Abstract
.Multiphoton microscopy provides a suitable technique for imaging biological tissues with submicrometer resolution. Usually a Gaussian beam (GB) is used for illumination, leading to a reduced power efficiency in the multiphoton response and vignetting for a square-shaped imaging area. A flat-top beam (FTB) provides a uniform spatial intensity distribution that equalizes the probability of a multiphoton effect across the imaging area. We employ a customized widefield multiphoton microscope to compare the performance of a square-shaped FTB illumination with that based on using a GB, for both two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging. The variation in signal-to-noise ratio across TPF images of fluorescent dyes spans for the GB and for the FTB illumination, respectively. For the GB modality, TPF images of mouse colon and Convallaria root, and SHG images of chicken tendon and human breast biopsy tissue showcase area that are not imaged due to either insufficient or lack of illumination. For quantitative analysis that depends on the illuminated area, this effect can potentially lead to inaccuracies. This work emphasizes the applicability of FTB illumination to multiphoton applications.
Highlights
Multiphoton microscopy (MPM) refers to an ensemble of imaging modalities that involve the simultaneous interaction of multiple photons with a material
For the final case of the Gaussian beam (GB) shown in column (c), we consider an enlarged GB beam where the diameter is 1.25× larger than the GB considered in the first case
For an incident GB diameter of 5 mm, flat-top beams (FTB) width of 4 mm, incident wavelength of 780 nm, and focal length of the focusing lens of 250 mm, we evaluate the value of β to be 645 as defined by Eq (4) of Sec. 2
Summary
Multiphoton microscopy (MPM) refers to an ensemble of imaging modalities that involve the simultaneous interaction of multiple photons with a material. In its simplest form, a rotating diffuser[12,13] has been used to obtain a time-averaged speckle pattern of approximately uniform intensity distribution to improve the accuracy in single molecule localization This method introduces moving components in the optical setup, leading to unwanted vibration in the imaging system. Another widely prevalent method for generating FTB involves the use of diffractive beam-shapers,[14,15,16,17] which redistributes the illumination intensity by interfering various diffracted orders. Diffractive optics create undesired diffraction orders, and their efficiency is strongly dependent on Journal of Biomedical Optics
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