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Optical Methods for Diabetic Foot Ulcer Screening
Published in Andrey V. Dunaev, Valery V. Tuchin, Biomedical Photonics for Diabetes Research, 2023
Robert Bartlett, Gennadi Saiko, Alexandre Yu. Douplik
The methodology for imaging is a straightforward fluorescent process in which the fluorophore (ICG) is excited by a wavelength of 750–820 nm. ICG absorbs a portion of the light within the blood vessels of the target tissue. The absorbed light undergoes quantum transformation and is released as fluorescent emissions at longer wavelengths around 820–900 nm. The change from an excited state back to a ground state occurs within a nanosecond. Emission filters at the camera sensor are used to prevent mixing the unabsorbed excitation light (strong), which is reflected, and the fluorescing light (weak) [39].
Cherenkov and Scintillation Imaging Dosimetry
Published in Arash Darafsheh, Radiation Therapy Dosimetry: A Practical Handbook, 2021
Rachael L. Hachadorian, Irwin I. Tendler, Brian W. Pogue
Scintillation is a luminescence process – molecular excitation caused by absorption of ionizing radiation results in emission of light during material electronic relaxation. Numerous studies have shown that under appropriate conditions, scintillation signal can be directly proportional to dose [9]. Plastic scintillators used for dosimetry purposes are often composed of a bulk medium and organic fluorophores, where light production is enabled through fluorescence resonance energy transfer between these two components, with the medium being the conduit for radiation interaction and electron liberation, and the fluorophore being the electron/energy acceptor. A secondary fluorophore (a material that will absorb light at the lower end of the visible light spectrum) is sometimes added to shift the emission spectra to higher wavelengths [10]. Specifically, scintillators emit light when the excited organic fluorophore de-excites via a fluorescence, phosphorescence, or some alternate delayed fluorescence pathway [11].
Scanning Angle Interference Microscopy (SAIM)
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
Cristina Bertocchi, Timothy J. Rudge, Andrea Ravasio
SAIM is feasible for fixed samples as well as live-cell imaging. To avoid loss of accuracy we do recommend special attention to the choice of fluorophores. Although SAIM does not require special fluorophores, the fluorophores should have high photostability to minimize photobleaching during the imaging scanning sequence and should be bright with high quantum yield to provide a good signal-to-noise ratio. Among the fluorescent proteins and synthetic dyes compatible with SAIM, genetically encoded fluorescent proteins have the principal advantage of being small and suitable for live-cell imaging and are capable of achieving maximal labeling specificity, removing any possible problems associated with nonspecific labeling. Furthermore, they do not require fixation or permeabilization procedures that could affect cellular nanostructure. Some fluorescent proteins successfully used in SAIM include green fluorescent proteins such as EGFP and mEmerald, red fluorescent protein mCherry,33 and photoconverted tdEOS25 that has excellent brightness and photostability. In addition, chromobodies (generated by the fusion of a fluorescent protein to a nanobody34), a novel species of extremely small antibodies that are endogenously synthesized within cultured cells, could possibly be used to prepare samples for SAIM.
Quantification methods for viruses and virus-like particles applied in biopharmaceutical production processes
Published in Expert Review of Vaccines, 2022
Keven Lothert, Friederike Eilts, Michael W. Wolff
Recent developments in imaging technology led to the application of direct stochastic optical reconstruction microscopy, often referred to as super resolution microscopy/imaging (STORM) [158,159]. Here, individual fluorophores are activated and detected. By subsequently spotting a large number of individual fluorophore molecules, and by statistical evaluations, the nanostructures, to which the fluorophore are bound to, can be revealed. Hence, the method enables the characterization of macromolecules on a nanometer scale. This does not only aid in the observation of the virus particle surface composition, but also allows a quantification of these particles. For example, the method was used to quantify HIV-1 GaG-GFP VLPs and, simultaneously, nucleic acid contents, while lipid membrane compositions were identified using an appropriate labeling [160]. However, the device set-up is not trivial, and throughput is limited. Hence, rather than for a nanoplex quantification, the technique is applied for understanding fundamental mechanisms, such as the VLP assembly on the cell surfaces [160], for VLP quality assessments [161], the characterization of binding properties [162], or for the evaluation of heterogenicities in the surface protein composition [163,164].
A mechanistic review on the dissolution phase behavior and supersaturation stabilization of amorphous solid dispersions
Published in Drug Development and Industrial Pharmacy, 2021
P. Ashwathy, Akshaya T. Anto, M. S. Sudheesh
A variety of methods have been used to analyze phase behavior during LLPS. NMR spectroscopy has been used as a method to determine LLPS by characterizing the broad peak obtained due to molecular proximity during nanoaggregate formation [28]. The peak intensity is mainly determined by the concentration of drug in the dispersed molecular phase. When the colloidal phase is generated during LLPS, peak intensity remains constant on further increase in drug concentration. UV extinction coefficient method is an easy method to observe LLPS [32,40]. The wavelength at which the drug molecule shows no absorbance is selected. A sudden change in the extinction coefficient represents light scattering due to phase separation. LLPS has also been studied using steady-state fluorescence spectroscopy, by monitoring change in fluorescent intensity and wavelength maxima of an environment-sensitive fluorophore when it partitions into a colloidal rich phase during LLPS [29,40,48]. Fluorescence lifetime is an intrinsic property of a fluorophore, which has also been used to study LLPS [48]. It is the time during which a fluorophore remains in an excited state before returning to the ground state by emitting photons. The advantage of fluorescence lifetime is that it is largely independent of the method of measurement (e.g. wavelength of excitation and duration of exposure) and on the intensity and concentration of the fluorophore (under certain constraints).
Phenotypic analysis of extracellular vesicles: a review on the applications of fluorescence
Published in Journal of Extracellular Vesicles, 2020
Maria S. Panagopoulou, Alastair W. Wark, David J S Birch, Christopher D. Gregory
Fluorescence provides information about a range of processes, such as the interaction of fluorophores with the solvent or rotational freedom and molecular distances [21]. Here we discuss in detail the potential of fluorescence in the detection and nanometrology of EVs. However, there are also limitations in the use of fluorescence-based techniques for the analysis of biological material that are mainly attributed to interference from an intrinsic fluorescence background signal, the need for labelling, as well as photobleaching and quenching by oxygen. Interestingly, many of these drawbacks are ameliorated by the development of stable fluorophores such as quantum dots (QDs) to minimize photobleaching, as well as the use of far-red dyes to improve tissue penetration and eliminate autofluorescence interference. Indeed, in the case of Stimulated Emission Depletion microscopy (STED) microscopy, photobleaching has been put to good effect in providing the very principle upon which the technique is founded [20,22].