SPY555-Golgi is a bright, far red & non toxic live cell golgi probe based on our SPYTM dyes series. Its optimized structure allows quick labeling of the golgi apparatus in live cells with high specificity and low background. SPY555-Golgi stains the golgi apparatus in live cells without the need for genetic manipulation or overexpression of fluorescent proteins. Its absorbance and emission spectra are similar to Cy5. SPY555-Golgi enables multicolor imaging with SPY505, SPY595, SPY650, SPY700 or GFP. SPY555-Golgi can be imaged with standard Cy3/TMR settings. It can be used for, confocal, SIM or STED imaging in living cells and tissue. Contains 1 vial of SPY555-Golgi (lyophilized). STED compatible with a 660 nm or 775 nm STED depletion line.
Absorbance maximum λabs | 555 nm |
Fluorescence maximum λfl | 580 nm |
Works on fixed cells? | Yes |
Probe Quantity | 100 stainings |
Fluorescence lifetime | 2.4 ns |
STED depletion wavelength | 660 or 775 nm |
Shipping | room temperature |
Storage | -20°C |
SPY555-Golgi and SPY650-Golgi are 2 new fluorescent live cell probes for the golgi apparatus. They are based on asparagusic acid golgi trackers developed by Saidjalolov et al.1 They are able to label the golgi apparatus in live cells as fast as 15 min after adding the probe. SPY-Golgi are bright, non toxic, cell permeable and highly specific live cell probes for fluorescence imaging of the Golgi apparatus. They do not require any transfection or genetic manipulation of the cells. SPY650-Golgi is highly suited for STED imaging
- Saidbakhrom Saidjalolov et Al. “Asparagusic Golgi Trackers”, JACS Au 2024.
Q1. Do the Golgi probes work on fixed cells?
A: Yes, cells need to be stained first and then fixed.
Q2. Do the Golgi probes work on plants?
A: not tested yet
Q3. Do the Golgi probes work in tissues?
A: not tested yet
Q4. Do the Golgi pobes work with STED or SIM superresolution microscopy?
A: yes, we recommend SPY650-Golgi for best results with superresolution techniques.
Q5. What is STED microscopy and how does it work?
A1. STED microscopy stands for Stimulated Emission Depletion microscopy. It is one type of super resolution microscopy which allows the capture of images with a higher resolution than conventional light microscopy which is constrained by diffraction of light. STED uses 2 laser pulses, one is the excitation pulse which excites the fluorophore, causing it to fluoresce. The second pulse, referred to as the STED pulse, de-excites the fluorophore via stimulated emission in an area surrounding a central focal spot that is not de-excited and thus continues to fluoresce. This is accomplished by focusing the STED pulse into a ring shape, a so-called donut, where the center focal spot is devoid of the STED laser pulse, conferring high resolution to the fluorescent area (Fig. 1; see Ref. 1 for more details on STED microscopy).
Figure 1. STED microscopic image of microtubules labeled with SiR-tubulin in human primary dermal fibroblasts.
Q6. Why is the SPY actin (or tubulin/DNA) probe good for STED microscopy?
A2. STED microscopy offers the ability to study cellular details on a nanometermolar scale in vivo. To take advantage of this super resolution microscopy, one must be able to select with high specificity the area to be examined using fluorescent probes. In addition, the fluorescent probes must be bright, photostable, exhibit no or little phototoxicity, be excited and emit in the far red spectrum. In addition, if the probe is to be used for live cell imaging (thus avoiding fixation artifacts that occur when cells are fixed), high cell permeability is necessary. The SPY actin and tubulin probes fulfill all of these requirements. In short, the combination of STED and SiR probes allows for unparalleled fluorescent visualization of subcellular actin and tubulin/microtubule structures and their physical characterization in living cells, (see Fig. 2 and Ref. 2).
Figure 2. STED images of cultured rat hippocampal neurons stained with SiR-actin. Bottom image is a close-up view of part of the top image to clearly visualize actin rings (stripes) with 180 nm periodicity. Courtesy Of Elisa D'Este, MPI Biophysical Chemistry, Göttingen.
Q7: Are the SPY probes stable at room temperature?
A3: Yes, the probes are stable at room temperature for a few days. However, it strongly depends on the probe and the solvent. Thus, it is recommended to store all of the probes or solutions at –20°C.
Q8: Are SPY-actin, SPY-DNA and SPY-tubulin toxic to cells?
A4: Yes, above a certain threshold both probes show some effect on cell proliferation and altered actin or microtubule dynamics. However, the probes are orders of magnitude less toxic than their parent drug. In HeLa cells, neither actin nor microtubule dynamics were altered at concentrations below 100 nM. At this concentration, SPY probes efficiently label microtubules and F-actin, allowing for the capture of high signal to noise images.
References
1. Hell S.W. and Wichmann J. 1994. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780-782.
2. D’Este E. et al. 2015. STED nanoscopy reveals the ubiquity of subcortical cytoskeleton periodicity in living neurons. Cell Rep. 10, 1246-1251.
3. Lukinavicius G. et al. 2013. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132-139.
4. Lukinavicius G. et al. 2014. Fluorogenic probes for live-cell imaging of the cytoskeleton.Nature Methods. 11, 731-733.
