Invited/Optics Frontier Conference: Advanced Photonics

1.Peking University

Here we will present two pieces of high-resolution fluorescence microscopy methods we invented for live sample imaging. The first one is for in vivo imaging, which is a fast, high-resolution, miniaturized two-photon microscope (FHIRM-TPM). With a headpiece weighing 2.15 g and a new type of hollow-core photonic crystal fiber to deliver 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels). It compares favorably with benchtop two-photon microscopy and miniature wide-field fluorescence microscopy in the structural and functional imaging of Thy1-GFP- or GCaMP6f-labeled neurons. Further, we demonstrate its unique application and robustness with hour-long recording of neuronal activities down to the level of spines in mice engaging in social interaction. The second method is for live cell long-term super-resolution (SR) imaging. We have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Hessian-SIM enables rapid imaging of moving vesicles or loops in the endoplasmic reticulum without motion artifacts and with a spatiotemporal resolution of 88 nm and 188 Hz. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling…