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Routine and Special Techniques in Toxicologic Pathology
Published in Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard, Toxicologic Pathology, 2018
Daniel J. Patrick, Matthew L. Renninger, Peter C. Mann
Naturally occurring fluorescent proteins (such as green fluorescent protein) and mutated derivatives allow for the labeling of a wide spectrum of intracellular processes in living organisms in addition to in vitro applications (Lang et al. 2006). These proteins can be fused to virtually any protein in living cells using recombinant complementary DNA cloning technology. Advantages of fluorescent proteins over the traditional organic and inorganic fluorochromes described above include a response to a wider variety of biologic events and signals, an ability to specifically target fluorescent probes in subcellular compartments, an extremely low or absent photodynamic toxicity, and widespread compatibility with tissues and intact organisms.
Preclinical evaluation of multimodality probes
Published in Yi-Hwa Liu, Albert J. Sinusas, Hybrid Imaging in Cardiovascular Medicine, 2017
Fluorescent proteins, e.g., green fluorescent protein (GFP) and red fluorescent protein (RFP), are among the earliest and well-established optical reporter probes that have been widely used primarily for in vitro gene expression identification and postmortem histological verification. GFP derived from jellyfish Aequorea victoria has been used to identify the presence of transplanted bone marrow- and adipose tissue-derived MSCs in the infarcted mouse myocardium (van der Bogt et al. 2009). However, fluorescent reporter proteins have inherent limitations, i.e., significantly high autofluorescence background and scattered photon attenuation. Although fluorescence techniques, such as fluorescence-mediated molecular tomography, which permits tomographic reconstruction, improved the detection depth up to 1 mm (Graves et al. 2003), it is unlikely that such limited penetration depth will be enough to allow in vivo cardiac imaging in large animals or man.
Application of Nonlinear Microscopy in Life Sciences
Published in Lingyan Shi, Robert R. Alfano, Deep Imaging in Tissue and Biomedical Materials, 2017
Zdenek Svindrych, Ammasi Periasamy
The most prevalent are tunable mode-locked Ti:sapphire oscillators, delivering sub-100 fs pulses at around 80 MHz repetition rate, covering the range of 680–1080 nm with up to 3 W of average output power. With 2P excitation these cover the range of 350–550 nm. To cover longer excitation wavelengths (to efficiently excite red fluorophores and to take advantage of the lower scattering and absorption in tissues), pulsed lasers covering 680–1300 nm, based on Yb-doped fibers, became available recently. These longer wavelengths also allow for three-photon excitation of some fluorescent proteins.
A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila
Published in Journal of Neurogenetics, 2021
Oliver Kobler, Aliće Weiglein, Kathrin Hartung, Yi-chun Chen, Bertram Gerber, Ulrich Thomas
The whole-body inspection of the expression patterns permitted by our protocol might do more than just enable a more comprehensive characterization of the larval anatomy. Rather, such whole-body assessment could have implications for functional analyses by means of, for example, RNAi or optogenetic effectors, because driver lines with well-characterized expression in any one organ, such as the brain, may also drive expression in cells or tissues elsewhere in the larval body. If such expression remains unrecognized, interpretation of phenotypes may unwittingly go astray. The present clearing protocol, we hope, will help to avoid such pitfalls. Particularly when combined with the relatively fast imaging on a light sheet microscope, this should be possible on a routine basis. While the equipment used for the present study allowed us to visualize neurons at cellular resolution, higher resolution can be achieved through thinner light sheets (Pende et al., 2018). Moreover, newly developed fluorescent proteins (Matlashov et al., 2020), especially those emitting in the far-red range, may well complement the present two- and three-color approaches to multi-fluorescent imaging.
The discovery and development of oncolytic viruses: are they the future of cancer immunotherapy?
Published in Expert Opinion on Drug Discovery, 2021
Shunchuan Zhang, Samuel D Rabkin
A number of reporter genes with different imaging modalities have been encoded in OVs [79]. E.coli LacZ has been inserted into oHSVs (G207, G47Δ) and oVVs (JX-594, GLV-1h68) (Tables 1 and 2). In addition to histochemical detection, it provided a unique sequence to distinguish administered oHSV from patients’ endogenous HSV in clinical trials [80]. Fluorescent proteins can be optically imaged noninvasively, with the most commonly used being GFP [79]. An oHSV encoding GFP (NV1066) has been used diagnostically for intraoperative detection of lymph node metastases [79] and micro-metastatic disease in peritoneal washes from pancreatic cancer patients [81]. Similarly, oVV GLV-1h68 and oAd OBP-301 (TelomeScan), derived from OBP-301 (Table 1), have detected metastases in vivo preclinically [82,83]. OVs expressing luciferase (oVV, oHSV, oAd, oVSV) have been used to noninvasively follow virus replication and biodistribution in animal models [79]. The sodium iodine symporter (NIS) is a useful radiotracer for noninvasive imaging of OV (oAd, oHSV, oVV, oMV, oVSV) spread in mice and patients after administration of 123I or 99mTcO4, which are approved for human use, and SPECT/CT imaging [84]. A number of clinical trials of NIS-expressing OVs, MV-NIS and VSV-IFNβ-NIS (Tables 1 and 2), revealed a threshold for detection and high variability [84]. In addition, NIS facilitates 131I accumulation and cytotoxicity, and thus can be used for radiovirotherapy [85].
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
Genetically encoded fluorescent redox sensors have been developed for H2O2 and to monitor the redox state of GSSG/2GSH, NADH/NAD+, NADPH/NADP+, and TRXSS/TRXSH2 [84–86]. These redox indicators include redox-sensing fluorescent proteins such as Redoxfluor, Hyper, Peredox, and redox-sensitive yellow fluorescent protein (rxYFP) and green fluorescent proteins (roGFPs) [87]. RoGFP is more commonly used than rxYFP for two reasons: 1) unlike rxYFP, roGFP is ratiometric and thus provides a more quantitative measurement; 2) roGFP variants are pH resistant, whereas rxYFP is pH sensitive in the physiological pH range. Importantly, redox potentials differ in subcellular compartments and these reporters can be genetically targeted to specific subcellular compartments. RoGFPs are not directly oxidized by ROS, but equilibrate with the glutathione redox couple (GSH/GSSG) through the action of endogenous glutaredoxins (GRXs) [88]. For example, the redox sensor Grx1-roGFP, a fusion protein consisting of roGFP and human glutaredoxin 1, allows for ratiometric measurements and qualifies it as reporter for imaging of compound-mediated effects in real-time [89]. Importantly, this sensor was used in MTS, providing a proof of concept for redox-based imaging in 3D culture [90].