D. R. acknowledges support from the German Research Foundation (DFG) Research Grant (RA 1848/1) and the European Research Council Starting Grant. V. N. acknowledges financial support from the European Research Council Advanced Investigator Award, and the BMBF&#39;s Innovation in Medicine Award.

1. Optoacoustic Characterization of the &#960;-FBG Detector
<ol>
	<li>Preparation of a microscopic sphere suspended in agar:
	<ol>
		<li>Mix agar powder (1.3% by weight) with distilled water in a glass beaker. Use a hot&#160;plate magnetic-stirrer device to heat the solution close to boiling temperature and dissolve the agar powder until the solution becomes clear and free of air bubbles. Alternatively, the agar solution may be heated using a conventional microwave with stirring performed manually using a glass stirrer. Pour the hot solution into a plastic mold, e.g. syringe with its tip cut out.</li>
		<li>Sprinkle a small amount of microscopic spheres on the agar solution and wait until the solution fully solidifies. Take the solid agar phantom out of the mold by pushing the plunger.</li>
		<li>View the phantom under a stereoscopic microscope cut a small piece of agar which contains a single microscopic sphere.</li>
		<li>Repeat step 1.1.1 and add to the agar solution the solid agar piece containing the single microscopic sphere.</li>
		<li>After solidification, cut the agar phantom under the microscope such that the microscopic sphere is located close to the phantom&#39;s surface.</li>
	</ol>
	</li>
	<li>Optoacoustic measurement
	<ol>
		<li>Use two v-groove fiber holders to hold the fiber tightly on both sides of the &#960;-FBG, and connected the holder to a three dimensional (XYZ) translation computer-operated stage. Ensure that the fiber is submerged to enable the propagation of ultrasound.</li>
		<li>Find the approximate location of the sensing &#960;-FBG element by illuminating different parts of the fiber with the high-power nanosecond-pulse laser beam. The optical absorption of the coating, however weak, will create a signal when the illumination is performed on the &#960;-FBG.</li>
		<li>Place the agar-embedded microscopic sphere directly underneath the &#960;-FBG. The microscopic sphere should be visible to the naked eye.</li>
		<li>Using the translation stage, perform a 2D scan of the &#960;-FBG in the plane parallel to the ground to find the location where the signal from the microscopic sphere is strongest and its corresponding time delay is shortest.</li>
		<li>Perform last adjustments to the illumination to deliver maximum power to the microscopic sphere.</li>
		<li>Using the translation stage perform a 3D scan of the &#960;-FBG and record the signal for each position.</li>
		<li>To obtain the spatially dependent frequency response of the ultrasound detector, perform the Fourier transform on the recorded time-domain ultrasound signal.</li>
	</ol>
	</li>
</ol>
2. Estimation of Robustness and Sensitivity of the &#960;-FBG Detector&#39;s Performance
<ol>
	<li>Use two v-groove fiber holders to hold the fiber tightly on both sides of the &#960;-FBG and submerge the &#960;-FBG.</li>
	<li>Place a dark plate or a graphite rod sturdily to face the &#960;-FBG and illuminate it with the high-power nanosecond-pulse laser beam to create a strong acoustic field.</li>
	<li>Place a water pump inside the water tank and turn it on in order to create rapid variations in the environmental conditions.</li>
	<li>To estimate the robustness of the system, measure the output with the locking circuit turned both on and off. When no locking is performed, it is not possible to accurately detect the ultrasound signal.</li>
	<li>Turn the water pump off.</li>
	<li>To estimate the benefit in sensitivity due to the high coherence of the source, replace the wideband pulse laser with a low coherence source and repeat the acoustic measurement. A decrease of over an order of magnitude in sensitivity is expected when the low-coherence source is used.</li>
</ol>

<ol>
	<li>Hunt, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+transducers+for+pulse-echo+medical+imaging.">Ultrasound transducers for pulse-echo medical imaging.</a> IEEE Transactions on Biomedical Engineering. 30, 453-481 (1983).</li><li>Razansky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+opto-acoustic+tomography+of+deep-seated+fluorescent+proteins+in+vivo.">Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo.</a> Nature Photon. 3, 412-417 (2009).</li><li>Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+deeper+than+microscopy:+the+optical+imaging+frontier+in+biology.">Going deeper than microscopy: the optical imaging frontier in biology.</a> Nature Methods. 7, 603-614 (2010).</li><li>Wang, L. H., Hu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+tomography:+In+vivo+imaging+from+organelles+to+organs.">Photoacoustic tomography: In vivo imaging from organelles to organs.</a> Science. 23, 1458-1462 (2012).</li><li>Sethuraman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+imaging+using+an+IVUS+imaging+catheter.">Photoacoustic imaging using an IVUS imaging catheter.</a> IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control. 54, 978-986 (2007).</li><li>Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optoacoustic+methods+for+frequency+calibration+of+ultrasonic+sensors.">Optoacoustic methods for frequency calibration of ultrasonic sensors.</a> IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control. 58, 316-326 (2011).</li><li>Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+compact+ultrasonic+detector+based+on+a+pi-phase-shifted+fiber+Bragg+grating.">High-sensitivity compact ultrasonic detector based on a pi-phase-shifted fiber Bragg grating.</a> Optics Letters. 36, 1833-1835 (2011).</li><li>Beard, P. C., Mills, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+optical+fibre+ultrasound+sensor+using+a+thin+polymer+film+as+a+low+finesse+Fabry-Perot+interferometer.">Extrinsic optical fibre ultrasound sensor using a thin polymer film as a low finesse Fabry-Perot interferometer.</a> Applied Optics. 35, 663-675 (1996).</li><li>Xiong Pernice, W. H. P., Tang, C., X, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Q+micro-ring+resonators+fabricated+from+polycrystalline+aluminum+nitride+films+for+near+infrared+and+visible+photonics.">High Q micro-ring resonators fabricated from polycrystalline aluminum nitride films for near infrared and visible photonics.</a> Optics Express. 20, 12261-12269 (2012).</li><li>Zhang, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Backward-mode+multiwavelength+photoacoustic+scanner+using+a+planar+Fabry-Perot+polymer+film+ultrasound+sensor+for+high-resolution+three-dimensional+imaging+of+biological+tissues.">Backward-mode multiwavelength photoacoustic scanner using a planar Fabry-Perot polymer film ultrasound sensor for high-resolution three-dimensional imaging of biological tissues.</a> Applied Optics. 47, 561-577 (2008).</li><li>Gr&#252;n, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+photoacoustic+imaging+using+fiber-based+line+detectors.">Three-dimensional photoacoustic imaging using fiber-based line detectors.</a> Journal of Biomedical Optics. 15, 021306-02 (2010).</li><li>Avino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Musical+instrument+pickup+based+on+a+laser+locked+to+an+optical+fiber+resonator.">Musical instrument pickup based on a laser locked to an optical fiber resonator.</a> Optics. Express. 19, 25057-25065 (2011).</li><li>Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wideband+optical+sensing+using+pulse+interferometry.">Wideband optical sensing using pulse interferometry.</a> Optics Express. 20, 19016-19029 (2012).</li><li>Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wideband+fiber-interferometer+stabilization+with+variable+phase.">Wideband fiber-interferometer stabilization with variable phase.</a> IEEE Photonics Technology Letters. 24, 1499-1501 (2012).</li><li>Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+characterization+of+the+response+of+a+silica+optical+fiber+to+wideband+ultrasound.">Spatial characterization of the response of a silica optical fiber to wideband ultrasound.</a> Optics Letters. 37, 15-3174 (2012).</li><li>Rosenthal, A., Razansky, D., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+optoacoustic+inversion+with+arbitrary-shape+detectors.">Model-based optoacoustic inversion with arbitrary-shape detectors.</a> Medical Physics. 38, 4285-4295 (2011).</li></ol>

The authors declare that they have no competing financial interests.

In conclusion, a new optical method for ultrasound detection is introduced, which is based on a combination of a &#960;-FBG and pulse interferometry. The technique is especially suited for optoacoustic imaging applications owing to the transparency of the sensing element, which enables almost arbitrary object illumination patterns. In contrast, standard piezoelectric based ultrasound detectors are opaque and thus block some of the optical paths to the imaged object, leading to bulky imaging setups. The developed optical ...

Ultrasound detectors play a key role in many imaging applications. Conventionally, ultrasound is detected by piezoelectric transducers, which transform pressure waves into voltage signals1. In optoacoustic imaging, ultrasound is generated via a process of thermal expansion by illuminating the object with high-power modulated light2-6. Although piezoelectric transducers are the method of choice in optoacoustic applications, their use often hinders miniaturization mainly because miniaturized piezoelectric transducers are often characterized by low sensitivity. Additionally, since piezoelectric transducers are optically opaque, they may severely interfere with light delivery to the imaged object, limiting possibilities for usable imaging configurations. Light that is back-scattered from the object to the transducer may also limit the proper detection of ultrasound and complicate the design of the imaging system due to optically induced parasitic signals in the transducer7.
Optical detectors of ultrasound have been recognized as a possible alternative to piezoelectric transducers that offers many benefits in optoacoustic imaging scenarios8-12: They are often transparent and can be usually miniaturized without loss of sensitivity. The working principle of optical detectors is interferometric detection of the minute deformation created in the optical medium due to the presence of ultrasound. Often, optical resonators are used to enhance detection sensitivity by trapping light in the perturbed medium for extended durations, thus increasing the effect of the deformation on the phase of the optical signal. In those cases, optical detection schemes are based on monitoring variations in the resonance wavelength, which directly relate to structure deformations in the resonator. Most commonly, narrow-linewidth continuous wave (CW) techniques are used in which a CW laser is tuned to the resonance wavelength. Small shifts in the resonance wavelength change the relative position of the laser&#8217;s wavelength within the resonance, thus causing variations in the intensity of the transmitted/reflected laser light, which can be readily monitored. However, if the resonance shifts are too strong, e.g. owing to large variations in pressure, temperature, or vibrations, the resonance may shift completely away from the laser&#8217;s wavelength, effectively saturating the detector13.
Pulse interferometry14 offers a solution to the limitation of signal saturation and enables ultrasound detection under volatile environmental conditions. In contrast to narrow-linewidth CW schemes, pulse interferometry employs a wideband pulse source to illuminate the resonator. In this case, the resonator acts as a bandpass filter, transmitting only those wavelengths that correspond to its resonance frequency, while the resonance shifts are detected by measuring the wavelength variations in the optical signal at the resonator&#8217;s output, e.g. by using a Mach-Zehnder interferometer locked to quadrature14,15. An automatic reset circuit is used to immediately restore the interferometer&#8217;s working point in the case it is lost due to extreme variation in environmental conditions. Because of the relatively broad bandwidth of the source, the resonance wavelength stays within the illuminated band even under strong perturbations, enabling stable detector operation even under harsh ambient conditions. The use of a coherent source for interrogation, i.e. optical pulses, facilitates low-noise detection.
The corresponding pulse interferometry system used in our experiments is shown in Figure&#160;1. The pulse laser used for interrogation produced 90 fsec pulses at a repetition rate of 100 MHz with output power of 60 mW and spectral width of over 100 nm. The optical filter had a FWHM spectral width of approximately 0.4 nm and was tuned to the frequency of the resonance. Following the filter, an optical amplifier was used to compensate for the significant loss in the filtering. Additional filtering was applied after the amplification stage to reduce amplified spontaneous emission from the amplifier. The resonator used in our experiments is a pi-phase-shifted fiber Bragg grating (&#960;-FBG)8, manufactured by Teraxion Inc. Particularly for the medical application of ultrasound sensing, &#960;-FBGs have the benefit of being all-fiber components, and thus robust and small. Figure 2 shows a comparison between the dimensions of the optical fiber used in this work and a 15 MHz miniaturized intravascular ultrasound (IVUS) piezoelectric transducer. Some alternative resonance-based detection approaches, such as micro-ring resonators fabricated in planar waveguides, require coupling fibers at the component&#8217;s input and output, either leading to more fragile devices or hindering miniaturization. In contrast, &#960;-FBGs are in-fiber components, and do not require additional fiber coupling. The resonance in &#960;-FBGs is created by the pi phase shift in their center; light is trapped around the pi phase shift over portion of the fiber which is considerably shorter than the length of the grating itself. In our experiments, the &#960;-FBG had a length of 4 mm and coupling coefficient of&#160;&#954; = 2 mm-1 and its sensitivity was distributed non-uniformly along its length, with the sensitivity exponentially decreasing from the grating&#8217;s center with a rate of &#954;. The full-width-half-maximum (FWHM) of the sensitivity distribution (SD) was approximately 350 &#181;m. The resonance width of the grating is determined by both its length and its coupling coefficient according to the following equation:
<img alt="Equation 1" fo:content-width="1.5in" src="/files/ftp_upload/50847/50847eq1.jpg" width="150" /> (1) 
where &#955; is the resonance wavelength and neff is the effective refractive index of the mode guided in the fiber8.
To assess whether the &#960;-FBG detector is appropriate for imaging applications, its spatially dependent response needs to be measured over a wide frequency band. However, this task is extremely challenging when conventional acoustic techniques are used. We therefore employ an optoacoustic method for ultrasound detector characterization16 in which a dark microscopic sphere embedded in transparent agar serves as an optoacoustic point source. In our experiment, the microscopic sphere has a diameter of approximately 100 &#181;m and is illuminated with high power nanosecond optical pulses with a repetition rate of 10 Hz, pulse duration of approximately 8 nsec, and average power of 200 mW. The optical energy deposited in the microscopic spheres generates broadband ultrasound signals owing to the optoacoustic effect. The &#960;-FBG detector is translated relatively to the microscopic sphere to obtain its spatially dependent acoustic response. Figure 3 shows an illustration of the optoacoustic experiment. Generally, this technique can be employed to characterize different kinds of ultrasound detectors.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">&#960;-FBG</td>
			<td style="width:71px;">Teraxion Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">&#160;Custom made device</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">microscopic sphere</td>
			<td style="width:71px;">Cospheric LLC</td>
			<td style="width:119px;">BKPMS 90-106um- 10g</td>
			<td style="width:167px;">100 &#181;m polyethylene microsphere</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Femto-second pulse laser used for interrogation&#160;</td>
			<td style="width:71px;">Menlo Systems GmbH</td>
			<td style="width:119px;">T-Light Femtosecond Laser</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Optical filter</td>
			<td style="width:71px;">Optoplex Corporation</td>
			<td style="width:119px;">2-Port Optical Tunable Filter (50 GHz)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Optical amplifier</td>
			<td style="width:71px;">Amonics</td>
			<td style="width:119px;">AEDFA-PM-PA-35-B-FC</td>
			<td style="width:167px;">Benchtop 35dB Gain Pre Amp Polarization Maintaining EDFA&#160;</td>
		</tr>
		<tr height="232">
			<td height="232" style="height:232px;width:216px;">50/50 coupler</td>
			<td style="width:71px;">OZ-Optics</td>
			<td style="width:119px;">FUSED-22-1550-8/125-50/50-3S3S3S3S-3-0.5-PM</td>
			<td style="width:167px;">Fused 2x2 fiber splitter with 0.5 meter long, 3mm OD PVC jacketed 1550nm 8/125&#956; PM fiber 
			pigtails, 50/50 split ratio in the slow axes of PM fibers and with super FC/PC connectors on all 
			ports.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fiber holder</td>
			<td style="width:71px;">Thorlabs</td>
			<td style="width:119px;">T711/M-250</td>
			<td style="width:167px;">Metric, Post-Mountable Fiber Clamp, 250 &#181;m&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Agar for microbiology</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">05039-500G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Nano-second pulse laser used for generating the optoacoustic signals</td>
			<td style="width:71px;">Opotek</td>
			<td style="width:119px;">VIBRANT Arrow 532 type I</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Graphite rod</td>
			<td style="width:71px;">&#160;Faber-Castell</td>
			<td style="width:119px;">120700</td>
			<td style="width:167px;">Faber-Castell Pencil Leads - 0.7 mm</td>
		</tr>
	</tbody>
</table>

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.

Figures 4a and 4b respectively show the signals and their corresponding spectra from the microscopic sphere at a distance of 1 mm from the fiber for three offsets from the center of the &#960;-FBG. The offsets are given in the z direction, as depicted in Figure&#160;3. Clearly, The optical detector&#8217;s sensitivity to high frequency ultrasound (f &#62; 6 MHz) is anisotropic and is highest when the center of the &#960;-FBG is directly above the microscopic sphere. Desp...

Watch this Scientific Journal Video about Wideband Optical Detector of Ultrasound for Medical Imaging Applications at JoVE.com

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

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 ...

wideband-optical-detector-ultrasound-for-medical-imaging

Research

JoVE Journal

Bioengineering

10.7K Views.  Technical University of Munich and Helmholtz Center Munich. 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.

Video: Wideband Optical Detector of Ultrasound for Medical Imaging Applications

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 ...

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Medical-grade Sterilizable Target for Fluid-immersed Fetoscope Optical Distortion Calibration

We have developed a calibration target for use with fluid-immersed endoscopes within the context of the GIFT-Surg (Guided Instrumentation for Fetal Therapy and Surgery) project. One of the aims of this project is to engineer novel, real-time image processing methods for intra-operative use in the treatment of congenital birth defects, such as spina bifida and the twin-to-twin transfusion syndrome. The developed target allows for the sterility-preserving optical distortion calibration of endoscopes within a few minutes. Good optical distortion calibration and compensation are important for mitigating undesirable effects like radial distortions, which not only hamper accurate imaging using existing endoscopic technology during fetal surgery, but also make acquired images less suitable for potentially very useful image computing applications, like real-time mosaicing. In this paper proposes a novel fabrication method to create an affordable, sterilizable calibration target suitable for use in a clinical setup. This method involves etching a calibration pattern by laser cutting a sandblasted stainless steel sheet. This target was validated using the camera calibration module provided by OpenCV, a state-of-the-art software library popular in the computer vision community.

This article describes the design and development of a sterilizable custom camera optical distortion calibration target for the peri-operative, fluid-immersed calibration of endoscopes during endoscopic interventions.

We have developed a calibration target for use with fluid-immersed endoscopes within the context of the GIFT-Surg (Guided Instrumentation for Fetal ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...