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Proceedings Paper

Simultaneous multiplane imaging for 3D confocal microscopy using high-speed z-scanning multiplexing
Author(s): Marti Duocastella; Giuseppe Vicidomini; Alberto Diaspro
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Paper Abstract

One of the key frontiers in optical imaging is to maximize the spatial information retrieved from a sample while minimizing acquisition time. Confocal laser scanning microscopy is a powerful imaging modality that allows real-time and high-resolution acquisition of two-dimensional (2D) sections. However, in order to obtain information from threedimensional (3D) volumes it is currently limited by a stepwise process that consists of acquiring multiple 2D sections from different focal planes by slow z-focus translation. Here, we present a novel method that enables the capture of an entire 3D sample in a single step. Our approach is based on an acoustically-driven varifocal lens integrated in a commercial confocal system that enables axial focus scanning at speeds of 140 kHz or above. Such high-speed allows for one or multiple focus sweeps on a pixel by pixel basis. By using a fast acquisition card, we can assign the photons detected at each pixel to their corresponding focal plane allowing simultaneous multiplane imaging. We exemplify this novel 3D confocal microscopy technique by imaging different biological fluorescent samples and comparing them with those obtained using traditional z-scanners. Based on these results, we find that image quality in this novel approach is similar to that obtained with traditional confocal methods, while speed is only limited by signal-to-noise-ratio. As the sensitivity of photodetectors increases and more efficient fluorescent labeling is developed, this novel 3D method can result in significant reduction in acquisition time allowing the study of new fundamental processes in science.

Paper Details

Date Published: 9 March 2015
PDF: 5 pages
Proc. SPIE 9330, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXII, 93300Q (9 March 2015); doi: 10.1117/12.2078914
Show Author Affiliations
Marti Duocastella, Istituto Italiano di Tecnologia (Italy)
Giuseppe Vicidomini, Istituto Italiano di Tecnologia (Italy)
Alberto Diaspro, Istituto Italiano di Tecnologia (Italy)

Published in SPIE Proceedings Vol. 9330:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXII
Thomas G. Brown; Carol J. Cogswell; Tony Wilson, Editor(s)

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