Design and analysis of multi-confocal spectral imaging microscopy

Abstract

Confocal microscopy has been a versatile tool for investigating various specimens in bioscience, material science, semi-conductor industry and etc. Recently, its applications are expanded to the bio-medical field along with the confocal endo-microscopy. Confocal fluorescence microscopy is an essential tool for modern biological research such as cellular and molecular biology. The increasing number of available fluorophore markers and labeling techniques allow the creation of increasingly complex multicolored samples; and then the confocal fluorescence microscopy is faced to measure the various fluorophores simultaneously. Multi-spectral fluorescence imaging techniques make use of the diversity of available markers to visualize different aspects of a specimen with specific fluorescent labels. Various spectral imaging microscopes have been proposed that combine spectrally decoding components such as prism or grating. Emission lights are spatially decoded as wavelengths and these lights are then simultaneously detected as selected wavelength bandwidths. In this way, the localization of several cellular proteins can be compared during processes such as cell division or secretion. The amount of information grows as the number of fluorophores increasing, leading to the need for complex quantitative analyses of the acquired data. However, fluorescence signals may not be completely separated into the different channels due to the effect of spectral bleed-through (SBT). SBT can occur when a specimen is multi-labeled and/or when there is significant auto-fluorescence. It is caused by the overlapping of fluorescence emission. As a result, the fluorophores cannot be distinguished, and a merged image can appear as co-localization if SBT exists. The SBT phenomenon can be reduced by narrowing the detection bandwidth, but this will also decrease the signal efficiency what is considered as a considerable trade-off in fluorescence applications. SBT can be eliminated using ...