Introduction
PKmito Orange FX (also known as PKMO FX) is a bright, photostable, mitochondrial probe based on the PKmito™ dyes developed in the lab of Zhixing Chen at Peking University1). PKmito Orange FX labels mitochondria in formaldehyde (FA) or glutaraldehyde (GA) fixed cells with very high specificity. The unique and unmatched feature of PKmito Orange FX is its ability to be retained nearly quantitatively after aldehyde fixation of stained cells. PKmito Orange FX is extremely well suited for STED and SIM superresolution imaging. It allows to perform nanoscopy of mitochondria with exquisite resolution and bright signal. PKmito Orange FX accumulates in the mitochondrial inner membrane (IM) and stays in place upon fixation. It is highly suited to image mitochonridal cristae structure by STED superresolution microscopy using a 775 nm depletion line. PKmito Orange FX does not require any genetic manipulation, transfection or overexpression of fluorescent proteins. PKmito Orange FX enables multicolor imaging with SPY505, SPY650, SPY700, SiR or GFP. It can be used for widefield, confocal, SIM or STED imaging in fixed cells and tissue. Contains 1 vial of PKmito Orange FX (lyophilized).
Absorbance maximum λabs (MeOH) | 584 nm |
Fluorescence maximum λfl (MeOH) | 604 nm |
Works on fixed cells? | Yes |
Probe quantity | 100 stainings* |
Fluorescence lifetime | n.d |
STED depletion wavelength | 775 nm |
Shipping | room temperature |
Storage | -20°C |
Cos7 cells co-labeled with PKmito-FX and SiR-DNA
z-stack STED images of mitochondrial cristae in a HeLa cell labeled with PKMO FX, fixed with 2% GA. Chen J. et al PNAS 2024. 121 (19) e2317703121
Z-stack STED images of mitochondrial cristae (STED, grey) and nascent DNA (confocal, green) in a HeLa cell labeled with PKMO FX and Click-iTTM AF488. Chen J. et al PNAS 2024. 121 (19) e2317703121
For product Datasheets and MSDSs please click on the PDF links below.
Spirochrome Technical Tips and Ex/Em spectra in graphical form (PDF)
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
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: Are the Spirochrome probes stable at room temperature?
A2: 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.
References
1. Liu Tianyan et al. “Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain” PNAS (2022):119 (52).
2. Yang, Zhongtian, et al. “Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging.” Chemical science 11.32 (2020): 8506-8516.
