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Demonstration of flat-top beam illumination in widefield multiphoton microscopy
- Source :
- Journal of Biomedical Optics
- Publication Year :
- 2019
-
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 ∼5.6 dB for the GB and ∼1.2 dB 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 ∼20% 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.
- Subjects :
- Paper
two-photon fluorescence
Materials science
Microscope
Optical fiber
flat-top beam
Optical Phenomena
Colon
Biomedical Engineering
Signal-To-Noise Ratio
01 natural sciences
law.invention
010309 optics
Biomaterials
Tendons
Mice
Optics
Signal-to-noise ratio
Special Section Celebrating Thirty Years of Multiphoton Microscopy in the Biomedical Sciences
law
0103 physical sciences
Animals
Humans
Computer Simulation
Breast
Fluorescent Dyes
Vignetting
business.industry
Resolution (electron density)
Optical Imaging
Second-harmonic generation
Equipment Design
Mathematical Concepts
Atomic and Molecular Physics, and Optics
Convallaria
Electronic, Optical and Magnetic Materials
Microscopy, Fluorescence, Multiphoton
multiphoton microscopy
uniform illumination
Female
business
Chickens
Beam (structure)
Gaussian beam
second-harmonic generation
Subjects
Details
- ISSN :
- 15602281
- Volume :
- 25
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Journal of biomedical optics
- Accession number :
- edsair.doi.dedup.....da98021d80619f4c4d301b714110d62c