KEYWORDS: Light sources and illumination, Speckle, Red blood cells, Speckle pattern, Photoacoustic spectroscopy, Microspheres, Diffusers, Ultrasonography, Signal detection, Imaging systems
We investigated application of speckle illumination to a heterogeneous sample where small optical absorbers are sparsely distributed. Although such sample is difficult to visualize by homogeneous illumination, speckle illumination has advantages. For example, speckle light has a heterogeneous and granular intensity distribution, which is similar to that of optical absorbers in a heterogeneous sample. In addition, speckle light has higher intensity spots compared to homogeneous illumination. These characteristics can be used for signal enhancement compared to homogeneous illumination. Moreover, speckle illumination enables us to perform heterogeneity evaluation of the heterogeneous sample since the signal is varied depending on spatial overlap between the speckle light and the optical absorbers. In this research, we used acoustic resolution photoacoustic microscope (AR-PAM) to investigate application of speckle illumination to a heterogeneous sample. In the AR-PAM system, speckle light is generated by using a diffuser. As heterogeneous samples, black-dyed microspheres and red blood cells (RBCs) in Matrigel were used. By illuminating speckle light to the sample and changing the speckle pattern, variation of the signal was observed. In the measurement of the microspheres, we confirmed that some speckle patterns provide higher signals compared to homogeneous illumination. In addition, we also confirmed that normalized signal variance is depended on concentration of RBCs. This can be used as heterogeneity evaluation.
KEYWORDS: Tumors, Cancer detection, Blood, Biological samples, In vivo imaging, Near infrared, Biomedical optics, Light sources, Signal intensity, Photoacoustic microscopy
SignificanceWe developed a high-speed optical-resolution photoacoustic microscopy (OR-PAM) system using a high-repetition-rate supercontinuum (SC) light source and a two-axes Galvano scanner. The OR-PAM system enabled real-time imaging of optical absorbers inside biological tissues with excellent excitation wavelength tunability.AimIn the near-infrared (NIR) wavelength range, high-speed OR-PAM faces limitations due to the lack of wavelength-tunable light sources. Our study aimed to enable high-speed OR-PAM imaging of various optical absorbers, including NIR contrast agents, and validate the performance of high-speed OR-PAM in the detection of circulating tumor cells (CTCs).ApproachA high-repetition nanosecond pulsed SC light source was used for OR-PAM. The excitation wavelength was adjusted by bandpass filtering of broadband light pulses produced by an SC light source. Phantom and in vivo experiments were performed to detect tumor cells stained with an NIR contrast agent within flowing blood samples.ResultsThe newly developed high-speed OR-PAM successfully detected stained cells both in the phantom and in vivo. The phantom experiment confirmed the correlation between the tumor cell detection rate and tumor cell concentration in the blood sample.ConclusionsThe high-speed OR-PAM effectively detected stained tumor cells. Combining high-speed OR-PAM with molecular probes that stain tumor cells in vivo enables in vivo CTC detection.
KEYWORDS: Biological imaging, Signal detection, Contrast agents, Tumors, Tissues, Real time imaging, Veins, In vivo imaging, Photoacoustic microscopy, Dyes
To understand various phenomena that occur in living tissue, high-speed volume imaging technology that visualizes the dynamics of cells and molecules in living tissue is desired. For this purpose, we have developed a reflective MS-PAI with high excitation wavelength flexibility using a supercontinuum light source and bandpass filters, and a fast B-scan rate of over 100 fps. The ability of the MS-PAM for cell dynamics imaging was demonstrated by performing time-lapse volume imaging of contrast agent-stained cells flowing through blood vessels. We plan to combine the MS-PAM technology with functionalized contrast agents (imaging probes) to analyze dynamic phenomena.
We have developed fluorescent dye-encapsulating nanoparticle reagents that can be administered to animal model, enabling both photoacoustic and fluorescence imaging. Two types of in vivo photoacoustic imaging systems were developed to visualize the imaging reagents administrated to tumor model mouse. Of the two imaging systems, one used a high frequency linear array ultrasound transducer and the other used a low frequency concave array ultrasound transducer. We also used an IVIS system for fluorescence imaging of the reagents administered to tumor model mouse. IVIS allows us to easily obtain the fluorescence distribution of the reagent on a two-dimensional plane of the whole body of mouse. We successfully obtained photoacoustic images of the distribution of reagents after intravenous administration. The photoacoustic image, as well as the fluorescence image, was able to visualize the tumor accumulation of the reagents due to the EPR (Enhanced Permeation and Retention) effect. Our fluorescent dye-encapsulated nanoparticle reagents can be used for the evaluation in vivo localization and accumulation over time. The reason for this success is that we took advantage of the ability of the reagents to simultaneously generate photoacoustic and fluorescence signals. This means that the measuring conditions for photoacoustic imaging could be determined based on the fluorescence data acquired with IVIS. In the case of in vivo photoacoustic imaging of the administered reagents such as the nanoparticle reagents developed in this study, whose distribution changes over time and has not previously been used as imaging targets, the simultaneous fluorescence signals must be effective in detecting photoacoustic signals of the reagents.
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