Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning

M. de Groot, C.L. Evans, J.F. de Boer

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Abstract

We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this method, the emitted fluorescence is collected in the backward direction and is sent through a phase plate that encodes the depth information into the phase of a spectrally resolved interference pattern. We demonstrate that decoding this phase information allows for depth localization accuracy better than 4 μm over a 500 μm depth-of-field. In a high numerical aperture configuration with a much smaller depth of field, a localization accuracy of tens of nanometers can be achieved. This approach is ideally suited for miniature endoscopes, where space limitations at the endoscope tip render depth scanning difficult. We illustrate the potential for 3D visualization of complex biological samples by constructing a threedimensional volume of the microvasculature of ex vivo murine heart tissue from a single 2D scan. © 2012 Optical Society of America.
Original languageEnglish
Pages (from-to)15253-15262
Number of pages10
JournalOptics Express
Volume20
Issue number14
DOIs
Publication statusPublished - 2012

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