Optimizing the performance of dual-axis confocal microscopes via Monte-Carlo scattering simulations and diffraction theory

Ye Chen,Jonathan T C Liu

doi:10.1117/1.jbo.18.6.066006

Abstract

Dual-axis confocal (DAC) microscopy has been found to exhibit superior rejection of out-of-focus and multiply scattered background light compared to conventional single-axis confocal microscopy. DAC microscopes rely on the use of separated illumination and collection beam paths that focus and intersect at a single focal volume (voxel) within tissue. While it is generally recognized that the resolution and contrast of a DAC microscope depends on both the crossing angle of the DAC beams, 2θ, and the focusing numerical aperture of the individual beams, α, a detailed study to investigate these dependencies has not been performed. Contrast and resolution are considered as two main criteria to assess the performance of a point-scanned DAC microscope (DAC-PS) and a line-scanned DAC microscope (DAC-LS) as a function of θ and α. The contrast and resolution of these designs are evaluated by Monte-Carlo scattering simulations and diffraction theory calculations, respectively. These results can be used for guiding the optimal designs of DAC-PS and DAC-LS microscopes.

Highlights

Confocal microscopy, using point illumination and pinhole detection to reject out-of-focus and multiply scattered light from the object, provides improved imaging resolution and contrast over traditional microscopy and has become one of the most widely used biomedical optical imaging techniques.[1]
In a conventional single-axis confocal microscope, both the illumination and collection beams travel a common path in tissue, causing a significant amount of out-of-focus and multiply scattered light to be collected by the high-numerical aperture (NA) objective as background, decreasing imaging contrast and depth.[16,17,18,19]
Since we aim to provide a guide to optimize the design of dual-axis confocal (DAC) microscopes, we vary θ and α in our Monte-Carlo simulations to analyze their effects on contrast and resolution

Summary

Introduction

Confocal microscopy, using point illumination and pinhole detection to reject out-of-focus and multiply scattered light from the object, provides improved imaging resolution and contrast over traditional microscopy and has become one of the most widely used biomedical optical imaging techniques.[1] In the past decade, various confocal microscope architectures have been developed for diverse biomedical applications.[2,3,4,5,6,7,8,9,10,11,12,13,14,15] A large objective lens with a high numerical aperture (NA) is generally utilized in a conventional confocal microscope to obtain highresolution images. A long working distance results from utilizing low-NA lenses, which allows for a scanning mirror to be placed at the distal end of the objective to provide a large field of view without introducing scanning-induced aberrations.[20]

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