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Optical Nanoprobes for Diagnosis
Published in D. Sakthi Kumar, Aswathy Ravindran Girija, Bionanotechnology in Cancer, 2023
R. G. Aswathy, D. Sakthi Kumar
Although fluorescent dyes have significant applications in biomedical imaging, the present status of them for visualizing biological tissue is seriously restricted, owing to autoflourescence stemming from fluorescent dyes. The design and development of next-generation fluorescence probes based on nanoscience and technology is crucial in current technology. With the advancement of nanotechnology, semiconductor QDs have developed as a substitute and favorable fluorescent nano labels than organic dyes [210]. QDs have been studied in FRET analysis, imaging cells and tissues, and gene/drug delivery [211]. The rising concerns on chemical instability and intrinsic cytotoxicity of QDs have restricted the application for biological usage [212]. The major drawback of QDs includes the difficulty in deep tissue imaging, low signal-to-noise ratio (SNR), and possible impairment of biomolecules due to long-term irradiation. Hence, there is an increasing need for the development of more proficient biological labels to overcome the above-explained limitations.
In Vivo Observations of Tumor Blood Flow
Published in Hans-Inge Peterson, Tumor Blood Circulation: Angiogenesis, Vascular Morphology and Blood Flow of Experimental and Human Tumors, 2020
These effects can, however, be completely avoided when one uses water-soluble dyes that do not bind in the body. Fluorescent dyes are generally preferred because they combine the advantages of a high contrast rate with a high (light) yield at very low concentrations. This means that for ophthalmological examinations fluorescent angiography is generally used.23 Fluorescein does not bind to proteins, and therefore, the bolus of the fluorescein solution, used for angiography, is readily excreted. Moreover, the dye has a low toxicity, which explains its extensive use in ophthalmology.
Medication: Nanoparticles for Imaging and Drug Delivery
Published in Harry F. Tibbals, Medical Nanotechnology and Nanomedicine, 2017
Imaging agents such as fluorescent dye-doped silica nanoparticles, quantum dots, and gold nanoparticles overcome many of the limitations of conventional contrast agents (organic dyes) such as poor photostability, low quantum yield, and low in vitro and in vivo stability. For these reasons, nanoparticles with resonance in the visible and ultraviolet are a viable alternative to fluorescent dyes for cellular imaging for diagnosis of cancer [41,42]. In one study, 30 nm gold nanoparticles conjugated with cancer antibodies were compared to the same antibodies with fluorescent dye reporters. The emission intensity of the gold nanoparticles was about 10 times stronger than that of the autofluorescence of the Karpas-299 line of cancer cells at the same excitation power, and was significantly stronger than that for fluorescent dyes. The nanoparticles also had higher photostability than the dyes [43].
Flow cytometry can reliably capture gut microbial composition in healthy adults as well as dysbiosis dynamics in patients with aggressive B-cell non-Hodgkin lymphoma
Published in Gut Microbes, 2022
Maren Schmiester, René Maier, René Riedel, Pawel Durek, Marco Frentsch, Stefan Kolling, Mir-Farzin Mashreghi, Robert Jenq, Liangliang Zhang, Christine B. Peterson, Lars Bullinger, Hyun-Dong Chang, Il-Kang Na
FCM represents microbes’ morphologies by detecting light scatter from cell shape and subcellular structures. Fluorescent dyes targeting various biomolecules, such as nucleic acids, can be added for further characterization. In contrast to sequencing analysis, the technology allows for multivariate phenotyping, aggregating morphological and physiological characteristics of thousands of single cells into a fingerprint of the studied microbial community.12 Cytometric fingerprints can be used to calculate phenotypic diversity metrics akin to their well-established taxonomic counterparts obtained from sequencing analysis, with alpha diversity describing the richness and/or evenness of the examined microbial community and beta diversity assessing the differences in community composition between samples. Cytometric profiles have been used to study changes in microbial composition in various ecological settings and murine models,13–15 but the technology has rarely been applied to human samples to date.16,17 As computational tools for the analysis of phenotypic microbiome profiles obtained from FCM are becoming more and more refined, even encompassing species recognition, they are paving the way for an exciting and accessible diagnostic tool with great potential for routine clinical application.18,19
Mapping densely packed αIIbβ3 receptors in murine blood platelets with expansion microscopy
Published in Platelets, 2022
Hannah S. Heil, Max Aigner, Sophia Maier, Prateek Gupta, Luise M.C. Evers, Vanessa Göb, Charly Kusch, Mara Meub, Bernhard Nieswandt, David Stegner, Katrin G. Heinze
Figure 1A shows the respective deconvolved confocal images of αIIbβ3 receptors on unexpanded versus 4x and 10x expanded platelets. The αIIbβ3 receptors are targeted by monoclonal antibodies carrying either an Alexa Fluor 594 (Figure 1, magenta) or an Alexa Fluor 488 marker (Figure 1, green). Thus, this assay is designed to show low colocalization if resolution is sufficient and clustering is low. For unexpanded platelets, however, low image resolution leads to a linear relationship in the intensity scatter plot for the two color channels (Figure 1B). Such largely overlapping color signals only provide a meaningless high degree of – potentially false-positive – co-localization. In contrast, with 4x and 10x expanded platelets this linear relationship vanishes and colocalization analysis becomes discernible for different scenarios (Figure 1C, D). Unfortunately, the resolution gain is accompanied by a loss in contrast due to reduced signal-to-noise ratio (Figure 1E). Moreover, the signal retention varies for different fluorescent dyes. While both antibodies were decorated with a similar number of fluorophores (see supplemental Table I), the signal retention is different for each color (Figure 1F, lower signal retention in the green channel than in the magenta channel). Low contrast and different degree of receptor visibility between channels require a careful preselection of the colocalization modality.
High-throughput screening in multicellular spheroids for target discovery in the tumor microenvironment
Published in Expert Opinion on Drug Discovery, 2020
Blaise Calpe, Werner J. Kovacs
Intracellular pH homeostasis is crucial for the maintenance of cellular metabolism and signaling. High glycolytic flux in cancer cells leads to acidification of the milieu as a result of lactate secretion while intracellular pH (pHi) becomes more alkaline [71]. Importantly, this reverse pH gradient is thought to contribute to drug resistance and metastasis [72]. Several pH-sensitive fluorescent dyes have been developed, the most widely used being derivatives of fluorescein and benzoxanthene [73]. These dyes can be utilized to measure the pH of the extracellular milieu [74] and are compatible with MTS [75]. On the other hand, genetically encoded pH-sensitive sensors are better suited for intracellular pH measurement. Tantama et al. developed pHRed, a genetically encoded pH sensor engineered by mutagenesis of the red fluorescent protein mKeima and the first ratiometric single-protein red fluorescent sensor of pH [76]. The pHRed ratio response is insensitive to oxidative stress (H2O2), temperature (21–37°C), and different ion concentrations (K+, Na+, Cl−, Mg2+, Ca2+, HCO3−) [76]. pHRed also allows the simultaneous imaging of intracellular ATP and pH because of its spectral compatibility with the GFP-based ATP sensor Perceval [77]. Shirmanova and colleagues successfully used the stably expressed fluorescent pH-sensitive ratiometric (dual-excitation) indicator SypHer2 [78] to image intracellular pH in MTS and xenografts [79].