Cells are the smallest unit of living organisms. Unicellular organisms consist of one cell, whereas multicellular organisms contain many cell types. Imaging cells allows information to be gathered about cells and how they interact with their environment. With this information, researchers can better understand a cell's structure, function, phenotype, and how it contributes to the whole organism.
Static cell imaging remains useful as preserved cells provide cellular information at a particular moment in time, and imaging can take place long after sample preparation. In contrast, live cell imaging monitors dynamic cellular processes, which yields information on the sequence of cellular events.
Cellular stains for live cell imaging
Fluorescent probes or fluorescent-labelled recombinant proteins can identify subcellular organelles and cell structures in live cell imaging. Using fluorescent-labelled recombinant proteins involves transfecting cells, which involves additional experimental steps with several cell types remaining difficult to transfect.
The alternative to transfection is using permeable fluorescent probes that cross the cell membrane to enter cells. LubioScience distribute a range of live cell imaging probes from Spirochrome for intracellular and membrane staining.
Image 1: HeLa cell expressing mcherry-H2B (red) stained with SiR-tubulin (green). Data collected by confocal imaging. Courtesy of Daniel Gerlich and Claudia Blaukopf, Institute of Molecular Biotechnology, Vienna.
How does intracellular and membrane staining work?
The first generation of Spirochrome products was based on silicon–rhodamine (SiR) probes. The SiR probes are directed to specific cellular targets by coupling the probes to ligands via linkers using different labelling techniques. The SiR structure is ideal for live cell imaging.
SiR derivatives exist in equilibrium between a non-fluorescent spirolactone closed off-state and a fluorescent open on-state containing an equal number of positive and negative charges (zwitterion). The hydrophobic nature of the non-fluorescent, off-state allows the dye to be cell permeable whilst the on-state imparts fluorescence to the probe.
The fluorescent SiR probes are biocompatible as they are excited and emit in the near-infrared region. This minimizes phototoxicity in time-dependent long-exposure microscopic techniques such as those employed in live cell imaging, which makes them ideal for high- and super-resolution imaging (Lukinavičius et al., 2013).
In addition, SiR probes are easy to use — no washing steps needed —they provide a high signal-to-noise ratio. Binding of the probe to the target shifts the equilibrium to the fluorescent zwitterion form, increasing fluorescence (Lukinavičius et al., 2014).
One of the first probes, SiR tubulin, uses the anticancer drug approved by the FDA, docetaxel, to target microtubules. The SiR-actin probe conjugates a SiR probe to ligand desbromo-desmethyl-jasplakinolide, which binds to filamentous actin (SiR actin).
Using reagents to image the cytoskeleton of difficult-to-transfect cells
LubioScience distribute several SiR probes. The SiR actin and SiR tubulin probes are ideal for staining the cytoskeleton of various cell types from different organisms. SiR probes work in cells that are difficult to transfect, such as human primary dermal fibroblasts (Lukinavičius et al., 2014). Red blood cells are also considered very difficult to transfect. The SiR probe's far-red excitation and emission wavelengths do not interfere with haemoglobin's absorbance spectrum. For this reason, SiR probes have been instrumental in staining the actin cytoskeleton of red blood cells from whole-blood samples (Lukinavičius et al., 2014). Also, SiR actin probes stain the actin cytoskeleton of whole embryo cultures (Schmitz-Elbers et al., 2021).
Image 2: Embryonic development and gradual increase of SiR-actin fluorescence in chick embryos cultured ex ovo, representative images. Time-points indicate incubation time with SiR-actin. Anterior is always to the left. Scale bars, 1 mm (A,B,D,E), 250 µm (I,K). (A) Darkfield (DF) microscopic time-lapse frames of 3-somite stage embryo (HH7), cultured in presence of 240 nM SiR-actin. Development progressed up to the 10-somite stage (HH10) during 12 h of incubation. (B) Corresponding widefield fluorescence (WF) time-lapse frames of embryo shown in (A). Dashed lines indicate line region of interest (ROI) used to measure fluorescence intensities of embryonic midline (white) and surrounding structures (background, cyan). (Schmitz-Elbers et al., 2021).
SiR probes label DNA, lysosomes or can be conjugated to your molecule of interest
The SiR probes can label DNA (SiR-DNA) and lysosomes (SiR-Lysosome). If specific applications are needed, Spirochrome provide SiR reagents for conjugating peptides, proteins, nucleic acids or small molecules to SiR probes using different conjugation chemistries.
SiR700 probes are available for DNA, actin, microtubules and lysosomes. The difference between SiR700 probes and SiR is the shift in the absorption and emission by approximately 50 nm. This allows dual colour imaging of live cells using SiR and SiR700 probes following careful setup of microscope optics and settings.
Imaging SPY probes using different fluorescence channels
SPY™ probes are also live cell imaging probes. Like the SiR probes, the SPY probes are bright, cell-permeable, photostable and exhibit no cytotoxicity. In addition to probes in the far-red channel, SPY probes are available in green, orange, and red channels.
SPY probes targeting DNA have an advantage over the popular Hoechst nucleic acid stain, as Hoechst is cytotoxic, even below the recommended concentration. In contrast, SPY-DNA probes show no toxicity at a range of working concentrations.
Leica Microsystems recently employed SiR-actin and SPY555-tubulin to detect multiple targets simultaneously using super-resolution microscopy. (Alvarez et al., 2021)
Image 3: Tracking fast spatial distribution changes of multiple subcellular structures in live cells helps you better understand their localization changes under physiological and pathological conditions.
Live-cell TauSTED showing three distinct cellular structures in U2OS cells. Labels were used for actin (SiR-actin, glow), microtubules (SPY555-tubulin, cyan), and membranes (WGA-CF4 88, green). TauSTED was performed with three dif ferent STED lines (775, 660, and 592 nm, respectively). Scale bar is 5 μm. SiR and SPY probes are available from Spirochrome. (leica-microsystems.com)
Measuring membrane tension using fluorescent Flipper-TR®
Changes constantly occur at the plasma membrane due to external stimuli at the cell surface or internal processes, leading to changes in membrane tension and consequent cellular processes, such as cell migration and membrane trafficking. Quantifying plasma membrane tension involves specialist equipment (atomic force spectroscopy and optical or magnetic tweezers) as well as the expertise needed to use the equipment successfully. Spirochrome's Flipper-TR® probes have made measuring membrane tension accessible to researchers.
Spirochrome's Flipper-TR probes spontaneously insert into the plasma membrane and fluoresce when they do. The twist angle and polarization between two twisted dithienothiophenes in the probe's structure are sensitive to membrane tension. These structural changes modify how long the fluorescent molecule remains excited before returning to the ground state – the fluorescence lifetime change. Fluorescence-lifetime imaging microscopy measures lifetime changes, which can be correlated to membrane tension using a calibration procedure. In a rigid state, the emission lifetime is longer, and the dithienothiophenes align. In a relaxed state, the lifetime is shorter, and the dithienothiophenes are twisted.
A recent study published in Nature Cell Biology (Claude-Taupin et al., 2021) added Flipper-TR probe (SC020) to the cell culture medium and incubated cells for 30 mins. The Flipper-TR probe assessed the degree of damage to the plasma membrane in different cellular contexts and under different conditions.
Flipper-TR responds to membrane tension by planarisation of the mechanophore
Learn more about LubioScience's range of live cell imaging probes
LubioScience are a trusted provider of live cell imaging probes. We would be happy to help you choose the ideal reagent for your research application.
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Spirochrome - live imaging probes
Spirochrome offers a broad range of fluorescent probes for live cell bioimaging. These include actin, tubulin, DNA membrane tension, cytoskeleton and lysosome probes.
References
Claude-Taupin, A., Jia, J., Bhujabal, Z., Garfa-Traoré, M., Kumar, S., da Silva, G. P. D., Javed, R., Gu, Y., Allers, L., Peters, R., Wang, F., da Costa, L. J., Pallikkuth, S., Lidke, K. A., Mauthe, M., Verlhac, P., Uchiyama, Y., Salemi, M., Phinney, B., … Deretic, V. (2021). ATG9A protects the plasma membrane from programmed and incidental permeabilization. Nature Cell Biology, 23(8). https://doi.org/10.1038/s41556-021-00706-w
Lukinavičius, G., Reymond, L., D’Este, E., Masharina, A., Göttfert, F., Ta, H., Güther, A., Fournier, M., Rizzo, S., Waldmann, H., Blaukopf, C., Sommer, C., Gerlich, D. W., Arndt, H. D., Hell, S. W., & Johnsson, K. (2014). Fluorogenic probes for live-cell imaging of the cytoskeleton. Nature Methods, 11(7). https://doi.org/10.1038/nmeth.2972
Lukinavičius, G., Umezawa, K., Olivier, N., Honigmann, A., Yang, G., Plass, T., Mueller, V., Reymond, L., Corrêa, I. R., Luo, Z. G., Schultz, C., Lemke, E. A., Heppenstall, P., Eggeling, C., Manley, S., & Johnsson, K. (2013). A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nature Chemistry, 5(2). https://doi.org/10.1038/nchem.1546
Schmitz-Elbers, M., Lukinavičius, G., & Smit, T. H. (2021). Live fluorescence imaging of f-actin organization in chick whole embryo cultures using sir-actin. Cells, 10(7). https://doi.org/10.3390/cells10071578
https://www.leica-microsystems.com/science-lab/live-cell-imaging-gallery/
Luis A. J. Alvarez, Ulf Schwarz, Lars Friedrich, Jonas Foelling, Frank Hecht & M. Julia Roberti. Application Note: Pushing STED beyond its limits with TauSTED. (2021) https://www.nature.com/articles/d42473-021-00241-0