Spectroscopic single-molecule localization microscopy (sSMLM) combines super-resolution microscopy and spectroscopy. Its single molecule sensitivity and high spectral precision have made it uniquely valuable for several applications, including multicolor imaging, chemical characterization, polarity sensing, and multiplexed single particle tracking. However, widespread adoption is hampered by a lack of standardization in optical implementation, calibration techniques, and image processing. We demonstrate our lab’s efforts to develop tools that simplify adoption and optimize photon efficiency, including protocols for calibration techniques, a user-friendly imageJ plugin for image processing, and a fabricated monolithic beam splitter and prism designed to fit into a microscope body with minimal optical alignment.
KEYWORDS: Super resolution, Photoacoustic microscopy, Red blood cells, Signal detection, Ultrasonography, Laser soldering, Blood circulation, Biological imaging, 3D tracking, Two photon excitation microscopy
Exploiting the optical absorption of hemoglobin, photoacoustic microscopy (PAM) has achieved label-free imaging of the microvasculature in vivo and enabled simultaneous quantification of blood oxygenation and flow. However, the axial resolution of PAM is limited to the mesoscopic level due to the finite bandwidth of detected ultrasound signals. To address this limitation, we have developed a super-resolution functional PAM technique based on spatiotemporal tracking of red blood cells, which enables label-free functional microvascular imaging in 3D at the single-cell level. We have demonstrated the utility of this technique by imaging the mouse brain’s responses to a single-vessel stroke in 3D.
Polymer micro-ring resonators (MRRs) have been proven to be one of the most versatile choices for ultrasonic detections, largely due to their high detection sensitivity over a wide frequency range, optical transparency, and at a significantly reduced form-factor. However, precisely controlling the resonance mode of individual MRRs became increasingly difficult due to the stringent requirements in their dimensional tolerance. Here we report a nanofabrication strategy to substantially improve the dimensional accuracy of the MRR to precisely control its resonance modes. We experimentally demonstrate polymer MRR in an array format, in enabling rapid and parallel ultrasound detection.
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