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

Abstract

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).

Redox-Magnetohydrodynamically Controlled Fluid Flow with Poly(3,4-ethylenedioxythiophene) Coupled to an Epitaxial Light Sheet Confocal Microscope for Image Cytometry Applications

“…The image aspect ratio was quantified as a function of applied current that drives the R-MHD microfluidics. We have previously demonstrated the importance of synchronization of a push broom scanning source with data acquisition of the linear sensor to ensure image quality Figure a shows the convergence of aspect ratio to an R-MHD current of 850 μA when a line exposure period was set to 300 μs.…”

“…We have previously demonstrated the importance of synchronization of a push broom scanning source with data acquisition of the linear sensor to ensure image quality. 21 Figure 4a shows the convergence of aspect ratio to an R-MHD current of 850 μA when a line exposure period was set to 300 μs. Figure 4b shows resulting images of fluorescent microspheres with high lateral resolution.…”

“…Line scanning confocal microscopy, which both illuminates and collects light across a one-dimensional path simultaneously, offers combined advantages of the other two approaches: , a greater speed than point scanning and suitability for compact instrumentation for clinical applications. , An important requirement, however, is that all points of the sample passing beneath the imaging line must exhibit a uniform linear speed perpendicular to the linear image sensor. Continuous imaging using a linear sensor (also known as a “push broom” geometry) has been previously demonstrated for image cytometry . The approach presented in this paper is aligning a light sheet to a linear image sensor in an epitaxial configuration.…”

“…Continuous imaging using a linear sensor (also known as a "push broom" geometry) has been previously demonstrated for image cytometry. 21 The approach presented in this paper is aligning a light sheet to a linear image sensor in an epitaxial configuration. Optical sectioning is provided by the inherently small pixel size (5 μm) of the camera sensor itself, which is well suited to rejecting out-of-plane emission light.…”