Abstract
Multifocal multiphoton microscopy (MMM) enhances imaging speed by parallelization. It is not well understood why the imaging depth of MMM is significantly shorter than conventional single-focus multiphoton microscopy (SMM). In this report, we show that the need for spatially resolved detectors in MMM results in a system that is more sensitive to the scattering of emission photons with reduced imaging depth. For imaging depths down to twice the scattering mean free path length of emission photons (2xl (s) (em)), the emission point spread function (PSF(em)) is found to consist of a narrow, diffraction limited distribution from ballistic emission photons and a broad, relatively low amplitude distribution from scattered photons. Since the scattered photon distribution is approximately 100 times wider than that of the unscattered photons at 2xl (s) (em), image contrast and depth are degraded without compromising resolution. To overcome the imaging depth limitation of MMM, we present a new design that replaces CCD cameras with multi-anode photomultiplier tubes (MAPMTs) allowing more efficient collection of scattered emission photons. We demonstrate that MAPMT-based MMM has imaging depth comparable to SMM with equivalent sensitivity by imaging tissue phantoms, ex vivo human skin specimens based on endogenous fluorophores, and green fluorescent protein (GFP) expressing neurons in mouse brain slices.
Highlights
Multiphoton microscopy is a three dimensional (3D) imaging technique based on nonlinear excitation of fluorophores [1]
We show that the need for spatially resolved detectors in Multifocal multiphoton microscopy (MMM) results in a system that is more sensitive to the scattering of emission photons with reduced imaging depth
We demonstrate that multi-anode photomultiplier tubes (MAPMTs)-based MMM has imaging depth comparable to single-focus multiphoton microscopy (SMM) with equivalent sensitivity by imaging tissue phantoms, ex vivo human skin specimens based on endogenous fluorophores, and green fluorescent protein (GFP) expressing neurons in mouse brain slices
Summary
Multiphoton microscopy is a three dimensional (3D) imaging technique based on nonlinear excitation of fluorophores [1]. The imaging volume of conventional multiphoton microscopy is limited to about several hundred microns on a side using typical high numerical aperture objectives [1, 4, 6]. While this volume is sufficient for cellular imaging, many tissues have physiologically relevant structures that are significantly larger. Traditional 3D microscopes with frame rate on the second scale can realistically study only a few hundreds of cells These microscopes cannot hope to provide comparable statistical accuracy and precision of quantitative assays such as flow cytometry. Large volume imaging has been applied to elucidate receptor dependent cancer metastasis processes [11]
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