The University of Arizona

Leilei Peng

Jun Ding

Yu Li

Ming Zhao

Han Zhang

Dongli Xu

A microscopic technique for generating high-clarity, large volume 3D images of fluorescent tissue structure at subcellular resolution and capture transient activities. The technique includes capturing two orthogonal 2D projection of the sample volume by performing a projection scan with an excitation laser sweeping through the volume at up to 100 vps, tracking the scan depth using an electrically tuned lens to keep the emission image in focus and generate an xy plane volume projection image at the camera; and placing a PMT behind the excitation lens to collect emission passed through the excitation lens, wherein signals from the PMT form a focus scan projection at the yz plane; and then merging the xy and yz projections.

Fast volumetric imaging system and process for fluorescent tissue structures and activities

A method for measuring distorted illumination patterns and correcting image artifacts in structured illumination microscopy. The method includes the steps of generating an illumination pattern by interfering multiple beams, modulating a scanning speed or an intensity of a scanning laser, or projecting a mask onto an object; taking multiple exposures of the object with the illumination pattern shifting in phase; and applying Fourier transform to the multiple exposures to produce multiple raw images. Thereafter, the multiple raw images are used to form and then solve a linear equation set to obtain multiple portions of a Fourier space image of the object. A circular 2-D low pass filter and a Fourier Transform are then applied to the portions. A pattern distortion phase map is calculated and then corrected by making a coefficient matrix of the linear equation set varying in phase, which is solved in the spatial domain.

System and method for measuring distorted illumination patterns and correcting image artifacts in structured illumination imaging

A superresolution STED-NSIM apparatus having an epifluorescence architecture utilizing a 2D structured STED pattern having a N.A. less than a N.A. of the microscope objective and no surface plasmon resonance (SPR) effects. A superresolution STED-NSIM imaging method using a fully deterministic imaging processing method, in which a pre-calibrated set of parameters are used to process all image data.

Stimulated emission depletion nonlinear structured illumination microscopy (STED-NSIM) apparatus, methods, and applications

A fast fluorescence lifetime microscopic system images FRET between multiple labels in live cells and deep tissue, using a quantitative analysis method to reconstruct the molecular machinery behind the multiplexed FRET phenomenon. The system measures fluorescence lifetime, intensity and anisotropy as images of excitation-emission matrices (EEM) in real time and high speed within a single image scan, performs high-resolution deep-penetrating 3D FRET imaging in live samples, and fully analyzes all possible photon pathways of multiplexed FRET. The system provides a way for systematic and dynamic imaging of biochemical networks in cells, tissue and live animals, which will help to understand mechanisms of genetic disorders, cancers, and more.

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Tucson, AZ, United States of America

Leilei Peng