In an institutional review board-approved study, 25 pediatric subjects diagnosed with chronic or recurrent otitis media were observed over a period of six months with optical coherence tomography (OCT). Subjects were followed throughout their treatment at the initial patient evaluation and preoperative consultation, surgery (intraoperative imaging), and postoperative follow-up, followed by an additional six months of records-based observation. At each time point, the tympanic membrane (at the light reflex region) and directly adjacent middle-ear cavity were observed in vivo with a handheld OCT probe and portable system. Imaging results were compared with clinical outcomes to correlate the clearance of symptoms in relation to changes in the image-based features of infection. OCT images of most all participants showed the presence of additional infection-related biofilm structures during their initial consultation visit and similarly for subjects imaged intraoperatively before myringotomy. Subjects with successful treatment (no recurrence of infectious symptoms) had no additional structures visible in OCT images during the postoperative visit. OCT image findings suggest surgical intervention consisting of myringotomy and tympanostomy tube placement provides a means to clear the middle ear of infection-related components, including middle-ear fluid and biofilms. Furthermore, OCT was demonstrated as a rapid diagnostic tool to prospectively monitor patients in both outpatient and surgical settings.
Influenced by both the intrinsic viscoelasticity of the tissue constituents and the time-evolved redistribution of fluid within the tissue, the biomechanical response of skin can reflect not only localized pathology but also systemic physiology of an individual. While clinical diagnosis of skin pathologies typically relies on visual inspection and manual palpation, a more objective and quantitative approach for tissue characterization is highly desirable. Optical coherence tomography (OCT) is an interferometry-based imaging modality that enables in vivo assessment of cross-sectional tissue morphology with micron-scale resolution, which surpasses those of most standard clinical imaging tools, such as ultrasound imaging and magnetic resonance imaging. This pilot study investigates the feasibility of characterizing the biomechanical response of in vivo human skin using OCT. OCT-based quantitative metrics were developed and demonstrated on the human subject data, where a significant difference between deformed and nondeformed skin was revealed. Additionally, the quantified postindentation recovery results revealed differences between aged (adult) and young (infant) skin. These suggest that OCT has the potential to quantitatively assess the mechanically perturbed skin as well as distinguish different physiological conditions of the skin, such as changes with age or disease.
We investigate and demonstrate the feasibility of using a combined Raman scattering (RS) spectroscopy and low-coherence interferometry (LCI) probe to differentiate microbial pathogens and improve our diagnostic ability of ear infections [otitis media (OM)]. While the RS probe provides noninvasive molecular information to identify and differentiate infectious microorganisms, the LCI probe helps to identify depth-resolved structural information as well as to guide and monitor positioning of the Raman spectroscopy beam for relatively longer signal acquisition times. A series of phantom studies, including the use of human middle ear effusion samples, were performed to mimic the conditions of in vivo investigations. These were also conducted to validate the feasibility of using this combined RS/LCI probe for point-of-care diagnosis of the infectious pathogen(s) in OM patients. This work establishes important parameters for future in vivo investigations of fast and accurate determination and diagnosis of infectious microorganisms in OM patients, potentially improving the efficacy and outcome of OM treatments, and importantly reducing the misuse of antibiotics in the presence of viral infections.
We performed ratiometric analysis of retinal optical coherence tomography images for the first time in multiple sclerosis (MS) patients. The ratiometric analysis identified differences in several retinal layer thickness ratios in the cohort of MS subjects without a history of optic neuritis (ON) compared to healthy control (HC) subjects, and there was no difference in standard retinal nerve fiber layer thickness (RNFLT). The difference in such ratios between HC subjects and those with mild MS-disability, without a difference in RNFLT, further suggests the possibility of using layer ratiometric analysis for detecting early retinal changes in MS. Ratiometric analysis may be useful and potentially more sensitive for detecting disease changes in MS.
Tympanic membrane (TM) thickness can provide crucial information for diagnosing several middle ear pathologies. An imaging system integrating low coherence interferometry (LCI) with the standard video otoscope has been shown as a promising tool for quantitative assessment of in-vivo TM thickness. The small field-of-view (FOV) of TM surface images acquired by the combined LCI-otoscope system, however, makes the spatial registration of the LCI imaging sites and their location on the TM difficult to achieve. It is therefore desirable to have a tool that can map the imaged points on to an anatomically accurate full-field surface image of the TM. To this end, we propose a novel automated mosaicking algorithm for generating a full-field surface image of the TM with co-registered LCI imaging sites from a sequence of multiple small FOV images and corresponding LCI data. Traditional image mosaicking techniques reported in the biomedical literature, mostly for retinal imaging, are not directly applicable to TM image mosaicking because unlike retinal images, which have several distinctive features, TM images contain large homogeneous areas lacking in sharp features. The proposed algorithm overcomes these challenges of TM image mosaicking by following a two-step approach. In the first step, a coarse registration based on the correlation of gross image features is performed. Subsequently, in the second step, the coarsely registered images are used to perform a finer intensity-based co-registration. The proposed algorithm is used to generate, for the first time, full-field thickness distribution maps of in-vivo human TMs.
Otitis media (OM) is a highly prevalent disease that can be caused by either a bacterial or viral infection. Because antibiotics are only effective against bacterial infections, blind use of antibiotics without definitive knowledge of the infectious agent, though commonly practiced, can lead to the problems of potential harmful side effects, wasteful misuse of medical resources, and the development of antimicrobial resistance. In this work, we investigate the feasibility of using a combined Raman scattering spectroscopy and low coherence interferometry (LCI) device to differentiate OM infections caused by viruses and bacteria and improve our diagnostic ability of OM. Raman spectroscopy, an established tool for molecular analysis of biological tissue, has been shown capable of identifying different bacterial species, although mostly based on fixed or dried sample cultures. LCI has been demonstrated recently as a promising tool for determining tympanic membrane (TM) thickness and the presence and thickness of middle-ear biofilm located behind the TM. We have developed a fiber-based ear insert that incorporates spatially-aligned Raman and LCI probes for point-of-care diagnosis of OM. As shown in human studies, the Raman probe provides molecular signatures of bacterial- and viral-infected OM and normal middle-ear cavities, and LCI helps to identify depth-resolved structural information as well as guide and monitor positioning of the Raman spectroscopy beam for relatively longer signal acquisition time. Differentiation of OM infections is determined by correlating in vivo Raman data collected from human subjects with the Raman features of different bacterial and viral species obtained from cultured samples.
Otolaryngologists utilize a variety of diagnostic techniques to assess middle ear health. Tympanometry, audiometry, and otoacoustic emissions examine the mobility of the tympanic membrane (eardrum) and ossicles using ear canal pressure and auditory tone delivery and detection. Laser Doppler vibrometry provides non-contact vibrational measurement, and acoustic reflectometry is used to assess middle ear effusion using sonar. These technologies and techniques have advanced the field beyond the use of the standard otoscope, a simple tissue magnifier, yet the need for direct visualization of middle ear disease for superior detection, assessment, and management remains.
In this study, we evaluated the use of portable optical coherence tomography (OCT) and pneumatic low-coherence interferometry (LCI) systems with handheld probe delivery to standard tympanometry, audiometry, otoacoustic emissions, laser Doppler vibrometry, and acoustic reflectometry. Comparison of these advanced optical imaging techniques and current diagnostics was conducted with a case study subject with a history of unilateral eardrum trauma. OCT and pneumatic LCI provide novel dynamic spatiotemporal structural data of the middle ear, such as the thickness of the eardrum and quantitative detection of underlying disease pathology, which could allow for more accurate diagnosis and more appropriate management than currently possible.
We report the development of a low-cost hand-held optical coherence imaging system. The proposed system is based on the principle of linear optical coherence tomography (Linear OCT), a technique which was proposed in the early 2000s as a simpler alternative to the conventional time-domain and Fourier-domain OCT. In our design, as in the traditional Michaelson interferometer, light from a broadband source is split into sample and reference beams. Unlike in a Michaelson interferometer though, upon return, a tilt is introduced to the reference beam before it is combined with the sample beam to illuminate a detector array. The resulting fringe pattern encodes information about the relative time-of-flight of photons between the sample and reference arms, which can be decoded by standard signal processing techniques to obtain depth resolved reflectivity profiles of the sample. The axial resolution and the SNR of our system was measured to be approximately 5.2 μm and 80 dB, respectively. The performance of the proposed system was compared with a standard state-of-the-art Fourier-domain low coherence interferometry (LCI) system by imaging several biological and non-biological samples. The results of this study indicate that the proposed low-cost system might be a suitable choice for applications where the imaging depth and SNR can be traded for lower cost and simpler optical design. Two potentially useful applications of the proposed imaging system could be for imaging the human tympanic membrane (TM) for diagnosing middle ear pathologies, and to visualize the sub-surface features of materials for non-destructive evaluation and quality inspection.
KEYWORDS: Field programmable gate arrays, Demodulation, Clocks, Signal processing, Image processing, Transducers, Photoacoustic microscopy, Heart, Real time imaging, Imaging systems
The ability to collect data in real time is important in all biological imaging modalities that aim to image dynamic
processes. Photoacoustic Microscopy (PAM) is a rapidly growing biomedical imaging technique that is often used to
image microvasculature and melanoma, and is capable of fully rendering three-dimensional images. However, due to the
bi-polar nature of the PAM signal, post processing through demodulation is required to accurately display morphological
data. Typically, demodulation requires post processing of the data, limiting its use in real-time applications. This results
in many PAM systems displaying data through maximum amplitude projection (MAP) images, completely ignoring the
axial dimension of their scans and throwing away useful data. We overcome this processing limit by utilizing a
configurable integrated circuit known as a Field Programmable Gate Array (FPGA). The FPGA allows us to perform
quadrature demodulation of the photoacoustic signal as it is being collected. The result is a PAM system capable of
producing continuous, morphologically accurate B-scans and volumes at a rate limited only by the repetition rate of the
laser. This allows us to generate accurately rendered volumes at the same speed as MAP images. With a 100 KHz
actively q-switched laser we are able to generate 200 by 200 pixel b-scans at a rate of 500 Hz. The imaging potential of
the system has been demonstrated in volumes of human hair phantoms and chick embryo vasculature. This system is
capable of 50 x 50 x 50 volume stacks processed and displayed at better than video rate.
Confocal microscopy has shown potential as an imaging technique to detect precancer. Imaging cellular features throughout the depth of epithelial tissue may provide useful information for diagnosis. However, the current in vivo axial scanning techniques for confocal microscopy are cumbersome, time-consuming, and restrictive when attempting to reconstruct volumetric images acquired in breathing patients. Chromatic dispersion confocal microscopy (CDCM) exploits severe longitudinal chromatic aberration in the system to axially disperse light from a broadband source and, ultimately, spectrally encode high resolution images along the depth of the object. Hyperchromat lenses are designed to have severe and linear longitudinal chromatic aberration, but have not yet been used in confocal microscopy. We use a hyperchromat lens in a stage scanning confocal microscope to demonstrate the capability to simultaneously capture information at multiple depths without mechanical scanning. A photonic crystal fiber pumped with a 830nm wavelength Ti:Sapphire laser was used as a supercontinuum source, and a spectrometer was used as the detector. The chromatic aberration and magnification in the system give a focal shift of 140μm after the objective lens and an axial resolution of 5.2-7.6μm over the wavelength range from 585nm to 830nm. A 400x400x140μm3 volume of pig cheek epithelium was imaged in a single X-Y scan. Nuclei can be seen at several depths within the epithelium. The capability of this technique to achieve simultaneous high resolution confocal imaging at multiple depths may reduce imaging time and motion artifacts and enable volumetric reconstruction of in vivo confocal images of the epithelium.
We have recently developed Transient Absorption Ultrasonic Microscopy (TAUM) as an ultrahigh-resolution
photoacoustic microscopy technique. The amplitude of the multiphoton pump-probe interaction is dependent on the
interpulse delay between the pump and probe pulses. Measuring the interpulse delay dependent TAUM amplitude maps
out the ground state recovery time of the chromophore. The ground state recovery time is a molecular signature that may
be used to differentiate multiple chromophores, analogous to fluorescence lifetime. We have used TAUM to measure the
ground state recovery time of Rhodamine 6G to be 3.65 ns, which matches well with known literature values. Whole
blood is also investigated, with measured ground state recovery times of 3.74 ns for oxygenated blood and 7.9 ns for
deoxygenated blood. The distinct difference in lifetimes for the oxidized and reduced forms suggests the feasibility of
subcellular SO2 images maps in future iterations of TAUM.
Optical coherence tomography (OCT) is becoming a popular tool for imaging morphology in the
middle and inner ear. Vibratory measurements of the structures of the ear facilitate better
understanding of the function and limitations of the ear. We have developed an algorithm that
enables a standard spectrometer based OCT system to measure the full spectrum (90 kHz) frequency
response of the mouse ear by incorporating coherently interleaved sampling, increasing the effective
Nyquist rate of the system by a factor of 5+. The algorithm is evaluated by measuring the frequency
response of a mouse tympanic membrane to a pure tone stimulus.
We present chromatic confocal microscopy as a technique to axially scan the sample by spectrally encoding depth
information to avoid mechanical scanning of the lens or sample. We have achieved an 800 μm focal shift over a range of
680-1080 nm using a hyperchromat lens as the imaging lens. A more complex system that incorporates a water
immersion objective to improve axial resolution was built and tested. We determined that increasing objective
magnification decreases chromatic shift while improving axial resolution. Furthermore, collimating after the
hyperchromat at longer wavelengths yields an increase in focal shift.
We have developed a novel, hybrid imaging modality, Transient Absorption Ultrasonic Microscopy
(TAUM), which fuses photoacoustic microscopy with non-linear microscopy. Photoacoustic
microscopy is well known for its ability to image chromophores deep (> 1 mm) in scattering media
with spatial resolutions in the 10s of microns. Non-linear microscopy is well known for its exquisite
spatial resolution in three dimensions. This superior spatial resolution is attributed to the fact that
the collected signal has a non-linear dependence on the light intensity. This dependence confines the
signal to a very small focal volume, producing optically resolved voxels. Transient absorption is a
non-linear process often used to map the excited state lifetimes of molecules exhibiting low
fluorescence quantum efficiency. This sensitivity to non-radiative transitions makes transient
absorption an attractive process to combine with photoacoustic imaging. We have built a prototype
transient absorption ultrasonic microscope, implementing off-axis photoacoustic detection to allow
the use of a high-quality, high numerical aperture objective. This high-quality, commercial lens is
required to provide the tight focusing needed to optimize non-linear effects. We have demonstrated
the increased spatial resolution of TAUM by imaging Rhodamine 6G in a capillary tube. The
capillary cross-section is fully resolved, suggesting an axial resolution of < 10 microns. A 6 MHz
transducer was used in this experiment, which results in an axial resolution of ~ 400 microns when
used in a traditional photoacoustic microscope. Boasting the superior penetration depth and
absorption contrast offered by photoacoustic emission and complemented by spatial resolutions
comparable to confocal microscopy, we believe that Transient Absorption Ultrasonic Microscopy
has excellent potential for producing volumetric images with cellular/subcellular resolution in three
dimensions deep inside living tissue.
Current medical imaging modalities, such as MRI and CT, do not provide high enough resolution to detect many
changes within the cochlea that cause hearing loss. We sought to develop the technique of optical coherence
tomography (OCT) to image the cochlea noninvasively and within its native environment. We used spectral domain
OCT with 950 nm as the center wavelength and a bandwidth of ~100 nm to image freshly excised normal mouse cochlea
at different developmental ages. The OCT system has an axial resolution of ~4 μm (in air) and a lateral resolution of
~10 μm. When we imaged normal adult mouse cochleae through the round window membrane, Reissner's membrane,
the basilar membrane, the tectorial membrane, the spiral ligament, the spiral limbus, and the modiolus could be clearly
identified. When we imaged intact adult cochleae, we were able to image through ~130 μm of bone and tissue to see up
to a depth of ~600 μm, and all of the previously identified structures were still visible. Imaging of early postnatal mice
during the timeline of cochlear development permitted visualization of the expected structural differences from adult
cochleae. Therefore, we conclude that spectral domain OCT is an effective technique for noninvasive imaging of the
murine cochlea.
Photoacoustic microscopy (PAM) is a high-contrast, high-resolution imaging modality used primarily for imaging
hemoglobin and melanin. Important applications include mapping of the microvasculature and melanoma tumor
margins. We have developed a novel photoacoustic microscope design, which substantially simplifies construction by
enabling the use of unmodified commercial optics and ultrasonic transducers. Moreover, the simple design may be
readily incorporated into a standard light microscope, thus providing a familiar imaging platform for clinical researchers.
A proof-of-concept Off-Axis PAM system with a lateral resolution of 26 μm and a modest axial resolution of 410 μm
has been assembled and characterized using tissue samples. We have derived the appropriate equations to describe the
relevant design parameters and verified the equations via measurements made on our prototype Off-Axis PAM system.
A consequence of the simple design is a reduction in axial resolution compared to coaxial designs. The reduction is
inversely proportional to the cosine of the angle between excitation and detection and equal to 15% and 41% for angles
of 30º and 45º, respectively. While resolution is negatively affected by off-axis detection, the ability to measure weak
signals at depth is enhanced. Off-axis detection has an inherent dark-field quality; chromophores excited outside the
numerical aperture of the ultrasonic detector will not be detected. The physical geometry of Off-Axis PAM enables the
placement of the ultrasonic transducer at the minimum distance from the sample with no obstructions between the
sample and transducer. This may prove to be an additional advantage of Off-Axis PAM over designs that incorporate
long working distance ultrasonic transducers and/or require the propagation of the acoustic wave through the laser
excitation optics to achieve co-axial detection.
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