The spectroscopic information and the corresponding polarization states of a single-molecule emission possess wealth molecule-specific signatures that can be used to reveal the unique molecular electronic state, conformation, and its interactions with the host media. However, existing spectroscopic methods and advanced image analysis techniques, which can potentially provide quantitative analytical tools for the study of cellular dynamics, are yet limited by the diffraction limit. Therefore, developing a nanoscopic imaging platform for simultaneous acquisition of multiple molecular specific properties is highly desirable. Here we report a three-dimensional (3D), polarization-sensitive, spectroscopic photon localization microscopy (3D-Polar-SPLM) that simultaneously captures nanoscopic location of individual fluorescent emitters and their corresponding optical spectra and polarization states. To evaluate the capability of the imaging system, we imaged model system consisting quantum rods (QRs). Using 3D-Polar-SPLM, we spatially localized individual QRs with a lateral localization precision of 8 nm and an axial localization precision of 35 nm. In addition, we achieved a spectral resolution of 2 nm and a polarization angle measuring precision of 8 degrees. The spectral profile of the fluorescence emission provided a particle-specific signature for identifying individual QRs among the heterogeneous population, which significantly improved the fidelity in parallel 3D tracking of multiple QRs at a temporal resolution of 10 ms. Except its versatility, 3D-Polar-SPLM further provides advantageous in practical applications since it only employs a single light-path and therefore, is compatible with existing PALM/STORM, potentially bringing immediate impact to the broader research community, across physics, chemistry, material science and biology.
Monitoring cortical hemodynamic response after ischemic stroke (IS) is essential for understanding the pathophysiological mechanisms behind IS-induced neuron loss. Functional optical coherence tomography (OCT) is an emerging technology that can fulfill the requirement, providing label-free, high-resolution 3D images of cerebral hemodynamics.
Unfortunately, strong tissue scattering pose a significant challenge for existing OCT oximetry techniques, as they either ignore the effect or compensate it numerically. Here we developed a novel dual-depth sampling and normalization strategy using visible-light OCT (vis-OCT) angiograms that can provide robust and precise sO2 estimations within cerebral circulation. The related theoretical formulation were established, and its implication and limitations were discussed.
We monitored mouse cortical hemodynamics using the newly-developed method. Focal ischemic stroke was induced through photothrombosis. The analysis on pre- and post-IS vis-OCT images revealed both vascular morphology and oxygenation altered substantially after the occlusion. First, the ischemic core could be clearly identified as angiographic intensity fell below the detection limit. In addition, vessel dilation presented universally in the penumbra region. Notably for pial arteriles, the percentage of increase demonstrated inverse relationship with their pre-occlusion, pre-dilation dimeter.
Vis-OCT oxygenation maps on intact cortex revealed spatial sO2 variations within pial vessels. Specifically, sO2 in arterioles decreased as it bifurcated and plunged into deeper tissue. Similarly, venous sO2 was higher in the larger, more superficial pial brunches. However, such difference was no longer appreciable after photothrombosis. Averaged arteriole sO2 dropped to 64% – 67% in the penumbra region.
We explored, both numerically and experimentally, whether OCT can be a good candidate to accurately measure retinal oxygen metabolism. We first used statistical methods to numerically simulate photon transport in the retina to mimic OCT working under different spectral ranges. Then we analyze accuracy of OCT oximetry subject to parameter variations such as vessel size, pigmentation, and oxygenation. We further developed an experimental OCT system based on the spectral range identified by our simulation work. We applied the newly developed OCT to measure both retinal hemoglobin oxygen saturation (sO2) and retinal retinal flow. After obtaining the retinal sO2 and blood velocity, we further measured retinal vessel diameter and calculated the retinal oxygen metabolism rate (MRO2). To test the capability of our OCT, we imaged wild-type Long-Evans rats ventilated with both normal air and air mixtures with various oxygen concentrations.
Our simulation suggested that OCT working within visible spectral range is able to provide accurate measurement of retinal MRO2 using inverse Fourier transform spectral reconstruction. We called this newly developed technology vis-OCT, and showed that vis-OCT was able to measure the sO2 value in every single major retinal vessel around the optical disk as well as in micro retinal vessels. When breathing normal air, the averaged sO2 in arterial and venous blood in Long-Evans rats was measured to be 95% and 72%, respectively. When we challenge the rats using air mixtures with different oxygen concentrations, vis-OCT measurement followed analytical models of retinal oxygen diffusion and pulse oximeter well.
We propose using noninvasive longitudinal optical-resolution photoacoustic microscopy (L-ORPAM) to quantify blood flow flux, oxygen saturation (sO2), and thereby the metabolic rate of oxygen (MRO2), for a renal tumor model in the same mouse over weeks to months. Experiments showed that the sO2 difference between the artery and vein decreased greatly due to the arteriovenous shunting effect during tumor growth. Moreover, hypermetabolism was exhibited by an increase in MRO2.
KEYWORDS: Signal to noise ratio, Raster graphics, Photoacoustic microscopy, Tumors, In vivo imaging, Tissue optics, Natural surfaces, Image acquisition, Ear, 3D acquisition
Accurate quantification of microvasculature remains of interest in fundamental pathophysiological studies and clinical trials. Current photoacoustic microscopy can noninvasively quantify properties of the microvasculature, including vessel density and diameter, with a high spatial resolution. However, the depth range of focus (i.e., focal zone) of optical-resolution photoacoustic microscopy (OR-PAM) is often insufficient to encompass the depth variations of features of interest—such as blood vessels—due to uneven tissue surfaces. Thus, time-consuming image acquisitions at multiple different focal planes are required to maintain the region of interest in the focal zone. We have developed continuous three-dimensional motorized contour-scanning OR-PAM, which enables real-time adjustment of the focal plane to track the vessels’ profile. We have experimentally demonstrated that contour scanning improves the signal-to-noise ratio of conventional OR-PAM by as much as 41% and shortens the image acquisition time by 3.2 times. Moreover, contour-scanning OR-PAM more accurately quantifies vessel density and diameter, and has been applied to studying tumors with uneven surfaces.
KEYWORDS: In vivo imaging, Tumors, Ear, Photoacoustic microscopy, Acoustics, Biomedical optics, Optical scanning, 3D image processing, 3D scanning, Transducers
Combined optical and mechanical scanning (COMS) in optical-resolution photoacoustic microscopy (OR-PAM) has provided five scanning modes with fast imaging speed and wide field of view (FOV). With two-dimensional (2D) galvanometer-based optical scanning, we have achieved a 2 KHz B-scan rate and 50 Hz volumetric-scan rate, which enables real-time tracking of cell activities in vivo. With optical-mechanical hybrid 2D scanning, we are able to image a wide FOV (10×8 mm2) within 150 seconds, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical-based contour scanning, we can maintain the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is ideal for fast and quantitative studies of tumors and the brain.
KEYWORDS: Raster graphics, Photoacoustic microscopy, Signal to noise ratio, Oxygen, 3D scanning, 3D photoacoustic microscopy, Blood circulation, In vivo imaging, Arteries, 3D image processing
We have developed three-dimensional arbitrary trajectory (3-DAT) scanning, which can rapidly image vessels of interest over a large field of view (FOV) and maintain a high signal-to-noise ratio (SNR) along the depth direction. The concept of 3-DAT scanning was demonstrated by imaging a human hair within a FOV of 3.5 × 2.0 mm2. Further, we showed that hemoglobin oxygen saturation (sO2) and blood flow can be measured simultaneously. The frame rate was 67 times faster than a traditional two-dimensional raster scan. We also observed sO2 dynamics in response to a switch between systemic hyperoxia and hypoxia.
We developed multi-contrast photoacoustic microscopy (PAM) for in vivo anatomical, functional, metabolic, and
molecular imaging. This technical innovation enables comprehensive understanding of the tumor microenvironment.
With multi-contrast PAM, we longitudinally determined tumor vascular anatomy, blood flow, oxygen saturation of
hemoglobin, and oxygen extraction fraction.
We have applied optical-resolution photoacoustic microscopy (OR-PAM) for longitudinal monitoring of cerebral
metabolism through the intact skull of mice before, during, and up to 72 hours after a 1-hour transient middle cerebral
artery occlusion (tMCAO). The high spatial resolution of OR-PAM enabled us to develop vessel segmentation
techniques for segment-wise analysis of cerebrovascular responses.
A major obstacle in understanding the mechanism of ischemic stroke is the lack of a tool to noninvasively or minimally
invasively monitor cerebral hemodynamics longitudinally. Here, we applied optical-resolution photoacoustic microscopy
(OR-PAM) to longitudinally study ischemic stroke induced brain injury in a mouse model with transient middle cerebral
artery occlusion (MCAO). OR-PAM showed that, during MCAO, the average hemoglobin oxygen saturation (sO2)
values of feeder arteries and draining veins within the stroke core region dropped ~10% and ~34%, respectively. After
reperfusion, arterial sO2 recovered back to the baseline; however, the venous sO2 increased above the baseline value by
~7%. Thereafter, venous sO2 values were close to the arterial sO2 values, suggesting eventual brain tissue infarction.
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