Abstract
In nonlinear microscopy, phase-only spatial light modulators (SLMs) allow achieving simultaneous two-photon excitation and fluorescence emission from specific region-of-interests (ROIs). However, as iterative Fourier transform algorithms (IFTAs) can only approximate the illumination of selected ROIs, both image formation and/or signal acquisition can be largely affected by the spatial irregularities of the illumination patterns and the speckle noise. To overcome these limitations, we propose an alternative complex illumination method (CIM) able to generate simultaneous excitation of large-area ROIs with full control over the amplitude and phase of light and reduced speckle. As a proof-of-concept we experimentally demonstrate single-photon and second harmonic generation (SHG) with structured illumination over large-area ROIs.
Highlights
In some recent introduced multiphoton microscopy techniques, simultaneous excitation and signal collection from multiple specific cell populations have become into key tools for monitoring the cellular activity [1,2,3]
The corresponding setup is basically composed of two consecutive optical modules that we refer throughout the whole manuscript as complex field encoding module (CFEM) and optical demagnifying module (ODM), respectively, see Fig. 1
The lack of significant dispersion contributions at the sample plane can be regarded as an advantage of our proposal in comparison with other illumination strategies e.g., computergenerated holograms (CGHs). In this manuscript we have proposed and tested an interferometric complex illumination method (CIM) mainly addressed to microscopy applications which is able to achieve simultaneous linear and/or non-linear excitation of user-defined ROIs
Summary
In some recent introduced multiphoton microscopy techniques, simultaneous excitation and signal collection from multiple specific cell populations have become into key tools for monitoring the cellular activity [1,2,3]. Under coherent illumination, the reconstruction of CGHs is currently unable to generate spatially homogeneous irradiance patterns over ROIs with dimensions on the order of the typical cellular-sizes, not to mention its inefficiency to precisely manipulate the amount of energy put into them e.g., to generate multiple-intensity level illumination patterns At this point, improved versions of the well-known GerchbergSaxton algorithm [21] have allowed both speckle reduction and phase control of 1-D illumination patterns [11,22].
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