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

Chen, Ye; Liu, Jonathan

doi:10.1117/1.jbo.18.6.066006

Cited by 14 publications

(13 citation statements)

References 37 publications

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“…According to diffraction theory, the FWHM width of the focal line in tissues is 1.4 μm. 32 Thus, we were sampling the x-direction at approximately the Nyquist frequency.…”

Section: Video-rate Line-scanned Dual-axis Confocalmentioning

confidence: 99%

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

et al. 2015

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Abstract. Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

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“…According to diffraction theory, the FWHM width of the focal line in tissues is 1.4 μm. 32 Thus, we were sampling the x-direction at approximately the Nyquist frequency.…”

Section: Video-rate Line-scanned Dual-axis Confocalmentioning

confidence: 99%

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

et al. 2015

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“…While ms2/LCI sought to use multiple scattering for imaging, other off-axis configurations have been developed to better separate ballistic and multiply scattered components of backscattered light [4, 5]. In dual-axis confocal microscopy, objectives with NA = 0.1–0.2 are used for illumination and detection with large offset angles (25–30°) between the two to create tight spatial gating for the rejection of out-of-focus background with improved depth of field [6, 7]. For interferometric techniques, low NA beams (NA < 0.02) with lower offset angles (3–4 °) can be used instead to create a larger imaging range for deep tissue imaging, because coherence gating is independent of NA and thus still provides diffuse background rejection [2].…”

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confidence: 99%

“…While larger crossing angles may improve the rejection of out-of-focus background [6], tighter spatial rejection will lead to a smaller imaging range, which is undesirable for deep imaging. Besides, larger offset angles also lead to greater signal attenuation due to longer photon paths within the tissue.…”

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confidence: 99%

Dual-axis optical coherence tomography for deep tissue imaging

et al. 2017

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We have developed dual-axis optical coherence tomography (DA-OCT) which enables deep tissue imaging by using a novel off-axis illumination/detection configuration. DA-OCT offers a 100-fold speed increase compared to its predecessor, multispectral multiple-scattering low coherence interferometry (ms2/LCI), by using a new beam scanning mechanism based on a microelectrical-mechanical system (MEMS) mirror. The data acquisition scheme was altered to take advantage of this scanning speed, producing tomographic images at a rate of 4 frames (B-scans) per second. DA-OCT differs from ms2/LCI in that the dual axes intersect at a shallower depth (~1mm). This difference, coupled with the faster scanning speed shifts the detection priority from multiply scattered to ballistic light. The utility of this approach was demonstrated by imaging both ex vivo porcine ear skin and in vivo rat skin from a Macfarlane flap model. The enhanced penetration depth provided by the DA-OCT system will be beneficial to various clinical applications in dermatology and surgery.

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“…The DAC architecture differs from a conventional confocal architecture (hereby referred to as a single-axis confocal, or SAC) in that the illumination and collection paths do not overlap except at the focus. From diffraction-theory-based calculations as well as Monte-Carlo scattering simulations performed previously [14, 16–18] , the DAC microscope has been shown to possess superior optical-sectioning capabilities in comparison to SAC microscopes, resulting in increased contrast and imaging depth.…”

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confidence: 99%

“…The software utilizes a Henyey-Greenstein approximation of Mie scattering theory and does not take into consideration diffraction, absorption, polarization, or beam steering events introduced by the heterogeneities inherent to real tissue. However, these simulations have been shown to provide an excellent first-order approximation of confocal microscope performance in homogeneous scattering media [17, 18] .…”

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confidence: 99%

Comparison of line-scanned and point-scanned dual-axis confocal microscope performance

et al. 2013

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The point-scanned dual-axis confocal (PS-DAC) microscope has been shown to exhibit a superior capability to reject out-of-focus and multiply scattered light in comparison to its conventional single-axis counterpart. However, the slow frame rate (typically < 5 Hz) resulting from point-by-point data collection makes these systems vulnerable to motion artifacts. While video-rate point-scanned confocal microscopy is possible, a line-scanned dual-axis confocal (LS-DAC) microscope provides a simpler means of achieving high-speed imaging through line-by-line data collection, but sacrifices contrast due to a loss of confocality along one dimension. Here we evaluate the performance tradeoffs between a LS-DAC and PS-DAC microscope with identical spatial resolutions. Characterization experiments of the LS-DAC and PS-DAC microscopes with tissue phantoms, in reflectance mode, are shown to match results from Monte-Carlo scattering simulations of the systems. Fluorescence images of mouse brain vasculature, obtained using resolution-matched LS-DAC and PS-DAC microscopes, demonstrate the comparable performance of LS-DAC and PS-DAC microscopy at shallow depths.

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

Cited by 14 publications

References 37 publications

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

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

Dual-axis optical coherence tomography for deep tissue imaging

Comparison of line-scanned and point-scanned dual-axis confocal microscope performance

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