Actin Test Kit Spirochrome Bioprobe
Fluorescent actin probes kit for F-actin live cell imaging.
Three far red actin probes in a single Kit!
Live cell actin imaging can be demanding and each experiment has its own requirements for many different parameters such as temporal resolution, fluorescence background tolerance, toxicity, bleaching rate, etc. Choosing the best performing probe for a specific is now simple and cost efficient with Actin_TestKit product It simply combines our 3 far red actin probes in a single product:
-SiR-actin our landmark actin probe, a true reference in the field
-SPY650-FastAct our 2nd generation actin with better performance with faster actin dynamics.
-SPY650-FastAct_X the last and 3rd generation with an optimized actin ligand that does not stabilize F-actin and fast exchange. highly effective for long term live cell and sensitive cells.
Each actin probe has a different design, specific pros and cons and their overall performance differ greatly from one experiment to the other.
| SIR-Actin | SPY650-FastAct | SPY-650-FastAct_X | |
| Absorbance maximum λabs | 652 nm | 652 nm | 652 nm |
| Fluorescence maximum λfl | 674 nm | 674 nm | 674 nm |
| Imaging "channel" | Cy5 | Cy5 | Cy5 |
| Works on fixed cells? | yes | yes | no |
| Probe quantity | 20 stainings | 20 stainings | 20 stainings |
| Exchange rate when F-actin bound | slow | fast | fastest |
| F-actin stabilization | mild | minimal | none |
| Shipping | room temperature | room temperature | room temperature |
| Storage | -20°C | -20°C | -20°C |
Cells Stained with SIR-Actin (Left) and SPY650-FastAct™ (Right)
HUVEC cells stained with SPY650-FastAct, SPY555-Tubulin, and SPY505-DNA
Check out these videos of SPY650-FastAct™ in action
For product Datasheets and MSDSs please click on the buttons on the top right of this product page.
The Actin Spirochrome Bioprobe Test Kit is designed to allow the researcher to rapidly characterize the best actin probe for their application and cell type.
Datasheet for the kit is here.
Datasheet for SiR-Actin is here.
Datasheet for SPY650-FastAct is here.
Datasheet for SPY650-FastAct-X is here.
The Excitation and Emission spectra for each probe in Excel format can be download here:
Raw data Excel file of SiR probes is here.
Raw data Excel file for SPY650 probes is here.
For more information about Spirochrome bioprobes in general click on the FAQs link above.
Q1. 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.
Q2. 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.
Q3: 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 the probe or the probe solution at –20°C.
Q4: 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.
