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The Impact of Photobleaching on Microarray Analysis
摘要： DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner’s laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube’s voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results.
关键词： photobleaching,microarray,cyanine dye,bioanalytics,fluorophore,bioinformatics,DNA
更新于2025-09-23 15:22:29
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Luminescence lifetime encoding in time-domain flow cytometry
摘要： Time-resolved flow cytometry represents an alternative to commonly applied spectral or intensity multiplexing in bioanalytics. At present, the vast majority of the reports on this topic focuses on phase-domain techniques and specific applications. In this report, we present a flow cytometry platform with time-resolved detection based on a compact setup and straightforward time-domain measurements utilizing lifetime-encoded beads with lifetimes in the nanosecond range. We provide general assessment of time-domain flow cytometry and discuss the concept of this platform to address achievable resolution limits, data analysis, and requirements on suitable encoding dyes. Experimental data are complemented by numerical calculations on photon count numbers and impact of noise and measurement time on the obtained lifetime values.
关键词： lifetime-encoded beads,multiplexing,Time-resolved flow cytometry,bioanalytics,time-domain measurements
更新于2025-09-11 14:15:04
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Label-Free Bioanalysis Based on Low-Q Whispering Gallery Modes: Rapid Preparation of Microsensors by Means of Layer-by-Layer Technology
摘要： Low-Q-whispering gallery modes (low-Q-WGM) can be used for label-free detection of interactions between biomolecules, measuring their binding and release kinetics or for analysis of changes in the medium in real-time. The main advantage of the low-Q-WGM approach over other label-free methods is the possibility of measurements in small cavities as the method uses microparticles down to 6 μm as sensors. Commercially available dye-doped microparticles that are used as low-Q-WGM sensors exhibit several drawbacks. Therefore, alternative particle types are developed and optimized as low-Q-WGM sensors. First, dye-doped particles made of different materials are screened. The most critical parameter for WGM performance is the refractive index (RI) of sensor particles. Furthermore, surface roughness of particles, determined by scanning electron microscopy and atomic force microscopy, affects their performance as WGM microsensors. In the second test, fluorescent dyes immobilized on nonfluorescent particles by means of nanometer thick layer-by-layer (LbL) films are shown to generate a strong WGM signal. The LbL-coated particles show remarkably less background fluorescence than dye-doped particles and are easier to prepare. Finally, this article proposes rapid preparation methods for WGM microparticle sensors based on various parameters such as material type, RI, surface roughness, and number of coated polymer layers.
关键词： fluorescent microparticles,whispering gallery mode,layer-by-layer coating,label-free bioanalytics
更新于2025-09-09 09:28:46