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.

Date Published: 4 March 2019
PDF
Proc. SPIE 10881, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII, 1088118 (4 March 2019); doi: 10.1117/12.2516234

Published in SPIE Proceedings Vol. 10881:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII
Daniel L. Farkas; Attila Tárnok; James F. Leary, Editor(s)