The first commercial confocal microscope was released in 1987. Since then, laser scanning confocal microscopy has been widely used in tissue, cell, developmental and molecular biology, and even pathology. Currently, there is a growing need to image multiple fluorescent (and autofluorescent) markers, simultaneously and cost effectively, to detect, discriminate and quantitate various components and their interactions (e.g. by fluorescence resonance energy transfer) in biological materials, with enhanced axial resolution. In addition, with high enough spectral resolution, even cell nuclear size estimations are possible, using Mie scattering. Current multispectral/hyperspectral scanning laser confocal microscopes are extremely expensive and there is huge need for a cost-effective and efficient add-on system to upgrade conventional microscopes to spectral laser scanning confocal microscopes.
We developed an affordable ultra-resolution spectral confocal add-on system and tested it on research-grade microscopes. Our system includes supercontinuum laser as a broadband light source, a confocal scanning system, and spectral selection using custom spectral tunable cavities (STCs) offering ultra-spectral (sub-nanometer) resolution. The STCs use picoliter volume Fabry–Perot-type optical cavities filled with liquid crystal for tuning and can be incorporated in excitation or emission optical paths as a single cell or an array format. The spectral selection is done with no moving parts and just applying voltage to the STC filter. We will present various fabrication methods of STCs with different geometrical and material selections (glass versus polymer substrates) to improve resolution, throughput and manufacturability.
We present system design, validated spectral and spatial resolution and testing the confocal system on histopathology slides.
We present a multimode hyperspectral imaging (HIS) system operating in fluorescence and reflectance modes for food quality and safety applications. The system uses spectral band sequential imaging on the detection side. To ensure constant, high S/N levels, the image acquisition time is optimized for each spectral band. The illumination module uses two independent light sources for fluorescence and reflectance measurements, based on three computer-controlled LED illumination rings. UVA (371 nm, FWHM 16nm, power = 91.7mW/cm²) and blue/violet (418 nm, FWHM = 21nm, power = 38.9mW/cm²) LEDs provide fluorescence excitation. White LEDs (power = 35.8mW/cm²) are used for reflectance. The spectral imaging system incorporated within the detection pathway is able to transition between wavelengths within microseconds over the full bandwidth of the device (450 nm - 800 nm). The system is configured as a tabletop platform with both the illumination and detection located above the food sample. Illumination uniformity is ~90%, spatial resolution is 89μm, and spectral resolution is 8nm.
The system was tested for food safety applications by imaging of pet food spiked with Salmonella enterica, where the ability to identify the bacteria in these samples was compared to existing detection methodologies. As an example of food authentication applications, images captured at 75 wavelengths over the range 450 nm to 810 nm with a 5 nm interval were collected from wild salmon and farmed salmon purchased locally. Wild salmon and farmed salmon were found to have distinctly different reflectance features from 515 nm to 650 nm.
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