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Developments in multifocal multiphoton microscopy with single element detection
Young, Michael Dennis
Young, Michael Dennis
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2015
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2016-06-01
Abstract
The goals of microscopy generally involve improvements to resolution and specificity or contrast in the sample. When microscopy intersects with the life sciences, these goals also include acquiring images at significant rates (30 fps), deep within a scattering sample -- at depths greater than 2 um -- and with good sectioning or axial resolution for volumetric images (~400 nm). Multiphoton microscopy is well suited to biological imaging as the fundamental excitation beam is at longer wavelength (~0.8--1.5 um) and is scattered less strongly in scattering media. Additionally, when coupled with single element detection, image aberration from scattering ambiguity is eliminated. However, multiphoton-microscopy images are typically acquired by serially measuring the nonlinear signal and reassembling the image on a computer. To increase the image acquisition rate, the laser focus can be spatially and temporally decorrelated so as to acquire the images in parallel. Abstract We present a "multifocal" microscope with an extended geometry focus, capable of imaging in scattering media with a single element detector at the diffraction limit of the objective lens. The extended geometry focus is scanned across a modulation pattern displayed on a spatial light modulator. Spatial frequencies in the modulation pattern uniquely encode carrier frequencies along the extended geometry focus as a function of position. This focus is image relayed to the the sample where, either absorption or fluorescence is measured by the detector. We have built upon previous work, where the resolution along the extent of the illumination was a function of the modulation pattern, by building a microscope where the resolution is a function of the objective lens. This is accomplished by relaying diffracted light from the modulation pattern to the back aperture of the objective. We demonstrate that the microscope is diffraction limited and provide examples of both absorptive and twophoton excitation fluorescence images.
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