KEYWORDS: Brain, Neuroimaging, Photoacoustic tomography, Calcium, Acquisition tracking and pointing, Neurons, Luminescence, Tissue optics, In vivo imaging, Signal detection
Significance: Optical imaging of responses in fluorescently labeled neurons has progressed significantly in recent years. However, there is still a need to monitor neural activities at divergent spatial scales and at depths beyond the optical diffusion limit.Aim: To meet these needs, we aim to develop multiscale photoacoustic tomography (PAT) to image neural activities across spatial scales with a genetically encoded calcium indicator GCaMP.Approach: First, using photoacoustic microscopy, we show that depth-resolved GCaMP signals can be monitored in vivo from a fly brain in response to odor stimulation without depth scanning and even with the cuticle intact. In vivo monitoring of GCaMP signals was also demonstrated in mouse brains. Next, using photoacoustic computed tomography, we imaged neural responses of a mouse brain slice at depths beyond the optical diffusion limit.Results: We provide the first unambiguous demonstration that multiscale PAT can be used to record neural activities in transgenic flies and mice with select neurons expressing GCaMP.Conclusions: Our results indicate that the combination of multiscale PAT and fluorescent neural activity indicators provides a methodology for imaging targeted neurons at various scales.
Colorectal cancer is the second leading cause of cancer death in the United States. According to American Cancer society, the overall lifetime risk of developing colorectal cancer is about 4.7% for men and 4.4% for women. We have developed a rigid, endoscopic photoacoustic microscopy (PAM) probe for imaging of in vivo human colorectal cancers. In order to accommodate colon sections with different size (typically from 50 to 70mm), our 10mm diameter rigid probe uses an off-optical-axis, external mechanical scanning mechanism with a speed of 35deg/s instead of an on-optical-axis, internal mechanical scan mechanism. 532-nm pulsed laser light enters the ridged probe through a photonic crystal single mode fiber before it is collimated and refocused by a water-immersed objective lens onto the colon surface. A focused ultrasound ring transducer (40.5 MHz, 6.5mm focal length) receives photoacoustic signal from chromophores excited by laser beam. Imaging system performance specifications including resolution (6μm) and signal-to-noise ratio are quantified and verified from phantom imaging tests. Ex vivo human colon samples are studied to reveal microscopic features of normal colon, benign polyps, adenocarcinoma and cancer.
It is important to provide timely information to surgeons on diagnosis of a suspicious ovarian tissue before excision to avoid unnecessary surgery, especially for young women. In this report, we introduce a new 3-D surface mapping technique to map ovarian tissue scattering properties by fitting the swept-source optical coherence tomography (SS-OCT) signals to a scattering model. We observed that lower scattering coefficients and heterogeneous spatial distribution were associated with malignant ovarian tissues, and higher scattering coefficients and homogeneous spatial distribution indicated benign ovarian tissues. The initial results suggest that the 3-D scattering map has potential to be an effective tool to characterize normal and malignant ovarian tissues.
Neural scientists can benefit greatly from imaging tools that can penetrate thick brain tissue. Compared with traditional optical microscopy methods, photoacoustic imaging can beat the optical diffusion limit and achieve such deep tissue imaging with high spatial resolution. In this study, we used an optical-resolution photoacoustic microscope to image the odor-evoked neuronal activities in a drosophila model. Drosophila brain neurons stably express GCaMP5G, a calcium-sensitive fluorescent protein whose optical absorption coefficient changes with calcium influx during action potentials. We recorded an ~20% odor-evoked fractional photoacoustic signal increase at all depths of the drosophila brain in vivo, with and without removal of the brain cuticle, at a recording rate of 1 kHz. Our results were confirmed by concurrent fluorescent recordings. Furthermore, by performing fast 2D scanning, we imaged the antenna lobe region, which is of particular interest in neuroscience, at a volumetric rate of ~1 Hz with a sub-neuron resolution of 3 m. Unlike optical imaging, which requires surgical removal of the scattering brain cuticle, our photoacoustic system can image through the cuticle and measure neuronal signals of the whole drosophila brain without invasive surgery, enabling minimal disturbance to the animal’s behaviors. In conclusion, we have demonstrated photoacoustic imaging of calcium signals in drosophila brains for the first time. Utilizing the deep imaging capability of photoacoustic tomography, our methods could potentially be extended to in vivo imaging of neuronal activities from deep brains in other animal models.
Optical imaging of brain voltage signals is significantly limited in depth due to optical scattering and the absorptive property of brain tissue. Photoacoustic (PA) imaging promises to break this hard limit by utilizing both ballistic and diffused photons. To demonstrate the feasibility of PA, we used an in vivo mouse model. The brain cortex tissue was stained with dipicrylamine dye, electrically stimulated, and imaged with a customized dual-isosbestic-wavelength PA microscope (DIW-PAM). DIW-PAM separates voltage-induced PA signals from blood-induced PA signals and thereby allows recording the voltage response of mouse cortex tissue without interference from hemoglobin responses. The resting state PA voltage response signal exhibited a noise-like signal in the frequency domain. Upon 3 Hz electrical stimulation, the PA voltage response signal showed frequency peaks of 3.2 Hz and 6.3 Hz (Fig. 1). Although dipicrylamine dye is not fast enough for recording neuron action potentials, it served well for the purpose of this feasibility study. In conclusion, we successfully demonstrated in vivo photoacoustic imaging of mouse brain voltage signals for the first time. If a fast voltage-sensitive dye is available, using photoacoustic computed tomography (PACT) instead of PA microscopy could allow acquiring full-field PA action potential images at a speed limited only by the laser pulse repetition rate.
Intravital microscopy techniques have become increasingly important in biomedical research because they can provide unique microscopic views of various biological or disease developmental processes in situ. Here we present an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system that visualizes internal organs with a much finer resolution than conventional acoustic-resolution photoacoustic endoscopy systems. By combining gradient index (GRIN) lens-based optical focusing and ultrasonic ring transducer-based acoustic focusing, we achieved a transverse resolution as fine as ~10 μm at an optical working distance of 6.5 mm. The OR-PAEM system’s high-resolution intravital imaging capability is demonstrated through animal experiments.
The invention of green fluorescent protein and other molecular fluorescent probes has promoted applications of confocal and two-photon fluorescence microscopy in biology and medicine. However, exogenous fluorescence contrast agents may affect cellular structure and function, and fluorescence microscopy cannot image nonfluorescent chromophores. We overcome this limitation by integrating optical-resolution photoacoustic microscopy into a modern Olympus IX81 confocal, two-photon, fluorescence microscope setup to provide complementary, label-free, optical absorption contrast. Automatically coregistered images can be generated from the same sample. Imaging applications in ophthalmology, developmental biology, and plant science are demonstrated. For the first time, in a familiar microscopic fluorescence imaging setting, this trimodality microscope provides a platform for future biological and medical discoveries.
Photoacoustic microscopy (PAM) offers label-free, optical absorption contrast. A high-speed,
high-resolution PAM system in an inverted microscope configuration with a laser pulse repetition
rate of 100,000 Hz and a stationary ultrasonic transducer was built. Four-dimensional in vivo
imaging of microcirculation in mouse skin was achieved at 18 three-dimensional volumes per
second with repeated two-dimensional raster scans of 100 by 50 points. The corresponding twodimensional
B-scan (50 A-lines) frame rate was 1800 Hz, and the one-dimensional A-scan rate
was 90,000 Hz. The lateral resolution is 0.23±0.03 μm for Au nano-wire imaging, which is 2.0
times below the diffraction limit.
Photoacoustic microscopy (PAM) utilizes short laser pulses to deposit energy into light
absorbers and sensitively detects the ultrasonic waves the absorbers generate in response.
PAM directly renders a three-dimensional spatial distribution of sub-surface optical absorbers.
Unlike other optical imaging technologies, PAM features label-free optical absorption contrast
and excellent imaging depths. Standard dental imaging instruments are limited to X-ray and
CCD cameras. Subsurface optical dental imaging is difficult due to the highly-scattering enamel
and dentin tissue. Thus, very few imaging methods can detect dental decay or diagnose dental
pulp, which is the innermost part of the tooth, containing the nerves, blood vessels, and other
cells. Here, we conducted a feasibility study on imaging dental decay and dental pulp with PAM.
Our results showed that PAM is sensitive to the color change associated with dental decay.
Although the relative PA signal distribution may be affected by surface contours and subsurface
reflections from deeper dental tissue, monitoring changes in the PA signals (at the same site)
over time is necessary to identify the progress of dental decay. Our results also showed that
deep-imaging, near-infrared (NIR) PAM can sensitively image blood in the dental pulp of an in
vitro tooth. In conclusion, PAM is a promising tool for imaging both dental decay and dental
pulp.
Optical-resolution photoacoustic microscopy (OR-PAM) provides superb optical absorption contrast for red blood cells
(RBCs), which makes it ideal for in vivo microvasculature imaging. In comparison, optical coherence tomography
(OCT), widely used for tissue microstructure imaging, provides high optical scattering contrast. The two contrast
mechanisms are highly complementary. In this work, we combined OR-PAM and OCT into a single, dual-modality
imaging instrument for in vivo mouse eye imaging. We demonstrated in vivo dual-modality imaging of the anterior
segment of mouse eyes with laser pulse energy within the ANSI laser safety standard.
Both iris fluorescein angiography (IFA) and indocyanine green angiography (ICGA) provide ophthalmologists imaging
tools in studying the microvasculature structure and hemodynamics of the anterior segment of the eye in normal and
diseased status. However, a non-invasive, endogenous imaging modality is preferable for the monitoring of
hemodynamics of the iris microvasculature. We investigated the in vivo, label-free ocular anterior segment imaging
with photo-acoustic microscopy (PAM) in mouse eyes. We demonstrated the unique advantage of endogenous contrast
that is not available in both IFA and ICGA. The laser radiation was maintained within the ANSI laser safety limit. The
in vivo, label-free nature of our imaging technology has the potential for ophthalmic applications.
We previously demonstrated that multimodal microscopy combining photoacoustic microscopy and
optical coherence tomography can provide comprehensive insight into biological tissue at μm-level
resolution by exploiting both optical absorption and scattering contrasts. Recently, we have developed a
second-generation integrated photoacoustic and optical-coherence microscope, which can potentially be
adapted for clinical applications. In this new system, we can perform photoacoustic and optical-coherence
imaging simultaneously at a speed of 5,000 A-lines per second with real-time on-screen display. Also, both
modalities now work in reflection mode instead of transmission mode, allowing easy access to various
anatomical locations of interest. Imaging of skin and eye has been demonstrated in living small animals.
KEYWORDS: Optical coherence tomography, Speckle, 3D image processing, Image processing, Signal attenuation, Signal processing, Signal to noise ratio, Interference (communication), Wavelets, Biomedical optics
Optical coherence tomography is an emerging non-invasive technology that provides high resolution, cross-sectional
tomographic images of internal structures of specimens. It holds great potentials for a wide variety of applications,
especially in the field of biomedical imaging. OCT images, however, are usually degraded by significant speckle noise.
Here we report a 3D approach to attenuating speckle noise in OCT images. This approach is based on the 3D curvelet
transform, and is conveniently controlled by a single parameter that determines the threshold in the curvelet domain.
Unlike 2D approaches which only consider information in individual images, 3D processing, by analyzing all images in
a volume simultaneously, has the advantage of also taking the information between images into account. This, coupled
with the curvelet transform's nearly optimal sparse representation of curved edges that are common in OCT images,
provides a simple yet powerful platform for speckle attenuation. We show the approach suppresses a significant amount
of speckle noise, and in the mean time preserves and thus reveals many subtle features that could get attenuated in other approaches.
We present a kind of rotational two photon mciroendoscopy for 1μm fiber
femtosecond laser. The fiber laser provide ultrashort femto-second pulses with center
wavelength at 1.034μm and repetition rate of 50MH. The rotational probe is based on
double cladding photonic crystal fiber (CD PCF) fiber, Grin lens, microprism and
rotational MEMS motor. The MEMS motor has diameter of 2.2mm and can provide
360 degree full view rotation. We experimentally show that the DC PCF fiber works
for 1μm fiber laser two photon system. Second harmonic generation (SHG) singnal
line profile of rat tail tendon and fish scale was taken with the endoscopy system.
Transplant technologies have been studied for the recovery of vision loss from retinitis pigmentosa (RP) and age-related macular degeneration (AMD). In several rodent retinal degeneration models and in patients, retinal progenitor cells transplanted as layers to the subretinal space have been shown to restore or preserve vision. The methods for evaluation of transplants are expensive considering the large amount of animals. Alternatively, time-domain Stratus OCT was previously shown to be able to image the morphological structure of transplants to some extent, but could not clearly identify laminated transplants. The efficacy of screening retinal transplants with Fourier-domain OCT was studied on 37 S334ter line 3 rats with retinal degeneration 6-67 days after transplant surgery. The transplants were morphologically categorized as no transplant, detachment, rosettes, small laminated area and larger laminated area with both Fourier-domain OCT and histology. The efficacy of Fourier-domain OCT in screening retinal transplants was evaluated by comparing the categorization results with OCT and histology. Additionally, 4 rats were randomly selected for multiple OCT examinations (1, 5, 9, 14 and 21days post surgery) in order to determine the earliest image time of OCT examination since the transplanted tissue may need some time to show its tendency of growing. Finally, we demonstrated the efficacy of Fourier-domain OCT in screening retinal transplants in early stages and determined the earliest imaging time for OCT. Fourier-domain OCT makes itself valuable in saving resource spent on animals with unsuccessful transplants.
Doppler Optical Coherence Tomography (DOCT) imaging of in-vivo retinal blood flow was widely studied as efforts of research community to push this technology into clinic. Spectral Doppler imaging of DOCT has been demonstrated as a quantification method of in-vivo pulsatile retinal blood flow in human eye. This technology has the all the advantages inherited from OCT comparing to Doppler ultrasound. Comparing to normal spatial-distributed color Doppler imaging of DOCT, spectral Doppler imaging can reveal more haemodynamics details on the time dimension. Although resistance index (RI) of a micro-vascular can be measured in vivo from human retina, the clinical significance of RI measurements still needs to be investigated. In vitro experiment conduced with ultrasound has demonstrated the higher vascular resistance value is associated with the higher RI measured assuming the constant compliance of vascular tube. In this study, the rodent window-chamber model (RWCM) was used as a platform to investigate the RI change as the micro-vasculature response to laser irradiation. The higher RI was measured after the occlusion of two veins (should it be arterials) that was verified with laser speckle imaging in our preliminary experiment results.
A functional Fourier domain optical coherence tomography instrument offering spectral Doppler imaging of in vivo pulsatile human retinal blood flow was constructed. An improved phase-resolved algorithm was developed to correct bulk motion artifacts. Spectral Doppler imaging provides complementary temporal flow information to the spatially distributed flow information of the color Doppler image by providing direct visualization of the Doppler spectrum of the flow whose pattern can be further quantified with various velocity envelope curves and their corresponding flow indices. The coefficient of repeatability on resistance index measurement was assessed by analyzing 14 measurements on two vessels within two normal subjects.
In this presentation, we demonstrate a novel optical tomography technique, thermoelastic
optical Doppler tomography (ODT). Short laser pulses are used to generate thermoelastic
waves in biological samples. Optical phase variations in response to wave propagation
are detected using ODT. It is shown that areas of different elastic property in the phantom
can be clearly resolved.
A technique to improve the signal-to-noise ratio (SNR) of a high speed 1300 nm swept source optical coherence tomography (SSOCT) system was demonstrated. A semiconductor optical amplifier (SOA) was employed in the sample arm to coherently amplify the weak light back-scattered from sample tissue without increasing laser power illuminated on the sample. The image quality improvement was visualized and quantified by imaging the anterior segment of a rabbit eye at imaging speed of 20,000 A-lines per second. The theory analysis of SNR gain is given followed by the discussion on the technologies that can further improve the SNR gain.
We used optical coherence tomography (OCT) for non-invasive imaging of the anterior segment of the eye for
investigating partial-thickness scleral channels created with a femtosecond laser. Glaucoma is associated with elevated
intraocular pressure (IOP) due to reduced outflow facility in the eye. A partial-thickness aqueous humor (AH) drainage
channel in the sclera was created with 1.7-&mgr;m wavelength femtosecond laser pulses to reduce IOP by increasing the
outflow facility, as a solution to retard the progression of glaucoma. It is hypothesized that the precise dimensions and
predetermined location of the channel would provide a controlled increase of the outflow rate resulting in IOP reduction.
Therefore, it is significant to create the channel at the exact location with predefined dimensions. The aim of this
research has two aspects. First, as the drainage channel is subsurface, it is a challenging task to determine its precise
location, shape and dimensions, and it becomes very important to investigate the channel attributes after the laser
treatment without disturbing the internal anterior structures. Second, to provide a non-invasive, image-based verification
that extremely accurate and non-scarring AH drainage channel can be created with femtosecond laser. Partial-thickness
scleral channels created in five human cadaver eyes were investigated non-invasively with a 1310-nm time-domain OCT
imaging system. Three-dimensional (3D) OCT image stacks of the triangular cornea-sclera junction, also known as
anterior chamber angle, were acquired for image-based analysis and visualization. The volumetric cutting-plane
approach allowed reconstruction of images at any cross-sectional position in the entire 3D volume of tissue, making it a
valuable tool for exploring and evaluating the location, shape and dimension of the channel from all directions. As a
two-dimensional image-based methodology, an image-processing pipeline was implemented to enhance the channel
features to augment the analysis. In conclusion, we successfully demonstrate that our image-based visualization tool is
appropriate for effective investigation and evaluation of femtosecond laser-created, partial-thickness aqueous humor
drainage channels in the sclera.
Photodisruption of femtosecond laser at 1700nm wavelength has been demonstrated
as a potential subsurface surgical method that can be used in trabeculectomy for
glaucoma treatment without causing failure due to scarring at the level of conjunctiva
and underlying tissue [1, 2]. In this study, Fourier domain optical coherence
tomography (FD-OCT) technology is used to demonstrate high speed non-invasive
imaging of high precision photodisruption in human sclera. Photodisruption cavities
of different size in human sclera can reveal itself in its 3D FDOCT image. Transclera
channel cut from back to surface and partial transclera channel are easily identified in
3D OCT image. The whole 3D data set acquired with high speed frequency domain
OCT system permits further quantitative analysis of subsurface phtodisruption
incisions. The preliminary results indicate that high speed frequency domain OCT
system is a good candidate for imaging subsurface photodisruption with femtosecond
laser and its 3D image may provide good guidance during surgical procedures when it
is integrated with laser ablation system.
Optical coherence tomography relies on the coherent gating and coherent amplification
from its reference light to produce high axial resolution, high sensitivity image. The
signal to noise ratio of the optical coherence tomography image is proportional to the
detected back-scattering photon numbers from sample beam. Thus, the gain of an optical
amplifier device can be added to the weak sample beam reflected signal while preserving
its coherence by the coherent amplification process when the weak back reflected signal
is amplified by the optical amplifier device. However, the optical amplifier device will
emit spontaneous emission in its coherent amplification process. In this study, we report
some preliminary results on the investigation of the coherent amplification and the side
effects caused by the spontaneous emission in a swept source OCT system.
A swept laser source at center wavelength of 1060 nm for Fourier domain optical coherence tomography (FDOCT) was
demonstrated. The laser is composed of a fiber-coupled SOA gain module, a fiber Fabry-Perot tunable filter, fiber
isolators and couplers to form a ring laser. The laser is capable of a scanning range of 64 nm and coherence length of
9.8 mm at 2 KHz sweep rate. With the built swept source, a FDOCT system was developed which can achieve 12 &mgr;m
axial resolution in tissue. Imaging of pig retina was demonstrated with the FDOCT system.
A novel swept laser based Fourier domain functional optical coherence tomography (FD-F-OCT) system using an electro-optic phase modulator was designed and developed. The imaging range was doubled by the cancellation of the mirror image. The elimination of low frequency noises due to DC and autocorrelation terms increased the sensitivity by 20 dB. 3-D volume imaging as well as Doppler and polarization imaging with high speed and high sensitivity is described and demonstrated.
In this paper, the conventional and polarization sensitive optical coherence tomography (PS-OCT) were used to image bovine articular cartilage and diagnose degenerative joint disease. The results showed that these 2D cross-sectional OCT images that can cover an area of 6×2.8mm2 (limited by scope size) with a traverse resolution of 12 μm and an axial resolution of 10 μm could assess the microstructure of articular cartilage and differentiate the abnormalities in structure. The results were confirmed by their histology. Compared to conventional OCT, PS-OCT could provide depth-resolved strokes parameter images, which reflect tissue birefringence. Both conventional and PS-OCT have high efficiency and sensitivity of osteoarthritis and cartilage injury and disease diagnosis.
Dynamic morphological changes of clear corneal cataract incisions are studied with Optical Coherence Tomography (OCT). Two opposite types of dynamic incision wound behaviors are documented. A stable incision angle range is found to be existent for single-planed, clear corneal cataract incisions. When well pressurized, incision angles within this stable range result in well-apposed incision edges that resist gapping while incision angles falling outside this range have a larger tendency for wound leakage. It is also shown that a two-planed incision can effectively expand the stable range. For incision angles outside the stable range, the farther the incision angle is away from stable range, the larger the gap between incision wound edges when well pressurized. Thus, incision construction method has a major impact on the self-sealing capability of the incision wounds. In this investigation, OCT has been demonstrated as an effective modality for imaging and monitoring corneal surgery.
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