Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.
We developed a simple and effective contrast for tissue characterization based on the recently proposed dual-pulse nonlinear photoacoustic technology. The new contrast takes advantage of the temperature dependence of Grüneisen parameter of tissue and involves a dual-pulse laser excitation process. A short pulse first heats the sample and causes a temperature jump, which then leads to the change of Grüneisen parameter and amplitude of the photoacoustic signal of the second pulse. For different tissues, the induced rate or trend of change is expected to be different, which constitutes the basis of the new contrast. Preliminary phantom experiment in blood and lipid mixtures and in vitro experiment in fatty rat liver have demonstrated that the proposed contrast has the capability of fast characterization of lipid-rich and blood-rich tissues.
For three-dimensional imaging of optical absorbance, the existing technology of photoacoustic microscopy (PAM) has quite poor axial resolution, the tens of microns to hundreds of microns. This is despite the fact that PAM has recently achieved lateral resolutions on the order of a micron or submicron, comparable to that of optical microscopy. In this paper, a pure optical photoacoustic microscopy (POPAM) with optical rastering of a focused excitation beam and optically sensing of the photoacoustic signal using a microring resonator was developed with the super broad bandwidth of the system more than 350MHz. With unprecedented broad bandwidth of POPAM, 3.8μm axial resolution was achieved without deconvolution processing. Sectioning imaging ability along axial direction presenting 3D morphologic features was shown based on imaging printed phantom. The impact of this approach will be similar to how confocal optical microscopy revolutionized the conventional optical microscopy by enabling the axial sectioning capability. Tissue imaging comparing POPAM and conventional PAM based on needle hydrophone demonstrated that though such broad bandwidth compromised the sensitivity of POPAM 4.35 times than that of conventional PAM, the noise equivalent detectable pressure (NEDP) was estimated as 74Pa, still able to get the tissue imaging.
We present the photoacoustic microscopy (PAM) for evaluation of angiogenesis inhibitors on a chick embryo model. Microvasculature in the chorioallantoic membrane (CAM) of the chick embryos was imaged by PAM, and the optical microscopy (OM) images of the same set of CAMs were also acquired for comparisons, serving for validation of the results from PAM. The angiogenesis inhibitors, Sunitinib, with different concentrations applied to the CAM result in the change in microvascular density, which was quantified by both PAM and OM imaging. Similar change in microvascular density from PAM and OM imaging in response to angiogenesis inhibitor at different doses was observed, demonstrating that PAM has potential to provide objective evaluation of anti-angiogenesis medication. Besides, PAM is advantageous in three-dimensional and functional imaging compared with OM so that the emerging PAM technique may offer unique information on the efficacy of angiogenesis inhibitors and could benefit applications related to antiangiogenesis treatments.
It is beneficial to study tumor angiogenesis and microenvironments by imaging the microvasculature and cells at the same time. Photoacoustic microscopy (PAM) is capable of sensitive three-dimensional mapping of microvasculature, while fluorescence microscopy may be applied to assessment of tissue pathology. In this work, a fiber-optic based PAM and confocal fluorescence microscopy (CFM) dual-modality imaging system was designed and built, serving as a prototype of a miniaturized dual-modality imaging probe for endoscopic applications. As for the design, we employed miniature components, including a microelectromechanical systems (MEMS) scanner, a miniature objective lens, and a small size optical microring resonator as an acoustic detector. The system resolutions were calibrated as 8.8 μm in the lateral directions for both PAM and CFM, and 19 μm and 53 μm in the axial direction for PAM and CFM, respectively. Images of the animal bladders ex vivo were demonstrated to show the ability of the system in imaging not only microvasculature but also cellular structure.
To view the individual cells and ambient microvasculature simultaneously will be helpful to study tumor angiogenesis
and microenvironments. To achieve this, two molecular contrast mechanisms were exploited simultaneously by
integrating two imaging modalities, confocal fluorescence microscopy (CFM) and photoacoustic microscopy (PAM).
These share the same scanning optical path and laser source. The induced photoacoustic (PA) signal was detected by a
highly sensitive needle hydrophone; while the back-traveling fluorescent photons emitted from the same sample were
collected by an avalanche photodetector. Experiments on ex vivo rat bladders were conducted. The CFM image depicted
the shape and size of the individual cells successfully. Besides large polygonal umbrella cells, some intracellular
components can also be discerned. With the CFM image presenting morphologic cellular information in the bladder wall,
the PAM image provides the complementary information, based on the endogenous optical absorption contrast, of the
microvascular distribution inside the bladder wall, from large vessels to capillaries. Such multimodal imaging provides
the opportunity to realize both histological assay and characterization of microvasculature using one imaging setup. This
approach offers the possibility of comprehensive diagnosis of cancer in vivo.
This photoacoustic volume imaging (PAVI) system is designed to study breast cancer detection and diagnosis in the
mammographic geometry in combination with automated 3D ultrasound (AUS). The good penetration of near-infrared
(NIR) light and high receiving sensitivity of a broad bandwidth, 572 element, 2D PVDF array at a low center-frequency
of 1MHz were utilized with 20 channel simultaneous acquisition. The feasibility of this system in imaging optically
absorbing objects in deep breast tissues was assessed first through experiments on ex vivo whole breasts. The blood
filled pseudo lesions were imaged at depths up to 49 mm in the specimens. In vivo imaging of human breasts has been
conducted. 3D PAVI image stacks of human breasts were coregistered and compared with 3D ultrasound image stacks of
the same breasts. Using the designed system, PAVI shows satisfactory imaging depth and sensitivity for coverage of the
entire breast when imaged from both sides with mild compression in the mammographic geometry. With its unique soft
tissue contrast and excellent sensitivity to the tissue hemodynamic properties of fractional blood volume and blood
oxygenation, PAVI, as a complement to 3D ultrasound and digital tomosynthesis mammography, might well contribute
to detection, diagnosis and prognosis for breast cancer.
We explored the potential of an emerging laser-based technology, photoacoustic imaging (PAI), for bladder cancer
diagnosis through high resolution imaging of microvasculature in the interior bladder tissues. Images of ex vivo canine
bladders demonstrated the excellent ability of PAI to map three-dimensional microvasculature in optically scattering
bladder tissues. By comparing the results from human bladder specimens affected by cancer to those from the normal
control, the feasibility of PAI in differentiating malignant from benign bladder tissues was explored. The reported
distinctive morphometric characteristics of tumor microvasculature can be seen in the images from cancer samples,
suggesting that PAI may allow in vivo assessment of neoangiogenesis that is closely associated with bladder cancer
generation and progression. By presenting subsurface morphological and physiological information in bladder tissues,
PAI, when performed in a similar way to that in conventional endoscopy, provides an opportunity for improved
diagnosis, staging and treatment guidance of bladder cancer.
It has been studied that a potential marker to obtain prognostic information about bladder cancer is tumor neoangiogenesis, which can be quantified by morphometric characteristics such as microvascular density. Photoacoustic microscopy (PAM) can render sensitive three-dimensional (3D) mapping of microvasculature, providing promise to evaluate the neoangiogenesis that is closely related to the diagnosis of bladder cancer. To ensure good image quality, it is desired to acquire bladder PAM images from its inside via the urethra, like conventional cystoscope. Previously, we demonstrated all-optical PAM systems using polymer microring resonators to detect photoacoustic signals and galvanometer mirrors for laser scanning. In this work, we build a miniature PAM system using a microelectromechanical systems (MEMS) scanning mirror, demonstrating a prototype of an endoscopic PAM head capable of high imaging quality of the bladder. The system has high resolutions of 17.5 μm in lateral direction and 19 μm in the axial direction at a distance of 5.4 mm. Images of printed grids and the 3D structure of microvasculature in animal bladders ex vivo by the system are demonstrated.
The concept of pure optical photoacoustic microscopy(POPAM) was proposed based on optical rastering of a focused
excitation beam and optically sensing the photoacoustic signal using a microring resonator fabricated by a
nanoimprinting technique. After some refinedment of in the resonator structure and mold fabrication, an ultrahigh Q
factor of 3.0×105 was achieved which provided high sensitivity with a noise equivalent detectable pressure(NEDP) value
of 29Pa. This NEDP is much lower than the hundreds of Pascals achieved with existing optical resonant structures such
as etalons, fiber gratings and dielectric multilayer interference filters available for acoustic measurement. The featured
high sensitivity allowed the microring resonator to detect the weak photoacoustic signals from micro- or submicroscale
objects. The inherent superbroad bandwidth of the optical microring resonator combined with an optically focused
scanning beam provided POPAM of high resolution in the axial as well as both lateral directions while the axial
resolution of conventional photoacoustic microscopy (PAM) suffers from the limited bandwidth of PZT detectors.
Furthermore, the broadband microring resonator showed similar sensitivity to that of our most sensitive PZT detector.
The current POPAM system provides a lateral resolution of 5μm and an axial resolution of 8μm, comparable to that
achieved by optical microscopy while presenting the unique contrast of optical absorption and functional information
complementing other optical modalities. The 3D structure of microvasculature, including capillary networks, and even
individual red blood cells have been discerned successfully in the proof-of-concept experiments on mouse bladders ex
vivo and mouse ears in vivo. The potential of approximately GHz bandwidth of the microring resonator also might allow
much higher resolution than shown here in microscopy of optical absorption and acoustic propagation properties at
depths in unfrozen tissue specimens or thicker tissue sections not now imageable with current optical or acoustic
microscopes of comparable resolution.
KEYWORDS: Photoacoustic spectroscopy, Capillaries, Picture Archiving and Communication System, Blood, In vivo imaging, Blood circulation, Spectroscopy, Particles, Photoacoustic imaging, Imaging systems
Photoacoustic imaging has been widely used in structural and functional imaging. Because of its safety, high resolution,
and high imaging depth, it has great potential for a variety of medical studies. Capillaries are the smallest blood vessels
and enable the exchange of oxygen and nutrients. Noninvasive flow speed measurement of capillaries in vivo can benefit
the study of vascular tone changes and rheological properties of blood cells in capillaries. Recently, there has been a
growing interest in photoacoustic velocimetry, such as photoacoustic Doppler and M-mode photoacoustic flow imaging.
Methods capable of high-resolution imaging and low-speed flow measurement are suitable to measure blood speeds in
capillaries. Previously we proposed photoacoustic correlation spectroscopy (PACS) and shown its feasibility for lowspeed
flow measurement. Here, in vivo measurement of blood speeds in capillaries in a chick embryo model by PACS
technique is demonstrated. The laser-scanning photoacoustic microscopy system is used for fast imaging acquisition and
high-resolution imaging. The measured speed in capillaries is similar to those found in literatures, which confirm the
feasibility of the PACS method for blood velocimetry. This technique suggests a fairly simple way to study blood flow
speeds in capillaries.
A photoacoustic (PA) imaging system was developed to achieve high sensitivity for the detection and characterization of
vascular anomalies in the breast in the mammographic geometry. Signal detection from deep in the breast was achieved
by a broadband 2D PVDF planar array that has a round shape with one side trimmed straight to improve fit near the
chest wall. This array has 572 active elements and a -6dB bandwidth of 0.6-1.7 MHz. The low frequency enhances
imaging depth and increases the size of vascular collections displayed without edge enhancement. The PA signals from
all the elements go through low noise preamplifiers in the probe that are very close to the array elements for optimized
noise control. Driven by 20 independent on-probe signal processing channels, imaging with both high sensitivity and
good speed was achieved. To evaluate the imaging depth and the spatial resolution of this system,2.38mm I.D. artificial
vessels embedded deeply in ex vivo breasts harvested from fresh cadavers and a 3mm I.D. tube in breast mimicking
phantoms made of pork loin and fat tissues were imaged. Using near-infrared laser light with incident energy density
within the ANSI safety limit, imaging depths of up to 49 mm in human breasts and 52 mm in phantoms were achieved.
With a high power tunable laser working on multiple wavelengths, this system might contribute to 3D noninvasive
imaging of morphological and physiological tissue features throughout the breast.
KEYWORDS: Absorption, Molecules, In vitro testing, Molecular aggregates, Mass attenuation coefficient, In vivo imaging, Luminescence, Tissue optics, Magnesium, Tumors
We have designed a protease-sensitive imaging probe for optoacoustic imaging whose absorption spectrum changes upon cleavage by a protease of interest. The probe comprises an active site, a derivative of chlorophyll or natural photosynthetic bacteriochlorophyll that absorbs in the near infrared, conjugated to a peptide backbone specific to the protease being imaged. The uncleaved molecules tend to aggregate in dimers and trimers, causing a change in the absorption spectrum relative to that of the monomer. Upon cleavage, the probe molecules deaggregate, giving rise to a spectrum characteristic of monomers. We show using photospectrometry that the two forms of the probe have markedly different absorption spectra, which could allow for in vivo optoacoustic identification using a multiwavelength imaging strategy. Optoacoustic measurements using a narrow-band dye laser find spectral peaks in the two forms of the probe at the expected location. The optoacoustic signal from the uncleaved probe is found to be considerably weaker than that of the cleaved probe, perhaps due to poor optical-acoustic coupling in the aggregated molecules. However, ultimately, it is detection of the cleaved probe that is of the greatest import, since it reports on the protease activity of interest.
KEYWORDS: Absorption, Molecules, Mass attenuation coefficient, Molecular aggregates, Signal attenuation, In vivo imaging, Tumors, In vitro testing, Dye lasers, Near infrared
We have designed a protease-sensitive imaging probe for optoacoustic imaging whose absorption spectrum changes
upon cleavage by a protease of interest. The probe comprises an active site, a derivative of chlorophyll or natural
photosynthetic bacteriochlorophyll that absorbs in the near infrared, conjugated to a peptide backbone specific to the
protease being imaged. The uncleaved molecules tend to aggregate in dimers and trimers causing a change in the
absorption spectrum relative to that of the monomer. Upon cleavage, the probe molecules de-aggregate giving rise to a
spectrum characteristic of monomers. We show using photospectrometry that the two forms of the probe have markedly
different absorption spectra, which could allow for in vivo optoacoustic identification using a multiwavelength imaging
strategy. Optoacoustic measurements using a narrow-band dye laser find spectral peaks in the two forms of the probe at
the expected location. The optoacoustic signal from the uncleaved probe is found to be considerably weaker than that of
the cleaved probe, perhaps due to poor optical-acoustic coupling in the aggregated molecules. However, ultimately, it is
detection of the cleaved probe that is of the greatest import since it reports on the protease activity of interest. PUBLISHERS NOTE 9/1/2010: The figure numbers are corrected. If you downloaded the incorrect version of Paper 75641T and no longer have access to download the correct paper, please contact CustomerService@SPIEDigitalLibrary.org for assistance.
We have developed a laser-scanning optical-resolution photoacoustic microscopy that can potentially be easily
integrated with several existing optical microscopic modalities. During data acquisition, the ultrasonic transducer is kept
stationary and only the laser light is raster-scanned by an x-y galvanometer scanner. In this configuration, the field-ofview
is limited by the beam diameter of the ultrasonic detector, which is related to the active element size, numerical
aperture, and the center frequency of the ultrasonic transducer. The spatial resolution is determined by the size of the
optical focus. A lateral resolution of 7.8 μm and a circular field-of-view with a diameter of 6 mm were achieved in an
optically clear medium. Using a laser system working at a pulse repetition rate of 1024 Hz, the data acquisition time for
an image consisting of 256×256 pixels was less than two minutes. In vivo imaging of microvasculature in mouse ears
were also achieved.
KEYWORDS: Tissue optics, Confocal microscopy, Beam shaping, Geometrical optics, Optical simulations, Monte Carlo methods, Convolution, Acoustics, Image quality, Signal to noise ratio
A modified MC convolution method for integration extension of MC simulation is developed for finite photon beam
with random shape of translational or rotational invariance, which is proven consistent with the conventional
convolution extension of MC simulation for normal incident finite beam. The method is applied to analyze the positions
of fluence foci and ratios of fluence at the focus and surface which are two key factors in the application of dark-field
confocal and some interesting points are presented including: 1) The fluence profile has a saddle-like shape with highest
peak in the bright field and low valley near the surface and a second rise in the center of dark field which is defined as
the effective optical focus; 2) Besides a little peak near zero inner radius, the ratio of fluences at the focus and surface
increases linearly with the inner radius, suggesting the large inner radius more advantageous to image at the effective
optical focus; 3) The position of effective optical foci deepens linearly with the increase of the inner radius, suggesting
that to get a high quality image of deeper target, a dark-field with larger size is more beneficial. But the position of
fluence foci are far away from the foci of geometrical laser beam in high scattering tissue, so aligning the foci of
geometrical laser beam and acoustic transducer doesn't guarantee that effective optical focus is accurately overlapping
with the acoustic focus. An MC simulation with integration extension presented in this paper maybe helpful to determine
where the acoustic focus should be to maximize the SNR in tissue imaging; 4) incident angle makes little difference to
ratio of fluences at the focus and surface and an incident angle between 30 and 50 degrees gives the highest fluence at
the effective optical focus; 5) the depth of fluence focus is insensitive to the incident angle.
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