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

Abstract

Various confocal microscope architectures have been developed for in vivo tissue imaging, including single-axis confocal (SAC) and dual-axis confocal (DAC) configurations utilizing both point-scanning (PS) and line-scanning (LS) approaches. While it is known that these design variations lead to tradeoffs in imaging performance, a quantitative comparison of the imaging performance of these configurations in highly turbid media would be of value. Here, we perform Monte-Carlo simulations to evaluate the optical-sectioning capability of these various confocal microscope architectures in reflectance mode. In particular, we investigate the axial and transverse responses of these configurations to reflective targets at various depths within a homogenous scattering medium. We find that the DAC-PS configuration results in superior rejection of multiply scattered background light compared to all other configurations, followed in performance by the SAC-PS, the DAC-LS, and then the SAC-LS. Line scanning with both the DAC and SAC configurations leads to photon crosstalk between pixels. However, at shallow depths, the axial and transverse resolution of all configurations is maintained in a homogeneous scattering medium.

Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology

“…Compared to a SAC microscope, a DAC microscope with comparable axial resolution (optical-section thickness) is capable of improved rejection of out-of-focus and multiply scattered background light, which enables high-contrast microscopy within highly scattering tissues [31][32][33]. The DAC architecture utilizes off-axis low-numerical aperture (NA) illumination and collection beams that intersect at their foci, which also defines the focal volume of the microscope [31][32][33].…”

“…Unlike a point-scanned confocal microscope, in which an illumination beam is focused to a tight spot within tissue and a pinhole is used for rejection (spatial filtering) of background light, a line-scanned confocal microscope utilizes a slit to reject background light from an illumination beam that is focused to a thin line within the tissue [34]. Line-scanned confocal microscopes thus sacrifice one dimension of confocality and typically exhibit reduced contrast (signal-to-background ratio, SBR) and tissue-imaging depth compared to point-scanned systems [31,34]. Nevertheless, simulations and experiments have demonstrated that a LS-DAC microscope is capable of achieving adequate contrast (SBR) when imaging near tissue surfaces (approximately 100-to 200-μm deep) [31,34,35].…”

“…Line-scanned confocal microscopes thus sacrifice one dimension of confocality and typically exhibit reduced contrast (signal-to-background ratio, SBR) and tissue-imaging depth compared to point-scanned systems [31,34]. Nevertheless, simulations and experiments have demonstrated that a LS-DAC microscope is capable of achieving adequate contrast (SBR) when imaging near tissue surfaces (approximately 100-to 200-μm deep) [31,34,35]. In addition, while the low-NA collection of light would be expected to limit the collection efficiency and signal-to-noise ratio (SNR) of DAC microscopes, the relatively isotropic resolution of the DAC microscope enables the collection of light from a larger focal volume compared to a SAC microscope with an equivalent optical-sectioning thickness.…”

Video-ratein vivofluorescence imaging with a line-scanned dual-axis confocal microscope

“…29 In addition, previous Monte-Carlo simulations suggest that a LS-DAC configuration provides superior contrast (signal to background ratio, SBR) in comparison to a line-scanned single-axis confocal microscope. 30 Recently, we have also introduced a variation on the LS-DAC technique called sheet-scanned dual-axis confocal (SS-DAC) microscopy, 31 in which a scientific complementary metal-oxide-semiconductor (sCMOS) camera enables an oblique light-sheet to be imaged at each scanned position. However, both the previous LS-DAC microscope and the recently developed SS-DAC microscope were slow stage-scanned prototypes (frame rate at 2 fps) that did not utilize a fast-scanning galvo (up to 30 Hz), high-speed camera acquisition (10-kHz line acquisition rate), and speed-optimized software.…”

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.…”