescent field photoacoustic excitation and traditional photoacoustic excitation. By plotting these two regimes on an angular spectrum graph, the transitional angle can be observed, which can then be used to derive the refractive index. Finally, in OTPAS, a spectrum of acoustic signals are obtained with laser irradiation beyond the critical angle &#952;c for both a thin film on a substrate and a bare substrate. By applying a non-linear curve-fitting algorithm to the data, the thin film thickness and refractive index can be derived. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54192/54192fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54192/54192fig2.jpg" /> 
Figure 2. EFPA schematic/photo. Left: To set up EFPA the laser beam must be expanded to overfill the sensing area covered by the latex rubber. The beam should initially be at a 45-degree angle to the prism as shown. Right: Photo of the setup showing the optical train. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54192/54192fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54192/54192fig3.jpg" /> 
Figure 3. Sample loading. Samples are loaded with the prism making optical contact via immersion oil to the substrate. In TIRPAS or PAS/TIRPAS refractometry, direct liquid contact is achieved with the sample on the substrate for testing. In OTPAS, the optical coupling through additional immersion oil between the substrate and red latex rubber allows for optical tunneling to occur. The mount is then clamped together using a torque wrench and mounting screws. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54192/54192fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54192/54192fig4.jpg" /> 
Figure 4. TIRPAS typical data. TIRPAS waveforms typically have a bipolar acoustic signal appearance that is characteristic of the TIRPAS method. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54192/54192fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54192/54192fig5.jpg" /> 
Figure 5. PAS/TIRPAS typical data. Left: Angular spectrum data that is obtained by irradiating the sample at various angles of incidence. Right: Numerical derivative of left figure that reveals a local minima indicating the transition from the PAS to TIRPAS regimes, which in turn corresponds with the position of the critical angle. Reprinted with permission.18 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54192/54192fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54192/54192fig6.jpg" /> 
Figure 6. Program flow chart. The program is run in a few iterative steps. The prism mount is set to zero degrees and then parameters are selected before running the program. Then the program is run to acquire an angular spectrum of both a substrate and a film. Finally, a curve is fit to the data to estimate the film refractive index and thickness. Reprinted with permission.6 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54192/54192fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54192/54192fig7.jpg" /> 
Figure 7. OTPAS typical data. Left: This figure shows the angular spectrum scans of an MgF2 film and an N-BK7 substrate respectively. Right: By dividing the MgF2 film angular spectrum scan by the N-BK7 substrate scan and multiplying by a constant factor beta, degree of optical tunneling (%) vs. angle of incidence can be obtained, which allows for the estimation of the refractive index and thickness of the thin film. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54192/54192fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>PAS/TIRPAS</td>
			<td>Sample 1</td>
			<td>Sample 2</td>
			<td>Sample 3</td>
			<td>Sample 4</td>
			<td>Sample 5</td>
			<td>Atago R-5000</td>
		</tr>
		<tr>
			<td>Direct red/PEG 
			125 &#181;g/ml</td>
			<td>1.395</td>
			<td>1.395</td>
			<td>1.395</td>
			<td>1.395</td>
			<td>1.395</td>
			<td>1.395-1.397</td>
		</tr>
		<tr>
			<td>Direct red/PEG 
			250 &#181;g/ml</td>
			<td>1.39</td>
			<td>1.39</td>
			<td>1.39</td>
			<td>1.39</td>
			<td>1.39</td>
			<td>1.390-1.396</td>
		</tr>
		<tr>
			<td>Direct red/PEG 
			500 &#181;g/ml</td>
			<td>1.388</td>
			<td>1.389</td>
			<td>1.389</td>
			<td>1.389</td>
			<td>1.389</td>
			<td>1.381-1.395</td>
		</tr>
		<tr>
			<td>Direct red/PEG 
			750 &#181;g/ml</td>
			<td>1.382</td>
			<td>1.382</td>
			<td>1.387</td>
			<td>1.387</td>
			<td>1.387</td>
			<td>1.372-1.395</td>
		</tr>
		<tr>
			<td>Myoglobin 
			460 &#181;g/ml</td>
			<td>1.33</td>
			<td>1.329</td>
			<td>1.331</td>
			<td>1.33</td>
			<td>1.331</td>
			<td>1.335</td>
		</tr>
	</tbody>
</table>
Table 1. PAS/TIRPAS results. The following table shows typical results for Direct Red dye with 50% PEG mixed in to raise the refractive index. Reprinted with permission.18
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Test type&#160;</td>
			<td>Technique&#160;</td>
			<td>Film type&#160;</td>
			<td>Refractive index&#160;</td>
			<td>Thickness (nm)&#160;</td>
		</tr>
		<tr>
			<td>Intrasample&#160;</td>
			<td>OTPAS&#160;</td>
			<td>MgF2 200 nm&#160;</td>
			<td>1.384&#177;0.004&#160;</td>
			<td>203&#177;6&#160;</td>
		</tr>
		<tr>
			<td>Intrasample&#160;</td>
			<td>Ellipsometry&#160;</td>
			<td>MgF2 200 nm&#160;</td>
			<td>1.393&#177;0.001&#160;</td>
			<td>192.4&#177;1.1&#160;</td>
		</tr>
		<tr>
			<td>Intersample&#160;</td>
			<td>OTPAS&#160;</td>
			<td>MgF2 200 nm&#160;</td>
			<td>1.395&#177;0.011&#160;</td>
			<td>220&#177;19&#160;</td>
		</tr>
		<tr>
			<td>Intersample&#160;</td>
			<td>Ellipsometry&#160;</td>
			<td>MgF2 200 nm&#160;</td>
			<td>1.392&#177;0.002&#160;</td>
			<td>195.2&#177;1.8&#160;</td>
		</tr>
	</tbody>
</table>
Table 2. OTPAS results. The following table shows typical results for 200 nm MgF2 thin films in OTPAS vs. spectroscopic ellipsometry. Intrasample refers to testing a single film ten times, whereas intersample refers to testing ten films independently. Reprinted with permission.6
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td># of averages</td>
			<td fo:width="0.6in" style="width:40px">1</td>
			<td>Start angle</td>
			<td fo:width="0.6in" style="width:35px">60</td>
			<td>Warmup (min)</td>
			<td style="width:90px">0</td>
			<td style="width:90px">Refractive index (prism)</td>
			<td>1.519</td>
		</tr>
		<tr>
			<td># scans</td>
			<td>1</td>
			<td>Step size</td>
			<td>0.1</td>
			<td>Save to</td>
			<td>&#8220;yourfilename&#8221;.csv</td>
			<td>Microstep #</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Set Q-switch</td>
			<td>275</td>
			<td>Stop angle</td>
			<td>80</td>
			<td>Laser select</td>
			<td>Surelite</td>
			<td>Velocity (rpm)</td>
			<td>500</td>
		</tr>
		<tr>
			<td>Error tolerance (%)</td>
			<td>5</td>
			<td>Laser startup</td>
			<td>on</td>
			<td>Low pass filter (program)</td>
			<td>1.00 x 107</td>
			<td>Acceleration (RPS)</td>
			<td>200</td>
		</tr>
	</tbody>
</table>
Table 3. Angular spectrum settings for PAS/TIRPAS refractometry. The following table shows the settings necessary for an angular spectrum in PAS/TIRPAS refractometry.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td># of averages</td>
			<td fo:width="0.6in" style="width:40px">64</td>
			<td>Start angle</td>
			<td fo:width="0.6in" style="width:35px">70</td>
			<td>Warmup (min)</td>
			<td style="width:90px">1</td>
			<td style="width:90px">Refractive index (prism)</td>
			<td>1.519</td>
		</tr>
		<tr>
			<td># scans</td>
			<td>1</td>
			<td>Step size</td>
			<td>0.1</td>
			<td>Save to</td>
			<td>&#8220;yourfilename&#8221;.csv</td>
			<td>Microstep #</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Set Q-switch</td>
			<td>275</td>
			<td>Stop angle</td>
			<td>72</td>
			<td>Laser select</td>
			<td>Surelite</td>
			<td>Velocity (rpm)</td>
			<td>500</td>
		</tr>
		<tr>
			<td>Error tolerance (%)</td>
			<td>5</td>
			<td>Laser startup</td>
			<td>on</td>
			<td>Low pass filter (program)</td>
			<td>1.00 x 107</td>
			<td>Acceleration (RPS)</td>
			<td>200</td>
		</tr>
	</tbody>
</table>
Table 4. Angular spectrum settings for OTPAS. The following table shows the settings necessary for an angular spectrum in OTPAS.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td style="width:65px">Lower refractive index</td>
			<td>1</td>
			<td style="width:72px">Tolerance</td>
			<td>1.00 x 10-12</td>
			<td style="width:80px">Coupler RI</td>
			<td style="width:83px">1.519</td>
			<td style="width:65px">Substrate data</td>
			<td style="width:83px">Select yourfilename.csv</td>
		</tr>
		<tr>
			<td>Lower thickness</td>
			<td>0 nm</td>
			<td>Refractive index guess</td>
			<td>1.3</td>
			<td>Wavelength</td>
			<td>532 nm</td>
			<td>Multiple files save to</td>
			<td>Select yourfilename.csv</td>
		</tr>
		<tr>
			<td>Upper thickness</td>
			<td>1,000 nm</td>
			<td>Thickness guess</td>
			<td>200 nm</td>
			<td>Polarization</td>
			<td>P polarized</td>
			<td>How many files</td>
			<td># of files you want to fit</td>
		</tr>
		<tr>
			<td>Max iteration</td>
			<td>5,000</td>
			<td>Substrate RI</td>
			<td>1.519</td>
			<td>Film data</td>
			<td>Select yourfilename.csv</td>
			<td>Type of fit</td>
			<td>Single fit/Batch fit</td>
		</tr>
	</tbody>
</table>
Table 5. Curve fitting parameters. The following table shows the curve fitting parameters necessary for correct parameter estimation.
Supplemental Code File: OTPAS thin film analyzer_USB-5133.vi. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54192/OTPAS_Thin_Film_Analyzer_USB-5133.vi" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces at JoVE.com

Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces

Here we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. This technique evanescent field-based photoacoustics can be used to create a photoacoustic metrology system to estimate materials&#39; thicknesses, bulk and thin film refractive indices, and explore their optical properties.

Here, we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. ...

evanescent-field-based-photoacoustics-optical-property-evaluation-at

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

11.6K Views.  Duquesne University. Here we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. This technique evanescent field-based photoacoustics can be used to create a photoacoustic metrology system to estimate materials' thicknesses, bulk and thin film refractive indices, and explore their optical properties.

Video: Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Bioengineering

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy

High resolution optical spectroscopy methods are demanding in terms of either technology, equipment, complexity, time or a combination of these. Here we demonstrate an optical spectroscopy method that is capable of resolving spectral features beyond that of the spin fine structure and homogeneous linewidth of single quantum dots (QDs) using a standard, easy-to-use spectrometer setup. This method incorporates both laser and photoluminescence spectroscopy, combining the advantage of laser line-width limited resolution with multi-channel photoluminescence detection. Such a scheme allows for considerable improvement of resolution over that of a common single-stage spectrometer. The method uses phonons to assist in the measurement of the photoluminescence of a single quantum dot after resonant excitation of its ground state transition. The phonon&#39;s energy difference allows one to separate and filter out the laser light exciting the quantum dot. An advantageous feature of this method is its straight forward integration into standard spectroscopy setups, which are accessible to most researchers.

The manuscript describes a method of phonon-assisted quasi-resonant fluorescence spectroscopy that incorporates both laser-limited resolution and photoluminescence (PL) spectroscopy. This method utilizes optical phonons to provide linewidth-limited resolution spectra of atom-like semiconductor structures in the energy domain. The method is also easily realized with a single spectrometer optical spectroscopy setup.

High resolution optical spectroscopy methods are demanding in terms of either technology, equipment, complexity, time or a combination of these. Here we ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

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Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

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Rapid and Non-Destructive Characterization of Biofilm Elastic Properties

Quantifying Elastic Properties of Environmental Biofilms using Optical Coherence Elastography

Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances (EPS). This paper presents a detailed account of the implementation of optical coherence elastography (OCE) measurements tailored for the elastic characterization of biofilms. OCE is a non-destructive optical technique that enables the local mapping of the microstructure, morphology, and viscoelastic properties of partially transparent soft materials with high spatial and temporal resolution. We provide a comprehensive guide detailing the essential procedures for the correct implementation of this technique, along with a methodology to estimate the bulk Young's modulus of granular biofilms from the collected measurements. These consist of the system setup, data acquisition, and postprocessing. In the discussion, we delve into the underlying physics of the sensors used in OCE and explore the fundamental limitations regarding the spatial and temporal scales of OCE measurements. We conclude with potential future directions for advancing the OCE technique to facilitate elastic measurements of environmental biofilms.

Author Spotlight: Characterizing Environmental Biofilm Mechanics Using Optical Coherence Elastography and its Applications in Wastewater Treatment

This paper highlights the optical coherence elastography (OCE) technique's efficacy in rapidly and non-destructively characterizing biofilm elastic properties. We elucidate critical OCE implementation procedures for accurate measurements and present Young's modulus values for two granular biofilms.

Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances ...

Environment

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...

Fabrication of a Stable, Biologically Relevant Phantom Material

A Stable Phantom Material for Optical and Acoustic Imaging

Establishing tissue-mimicking biophotonic phantom materials that provide long-term stability are imperative to enable the comparison of biomedical imaging devices across vendors and institutions, support the development of internationally recognized standards, and assist the clinical translation of novel technologies. Here, a manufacturing process is presented that results in a stable, low-cost, tissue-mimicking copolymer-in-oil material for use in photoacoustic, optical, and ultrasound standardization efforts.
The base material consists of mineral oil and a copolymer with defined Chemical Abstract Service (CAS) numbers. The protocol presented here yields a representative material with a speed of sound c(f) = 1,481 &#177; 0.4 m&#183;s-1 at 5 MHz (corresponds to the speed of sound of water at 20 &#176;C), acoustic attenuation &#945;(f) = 6.1 &#177; 0.06 dB&#183;cm-1 at 5 MHz, optical absorption &#181;a(&#955;) = 0.05 &#177; 0.005 mm-1 at 800 nm, and optical scattering &#181;s&#39;(&#955;) = 1 &#177; 0.1 mm-1 at 800 nm. The material allows independent tuning of the acoustic and optical properties by respectively varying the polymer concentration or light scattering (titanium dioxide) and absorbing agents (oil-soluble dye). The fabrication of different phantom designs is displayed and the homogeneity of the resulting test objects is confirmed using photoacoustic imaging.
Due to its facile, repeatable fabrication process and durability, as well as its biologically relevant properties, the material recipe has high promise in multimodal acoustic-optical standardization initiatives.

Author Spotlight: A Stable Phantom Material for Optical and Acoustic Imaging  

This protocol describes the fabrication of a stable, biologically relevant phantom material for optical and acoustic biomedical imaging applications, featuring independently tunable acoustic and optical properties.

Establishing tissue-mimicking biophotonic phantom materials that provide long-term stability are imperative to enable the comparison of biomedical imaging ...

Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces

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Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

A Stable Phantom Material for Optical and Acoustic Imaging