Two-photon scanned light sheet fluorescence microscopy with axicon imaging for fast volumetric imaging.

Po-Yen Lin,Bi-Chang Chen,Sheng-Ping L Hwang,Chi-Hon Lee

doi:10.1117/1.jbo.26.11.116503

Abstract

.Significance: Two-photon microscopy has become the standard platform for deep-tissue fluorescence imaging. However, the use of point scanning in conventional two-photon microscopy limits the speed of volumetric image acquisition.Aim: To obtain fast and deep volumetric images, we combine two-photon light sheet fluorescence microscopy (2p-LSFM) and axicon imaging that yields an extended depth of field (DOF) in 2p-LSFM.Approach: Axicon imaging is achieved by imposing an axicon lens in the detection part of LSFM.Results: The DOF with axicon imaging is extended more than 20-fold over that of a conventional imaging lens, liberating the synchronized scanning in LSFM. We captured images of dynamic beating hearts and red blood cells in zebrafish larvae at volume acquisition rates up to 30 Hz.Conclusions: We demonstrate the fast three-dimensional imaging capability of 2p-LSFM with axicon imaging by recording the rapid dynamics of physiological processes.

Highlights

Techniques to capture deep volumetric images at high speed are widely applicable in biological research and useful in neuroscience and developmental biology
We demonstrate the fast three-dimensional imaging capability of 2p-light sheet fluorescence microscopy (LSFM) with axicon imaging by recording the rapid dynamics of physiological processes
The signal-tonoise ratio of images acquired with the axicon was poor due to strong spherical aberrations induced by the axicon

Summary

Introduction

Techniques to capture deep volumetric images at high speed are widely applicable in biological research and useful in neuroscience and developmental biology. Optical signals that respond to neuronal activity can change on millisecond timescales and propagate dozens of micrometers in three dimensions.[1] studies on cardiac development in embryonic zebrafish typically require imaging rates of ∼10 volumes per second (volume acquisition rate of 10 Hz) to capture the 3D dynamics of a heart beating at 2 to 4 Hz.[2] Imaging such rapid processes deep within tissues is still challenging with current bioimaging techniques. Developed techniques for deep-tissue imaging typically utilize two-photon microscopy, which enables the visualization of fluorescence deep inside intact tissues.[3,4] In conventional twophoton microscopy, a near-infrared ultrashort pulsed laser is focused by an objective on a particular spot to excite fluorophores within the sample. After scanning the focal spot throughout the sample, a volumetric image is constructed by stacking a series of optical section images. The use of point scanning imposes a fundamental limitation on the volume imaging rate, according to the laser repetition

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