High-speed dual-beam, crossed line-scanning fluorescence microscope with a point confocal resolution

Jeong, Hyun Chul; Kim, Hyung Jin; Eun, Jung; Heo, Seungjin; Lim, Mikyoung; Cho, Yong-Hoon; Kim, Beop Min

doi:10.1364/ao.54.003811

Cited by 6 publications

(2 citation statements)

References 28 publications

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“…sharper features along the scanning direction. Although the degree of anisotropy is negligible in many cases [21], several methods have been suggested to obtain isotropic resolution, for example, by bi-directionally scanning and subsequent image processing [15,37]. Alternatively, the anisotropic PSF could be corrected through deep learning techniques, for instance, by isotropic 2D construction from anisotropic raw images [38].…”

Section: Discussionmentioning

confidence: 99%

Optimizing the performance of multiline-scanning confocal microscopy

Weng

Tang

Han

2020

J. Phys. D: Appl. Phys.

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Line-scanning (LS) confocal microscopy provides high imaging speed and moderate optical sectioning strength, which makes it a useful tool for imaging various biospecimens ranging from living cells to fixed tissues. Conventional LS systems have only used a single excitation line and slit, and thus have not fully exploited the benefits of parallelization. Here we investigate the optical performance of multi-LS confocal microscopy (mLS) by employing a digital micro-mirror device that provides programmable patterns of the illumination beam and the detection slit. Through experimental results and optical simulations, we assess the depth discrimination of mLS under different optical parameters and compare it with multi-point systems such as spinning disk confocal microscopy (SDCM). Under the same illumination duty cycle, we find that mLS has better optical sectioning than SDCM at a high degree of parallelization. The optimized mLS provides a low photobleaching rate and video-rate imaging while its optical sectioning is similar to single LS confocal microscopy.

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Section: Discussionmentioning

confidence: 99%

Optimizing the performance of multiline-scanning confocal microscopy

Weng

Tang

Han

2020

J. Phys. D: Appl. Phys.

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“…The limited imaging speed prevented their application in dynamic imaging. As a compromise strategy, line scanning confocal microscopy (LSCM) applied a narrow slit before the detector [24][25][26] or Rolling Shutter mode on camera [27]. Thus, LSCM could balance the frame-rate and out-of-focus rejection ability, which has been demonstrated in in vivo mouse brain.…”

Section: Introductionmentioning

confidence: 99%

Selective illumination on line-scanning confocal mesoscope enhances background rejection in cortex-wide mouse brain imaging

Xie

2024

Preprint

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Confocal microscope has optical sectioning that is accessible for structural and dynamic imaging in in vivo mouse brain. With the requirement of brain-wide field-of-view (FOV) in many neuroscience researches, existing confocal microscope fails to fulfill the requirement. Here, we proposed the brain-wide, high resolution line-scan confocal mesoscope (LSCM) for in vivo mouse imaging, achieving 6.6-mm-FOV, 3.2-μm-resolution and video-rate acquisition. To further enhance the background rejection ability, we introduced the selective illumination method into our system. We demonstrated that the proposed technique is s able to image the neurodynamics in in vivo mouse brain. Comparing to the LSCM and wide-field mesoscope, our selective illumination line-scan confocal mesoscope improves more than 25% and 3 times background rejection ability, respectively.

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A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

Hutcheson

Majid

Powless

et al. 2015

Review of Scientific Instruments

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Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min(-1) with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels(-1).

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High-speed dual-beam, crossed line-scanning fluorescence microscope with a point confocal resolution

Cited by 6 publications

References 28 publications

Optimizing the performance of multiline-scanning confocal microscopy

Optimizing the performance of multiline-scanning confocal microscopy

Selective illumination on line-scanning confocal mesoscope enhances background rejection in cortex-wide mouse brain imaging

A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

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