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High spatiotemporal resolution fluorescence imaging of biological samples in vivo| old_uid | 16476 |
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| title | High spatiotemporal resolution fluorescence imaging of biological samples in vivo |
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| start_date | 2018/10/05 |
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| schedule | 11h30 |
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| online | no |
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| location_info | Amphithéâtre |
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| details | Séminaire impromptu - Invitant : Daniel Choquet, Team Leader: Dynamic organization & Function of synapses / Directeur de l'IINS |
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| summary | 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 ºm laterally and 3.35 ºm 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 hour-long time-lapse SR imaging in live cells. |
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| responsibles | Deris |
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