Photochromic Fluorescent Probe Strategy for the Super-resolution Imaging of Biologically Important Biomarkers.
Chai Xianzhi,Han Hai-Hao,Sedgwick Adam C,Li Na,Zang Yi,James Tony D,Zhang Junji,Hu Xi-Le,Yu Yang,Li Yao,Wang Yan,Li Jia,He Xiao-Peng,Tian He
Journal of the American Chemical Society
Here, we report a β-galactosidase (β-Gal)-responsive photochromic fluorescent probe, , that was designed to prebind to human serum albumin (HSA) to form the probe/protein hybrid, . The formation of led to an increase in fluorescence emission (520 nm) corresponding to the binding of the fluorescent naphthalimide unit with HSA. In addition, this enabled visualization of the spiropyran fluorescence emission in aqueous media. Our probe/protein hybrid approach afforded a unique imaging platform with enhanced cell permeability and solubility that was capable of visualizing the cellular uptake of before its activation by β-Gal. The β-Gal-mediated cleavage of the galactose unit within the hybrid resulted in the formation of and an increase in red fluorescence emission (620 nm). The resultant merocyanine unit was then able to undergo photoisomerization (merocyanine ↔ spiropyran) to facilitate STORM (i.e., stochastic optical reconstruction microscopy) imaging with minimal phototoxicity and excellent photostability/reversibility. Using STORM, was able to determine the subcellular distribution of β-Gal activity between cell lines with nanoscale precision. We believe that this system represents a versatile imaging platform for the design of photochromic fluorescent probes suitable for illuminating the precise location of disease-specific biomarkers in various cellular processes.
10.1021/jacs.0c05379
Brief guide to senescence assays using cultured mammalian cells.
Molecules and cells
Cellular senescence is a crucial biological process associated with organismal aging and many chronic diseases. Here, we present a brief guide to mammalian senescence assays, including the measurement of cell cycle arrest, change in cellular morphology, senescence-associated β-galactosidase (SA-β-gal) staining, and the expression of senescence-associated secretory phenotype (SASP). This work will be useful for biologists with minimum expertise in cellular senescence assays.
10.1016/j.mocell.2024.100102
β-Galactosidase-Activatable Fluorescent and Photoacoustic Imaging of Tumor Senescence.
Analytical chemistry
β-Galactosidase (β-gal) is the gold standard marker of cellular senescence, which is linked with various age-related diseases. Therefore, it is essential to develop more excellent probes that can real-time monitor β-gal activity in cellular senescence . Fluorescent/photoacoustic (FL/PA) dual-modal imaging possesses excellent sensitivity and spatial resolution. To our knowledge, there has been no tumor-targeted FL/PA probe to image cellular senescence by monitoring the activity of β-gal . Therefore, we developed a tumor-targeted FL/PA probe () for β-gal-activatable imaging of tumor senescence. without tumor-targeted biotin is used as a control probe. is superior to due to the higher kinetic parameter of than . Moreover, biotin could help enter and accumulate in tumor cells with higher FL/PA signal. In detail, or could image senescent tumor cells with 4.6-fold or 3.5-fold FL enhancement and 4.1-fold or 3.3-fold PA enhancement. or could image tumor senescence with 2.9-fold or 1.7-fold FL enhancement and 3.8-fold or 1.3-fold PA enhancement. We envision that will be applied for FL/PA imaging of tumor senescence in clinic.
10.1021/acs.analchem.3c01656
Fidelity-oriented fluorescence imaging probes for beta-galactosidase: From accurate diagnosis to precise treatment.
Biotechnology advances
Beta-galactosidase (β-gal), a typical glycosidase catalyzing the hydrolysis of glycosidic bonds, is regarded as a vital biomarker for cell senescence and cancer occurrence. Given the advantages of high spatiotemporal resolution, high sensitivity, non-invasiveness, and being free of ionizing radiations, fluorescent imaging technology provides an excellent choice for in vivo imaging of β-gal. In this review, we detail the representative biotech advances of fluorescence imaging probes for β-gal bearing diverse fidelity-oriented improvements to elucidate their future potential in preclinical research and clinical application. Next, we propose the comprehensive design strategies of imaging probes for β-gal with respect of high fidelity. Considering the systematic implementation approaches, a range of high-fidelity imaging-guided theragnostic are adopted for the individual β-gal-associated biological scenarios. Finally, current challenges and future trends are proposed to promote the next development of imaging agents for individual and specific application scenarios.
10.1016/j.biotechadv.2023.108244
A Fully-Automated Senescence Test (FAST) for the high-throughput quantification of senescence-associated markers.
bioRxiv : the preprint server for biology
Cellular senescence is a major driver of aging and age-related diseases. Quantification of senescent cells remains challenging due to the lack of senescence-specific markers and generalist, unbiased methodology. Here, we describe the Fully-Automated Senescence Test (FAST), an image-based method for the high-throughput, single-cell assessment of senescence in cultured cells. FAST quantifies three of the most widely adopted senescence-associated markers for each cell imaged: senescence-associated β-galactosidase activity (SA-β-Gal) using X-Gal, proliferation arrest via lack of 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and enlarged morphology via increased nuclear area. The presented workflow entails microplate image acquisition, image processing, data analysis, and graphing. Standardization was achieved by i) quantifying colorimetric SA-β-Gal via optical density; ii) implementing staining background controls; iii) automating image acquisition, image processing, and data analysis. In addition to the automated threshold-based scoring, a multivariate machine learning approach is provided. We show that FAST accurately quantifies senescence burden and is agnostic to cell type and microscope setup. Moreover, it effectively mitigates false-positive senescence marker staining, a common issue arising from culturing conditions. Using FAST, we compared X-Gal with fluorescent CFDG live-cell SA-β-Gal staining on the single-cell level. We observed only a modest correlation between the two, indicating that those stains are not trivially interchangeable. Finally, we provide proof of concept that our method is suitable for screening compounds that modify senescence burden. This method will be broadly useful to the aging field by enabling rapid, unbiased, and user-friendly quantification of senescence burden in culture, as well as facilitating large-scale experiments that were previously impractical.
10.1101/2023.12.22.573123
Biomarkers of Cellular Senescence and Aging: Current State-of-the-Art, Challenges and Future Perspectives.
Advanced biology
Population aging has increased the global prevalence of aging-related diseases, including cancer, sarcopenia, neurological disease, arthritis, and heart disease. Understanding aging, a fundamental biological process, has led to breakthroughs in several fields. Cellular senescence, evinced by flattened cell bodies, vacuole formation, and cytoplasmic granules, ubiquitously plays crucial roles in tissue remodeling, embryogenesis, and wound repair as well as in cancer therapy and aging. The lack of universal biomarkers for detecting and quantifying senescent cells, in vitro and in vivo, constitutes a major limitation. The applications and limitations of major senescence biomarkers, including senescence-associated β-galactosidase staining, telomere shortening, cell-cycle arrest, DNA methylation, and senescence-associated secreted phenotypes are discussed. Furthermore, explore senotherapeutic approaches for aging-associated diseases and cancer. In addition to the conventional biomarkers, this review highlighted the in vitro, in vivo, and disease models used for aging studies. Further, technologies from the current decade including multi-omics and computational methods used in the fields of senescence and aging are also discussed in this review. Understanding aging-associated biological processes by using cellular senescence biomarkers can enable therapeutic innovation and interventions to improve the quality of life of older adults.
10.1002/adbi.202400079
Chromo-fluorogenic probes for β-galactosidase detection.
Lozano-Torres Beatriz,Blandez Juan F,Sancenón Félix,Martínez-Máñez Ramón
Analytical and bioanalytical chemistry
β-Galactosidase (β-Gal) is a widely used enzyme as a reporter gene in the field of molecular biology which hydrolyzes the β-galactosides into monosaccharides. β-Gal is an essential enzyme in humans and its deficiency or its overexpression results in several rare diseases. Cellular senescence is probably one of the most relevant physiological disorders that involve β-Gal enzyme. In this review, we assess the progress made to date in the design of molecular-based probes for the detection of β-Gal both in vitro and in vivo. Most of the reported molecular probes for the detection of β-Gal consist of a galactopyranoside residue attached to a signalling unit through glycosidic bonds. The β-Gal-induced hydrolysis of the glycosidic bonds released the signalling unit with remarkable changes in color and/or emission. Additional examples based on other approaches are also described. The wide applicability of these probes for the rapid and in situ detection of de-regulation β-Gal-related diseases has boosted the research in this fertile field.
10.1007/s00216-020-03111-8