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[IEEE 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Munich, Germany (2019.6.23-2019.6.27)] 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Anisotropic Fluorescence Emission at the Surface of 1D-Photonic Crystal Biochips
摘要： Novel disposable optical biochips based on one-dimensional photonic crystals (1DPC) sustaining Bloch surface waves (BSW) are a desirable tool for the detection of several disease-related biomarkers. In particular, cancer biomarkers have gained considerable attention, due to the increasing demand for cancer treatment. Within this framework, the herein proposed optical biochips can quantify low concentration (sub ng/mL) of the ERBB2 breast cancer biomarker in biological complex matrices. To discriminate ERBB2 levels in several different cell lysate samples, we made use of a biosensing platform based on 1DPC biochips and on a reading instrument that can work in both a label-free and a fluorescence detection mode. Such combined configuration provides the advantage of complementary information and lower limit of detection (LoD) in the fluorescence mode [1]. In the label-free configuration the BSW excitation is achieved by a prism coupling system (Kretschmann-Raether configuration), like in the surface plasmon resonance (SPR) technique, resulting in a dip in the angular reflectance spectrum, shifting its position due to the refractive index changes [2]. The fluorescence mode is carried out by making use of dye labeled antibodies bound at the 1DPC surface. Coupling between the dye labels and a BSW results in strongly directional fluorescence emission. The advantages brought by the 1DPC, when compared to metal structures, are smaller energy dissipation and narrower resonances [3]. Presently, there is no study about photobleaching in experiments with BSW sustained by 1DPC, while it is evident that such phenomenon cannot be neglected in biosensing assays carried out close to the LoD, where quantitative and accurate information is needed. Here we report for the first time on cancer detection assays carried out with our platform, in which the trustworthiness of the output is put in doubt by photobleaching, which not only affects the overall emission intensity but also its polarization distribution via the TE and TM BSW modes provided by the 1DPC. The experimental data is interpreted by means of a theoretical model for the orientational distribution of dye labels over time, taking into account the density of the optical states of the 1DPC, photobleaching and rotational diffusion of surface bound emitters. The approach permits to model anisotropic fluorescence emission and to manage photobleaching effects in biosensing assays, leading to their correct interpretation.
关键词： one-dimensional photonic crystals,optical biochips,Bloch surface waves,cancer biomarkers,fluorescence emission,photobleaching
更新于2025-09-12 10:27:22
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AIP Conference Proceedings [AIP Publishing HIGH-ENERGY PROCESSES IN CONDENSED MATTER (HEPCM 2019): Proceedings of the XXVI Conference on High-Energy Processes in Condensed Matter, dedicated to the 150th anniversary of the birth of S.A. Chaplygin - Novosibirsk, Russia (3–5 April 2019)] HIGH-ENERGY PROCESSES IN CONDENSED MATTER (HEPCM 2019): Proceedings of the XXVI Conference on High-Energy Processes in Condensed Matter, dedicated to the 150th anniversary of the birth of S.A. Chaplygin - Investigation of the photobleaching effect on the measurement of laser-induced fluorescence excitation-emission matrices of biological tissues
摘要： Photobleaching effect on laser-induced fluorescence excitation-emission matrices of biological tissues was investigated. Rat brain tissues were used as an object of study. Tunable laser was used as an excitation source with wavelength in 210-290 nm range with 10 nm steps. The fluorescence spectra were measured depending on irradiation dose and wavelength. The photobleaching rate was estimated as an absorbed radiation dose when peak fluorescence intensity decreases 2.7 times. The above level of intensity decrease is achieved at absorbed dose of 58 mJ/cm2 for the 210 nm wavelength, and at 2550 mJ/cm2 for 290 nm. The highest absorbed dose (4333 mJ/cm2) and respectively the lowest bleaching rate falls on 250 nm. It had been shown that higher photobleaching rate at shorter wavelengths in general has negligible effect on the excitation-emission matrices measurements.
关键词： excitation-emission matrices,laser-induced fluorescence,photobleaching,biological tissues
更新于2025-09-11 14:15:04
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Maximizing Explanatory Power in Stereological Data Collection: A Protocol for Reliably Integrating Optical Fractionator and Multiple Immunofluorescence Techniques
摘要： With the promise of greater reliability and replicability of estimates, stereological techniques have revolutionized data collection in the neurosciences. At the same time, improvements in immunohistochemistry and fluorescence imaging technologies have facilitated easy application of immunofluorescence protocols, allowing for isolation of multiple target proteins in one tissue sample. Combining multiple immunofluorescence labeling with stereological data collection can provide a powerful tool to maximize explanatory power and efficiency, while minimizing tissue use. Multiple cell classes, subtypes of larger populations, or different cell states can be quantified in one case and even in one sampling run. Here, we present a protocol integrating stereological data collection and multiple immunofluorescence using commonly employed widefield epifluorescence filter sets, optimized for blue (DAPI), green (FITC), and far red (CY5) channels. Our stereological protocol has been designed to accommodate the challenges of fluorescence imaging to overcome limitations like fixed filter sets, photobleaching, and uneven immunolabeling. To enhance fluorescence signal for stereological sampling, our immunolabeling protocol utilizes both high temperature antigen retrieval to improve primary antibody binding and secondary antibodies conjugated to optimally stable fluorophores. To illustrate the utility of this approach, we estimated the number of Ctip2 immunoreactive subcerebral projection neurons and NeuN immunoreactive neurons in rat cerebral cortex at postnatal day 10. We used DAPI (blue) to define the neocortex, anti-NeuN (far red) to identify neurons, and co-labeling of anti-Ctip2 (green) and anti-NeuN (far red) to isolate only subcerebral projection neurons. Our protocol resulted in estimates with low sampling error (CE < 0.05) and high intrarater reliability (ICC > 0.98) that fall within the range of published values, attesting to its efficacy. We show our immunofluorescence techniques can be used to reliably identify other cell types, e.g., different glial cell classes, to highlight the broader applications of our approach. The flexibility of the technique, increasingly reduced costs of fluorescence technologies, and savings in experimental time and tissue use make this approach valuable for neuroscientists interested in incorporating stereology to ask precise neurophysiological and neuroanatomical questions.
关键词： photobleaching,astrocytes,NeuN,microglia,oligodendrocytes,Ctip2,optical fractionator,neurons
更新于2025-09-09 09:28:46