- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Quantitative imaging of membrane micropolarity in living cells and tissues by spectral phasors analysis
摘要： Intracellular micropolarity is essential in several metabolic processes, as it controls membrane permeability, fluxes of molecules and energy. Here we describe a method for the determination of the micropolarity in living cells using spectral confocal microscopy. The method is based on a phasor analysis of spectrally resolved images of live cells, labelled with the solvatochromic probe Nile Red. An application is provided to extract a polarity profile from the acquired Spectral datasets, which represent the contribution of hyperpolar, polar and non-polar lipids, and to generate a micropolarity map at submicrometric spatial resolution. A metabolic parameter, representing a quantitative index of the fatty acid-triacylglycerol turnover, is also furnished. This method allows a functional profiling of cells and tissues and the detection of metabolic imbalances between lipid storage and usage.
关键词： Membranes micropolarity,Nile Red,Fatty acids,Triglycerides,Lipid droplets,Confocal microscopy,Metabolic imaging,Spectral phasors,Lipids
更新于2025-09-23 15:22:29
-
Super-resolution imaging of self-assembled nanocarriers using quantitative spectroscopic analysis for cluster extraction
摘要： Self-assembled nanocarriers have inspired a range of applications for bioimaging, diagnostics, and drug delivery. Non-invasive visualization and characterization of nanocarriers are important for understanding their structure to function relationship. However, quantitative visualization of nanocarriers in the sample’s native environment remains challenging using existing technologies. Single-molecule localization microscopy (SMLM) has the potential to provide both high-resolution visualization and quantitative analysis of nanocarriers in their native environment. However, non-specific binding of fluorescent probes used in SMLM can introduce artifacts, which impose challenges in quantitative analysis of SMLM images. We showed the feasibility of using spectroscopic point accumulation for imaging in nanoscale topography (sPAINT) to visualize self-assembled polymersomes (PS) with molecular specificity. Furthermore, we analyzed the unique spectral signatures of Nile Red (NR) molecules bound to the PS to reject artifacts from non-specific NR bindings. We further developed quantitative spectroscopic analysis for cluster extraction (qSPACE) to increase the localization density by 4-fold compared to sPAINT; thus, reducing variations in PS size measurements to less than 5%. Finally, using qSPACE we quantitatively imaged PS at various concentrations in aqueous solutions with ~20-nm localization precision and 97% reduction in sample misidentification relative to conventional SMLM.
关键词： nanocarriers,Nile Red,super-resolution imaging,single-molecule localization microscopy,spectroscopic analysis,polymersomes
更新于2025-09-23 15:19:57
-
Live cell imaging of <i>Plasmodiophora brassicae</i> -host plant interactions based on a two-step axenic culture system
摘要： Plasmodiophora brassicae, a parasitic protist, induces club‐shaped tumor‐like growth of host Brassicas roots and hypocotyls after infection. Due to its soil‐borne nature and intracellular, biotrophic parasitism the infection biology and early pathogenesis remains in doubt. In this study, we have established a new protocol, based on a two‐step axenic culture of P. brassicae with its host tissues, for easy and in planta observation of cellular interactions between P. brassicae and host plants: first, coculture of P. brassicae with infected canola root tissues, on growth‐medium plates, enables the propagation of P. brassicae that serves as pure inoculum for pathogenicity assays, and second, the pure inoculum is subsequently used for pathogenicity tests on both canola and Arabidopsis seedlings grown on medium plates in Petri dishes. During the first axenic culture, we established a staining protocol by which the pathogen was fluorescently labeled with Nile red and calcofluor white, thus allowing in planta observation of pathogen development. In the pathogenicity assays, our results showed that axenic cultures of P. brassicae, in calli, remains fully virulent and completes its life cycle in both canola and Arabidopsis roots grown in Petri dishes. Combining visualization of fluorescent probe‐labeled P. brassicae structures with fluorescent protein tagging of Arabidopsis cellular components, further revealed dynamic responses of host cells at the early stages of P. brassicae infection. Thus, established protocols for in planta detection of P. brassicae structures and the live cell imaging of P. brassicae—Arabidopsis interactions provide a novel strategy for improving our detailed knowledge of P. brassicae infection in host tissues.
关键词： DAPI,Nile red,resting spores,calcofluor white,canola,primary plasmodia,axenic culture,Plasmodiophora brassicae,Arabidopsis
更新于2025-09-10 09:29:36
-
Novel DRAQ5?/SYTOX? Blue Based Flow Cytometric Strategy to Identify and Characterize Stem Cells in Human Breast Milk
摘要： Human breast milk could be an important stem cell source for the development of newborn and preterm infants, but quantitative data on the stem cell content in breast milk at various gestational stages are needed to determine the clinical value of breast milk as a source of stem cells. Breast milk also contains milk fat globules, lipid droplets of different sizes, debris and dead cells and these components hamper flow cytometry analysis of human breast milk samples. Here, we originally used standard protocols for flow cytometry to characterize cell populations in human breast milk but failed to discriminate between cells and noncellular components. We then applied a centrifugation protocol to separate cream and skim milk from the cell-containing pellet and used a novel staining protocol with DRAQ5? and SYTOX? blue dye as well as antibodies to characterize cells within the pellet fraction. Flow cytometry analysis identified viable DRAQ5?+/SYTOX? Blue? cells and determined the content of CD11b+ monocytes and TRA-1-81+ putative stem cells in human breast milk samples. Hence, we developed a novel and reliable flow cytometry based-approach to quantify subpopulation of cells in human breast milk with a high content of milk fat globules, lipid droplets, and particles. This approach will improve the identification and quantification of breast milk cells and allow standardizing the flow cytometry-based evaluation of the stem cell content.
关键词： Nile red,viability,DRAQ5,flow cytometry,TRA-1-81,CD11b,SYTOX blue,human breast milk cells
更新于2025-09-10 09:29:36