Optimization of pupil design for point-scanning and line-scanning confocal microscopy

Abstract

Both point-scanning and line-scanning confocal microscopes provide resolution and optical sectioning to observe nuclear and cellular detail in human tissues, and are being translated for clinical applications. While traditional point-scanning is truly confocal and offers the best possible optical sectioning and resolution, line-scanning is partially confocal but may offer a relatively simpler and lower-cost alternative for more widespread dissemination into clinical settings. The loss of sectioning and loss of contrast due to scattering in tissue is more rapid and more severe with a line-scan than with a point-scan. However, the sectioning and contrast may be recovered with the use of a divided-pupil. Thus, as part of our efforts to translate confocal microscopy for detection of skin cancer, and to determine the best possible approach for clinical applications, we are now developing a quantitative understanding of imaging performance for a set of scanning and pupil conditions. We report a Fourier-analysis-based computational model of confocal microscopy for six configurations. The six configurations are point-scanning and line-scanning, with full-pupil, half-pupil and divided-pupils. The performance, in terms of on-axis irradiance (signal), resolution and sectioning capabilities, is quantified and compared among these six configurations.

Optical hyperspectral imaging in microscopy and spectroscopy - a review of data acquisition

“…4), thereby accelerating the datacube acquisition speed by a factor of N y with no loss in SNR. However, since the microscope operates with slits rather than pinholes, the gain in datacube acquisition speed is accomplished at the cost of a decreased spatial resolution and image contrast [45]. The spectral range of a hyperspectral line-scanning microscope varies from visible light [13, 46-49] to near infrared [50, 51].…”

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

“…Also, 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-NA objective as background, thus decreasing imaging contrast and depth. [16][17][18][19] In the dual-axis confocal (DAC) microscope, two off-axis low-NA beams are aligned such that the illumination and collection beams intersect and focus at a single location within tissue. 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 Furthermore, since the illumination and collection beams travel different paths in tissue, the detector collects less out-of-focus and multiply scattered background light, thus leading to improved imaging depth and contrast. 12,[16][17][18][19]21 In this paper, we are interested in optimizing the imaging performance of two distinct DAC architectures. In pointscanned DAC microscopy (DAC-PS), a focused point is scanned through tissue in two dimensions to reconstruct an image pixelby-pixel.…”

“…However, there is a tradeoff in performance since confocality is lost in one dimension for a line-scanned DAC, which results in greater pixel crosstalk and diminished rejection of out-of focus and multiply scattered background light. 10,[17][18][19]22,23 Contrast and spatial resolution are two major performance parameters to consider when assessing optical-sectioning microscopes. Previous diffraction theory studies have shown that spatial resolution depends on both the crossing half angle of the dual-axis beams, θ, and the focusing NA of the individual beams, α.…”

Assessing the tissue-imaging performance of confocal microscope architectures via Monte Carlo simulations

“…These variations include the dual-axis confocal (DAC) and single-axis confocal (SAC) architectures operating in both point-scanning (PS) and line-scanning (LS) modes [2–5]. In the past, these imaging devices have been characterized for spatial resolution and optical-sectioning ability in non-scattering media [6]. Quantitative analyses of sectioning ability in scattering media have also been made for individual configurations, both experimentally and numerically [2, 7].…”