The question: “I need to know where my protein is inside the cell — membrane, cytoplasm, nucleus, organelles — and watch it move. Which dyes and which microscope do I need?”
Compartment identification is the foundation of translocation assays. Whether you are tracking a transcription factor moving into the nucleus, a receptor internalising from the membrane, or a kinase shuttling to the mitochondria, you need reliable compartment references and a microscope that can image them in living cells.
Why Cellular Compartment Identification Matters
- Translocation assays: Most signalling events are localisation changes, not simple intensity changes.
- High-content screens: Automated compartment segmentation lets you classify cells as “activated” or “inactive” based on spatial distribution.
- Colocalisation: Knowing where an organelle is lets you assign puncta or a protein to the correct compartment.
- Quantification: Intensity ratios (nuclear/cytoplasmic, membrane/cytoplasmic) only make sense if the compartments are accurately segmented.
Reviews on organelle-targeted fluorescent probes and plasma-membrane staining summarise the chemical strategies that make these compartment markers possible.
EVOS M7000: Live Compartment Imaging on Dishes
The EVOS M7000 is a good fit for small-scale compartment and translocation work. It has up to five fluorescence channels, LED excitation with low phototoxicity, and the OSI-2 Onstage Incubator for long-term live-cell experiments.
- Image CellMask plus GFP-tagged protein plus nuclear dye in one field.
- Use transmitted light/phase for cell morphology context.
- Capture Z-stacks to improve organelle localisation in thicker cells.
- Export to ImageJ/CellProfiler for nuclear/cytoplasmic ratio analysis.
EVOS M7000 Review UK
CellInsight CX5 / CX7: Automated Multiwell Compartment Analysis
For translocation screens that use CellMask and organelle dyes across many conditions, the CellInsight CX5 and CX7 platforms automate acquisition and analysis.
- Segment nuclei, cytoplasm, membrane rings and punctate organelles per cell.
- Measure protein-of-interest intensity in each compartment and compute ratios.
- Run 96- or 384-well compound screens with consistent segmentation rules.
- With onstage incubator, extend to kinetic translocation assays.
CellInsight CX7 & HCS UK
Publications and Resources
These references cover plasma-membrane staining chemistry, organelle-targeted probes, quantitative translocation analysis and practical staining protocols.
Thermo Fisher — CellMask Plasma Membrane Stains User Guide
Official user guide covering CellMask Orange, Deep Red, Green and Near-IR plasma membrane stains, storage, protocols and spectral information for live-cell imaging.
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Yin et al. (2023) — Small-molecule plasma membrane probes
Coordination Chemistry Reviews. Comprehensive review of small-molecule fluorescent probes for plasma membrane staining, covering design, mechanisms and biological applications.
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Haucke & colleagues (2021) — Organelle membrane nanoscale imaging
Current Opinion in Chemical Biology. Discusses imaging organelle membranes in live cells at the nanoscale with lipid-based fluorescent probes.
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Lin et al. (2021) — Strategies for organelle-targeted probes
Organic & Biomolecular Chemistry. Reviews strategies for organelle targeting of fluorescent probes, including mitochondria, ER, lysosomes, Golgi and lipid droplets.
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Jia et al. (2014) — Quantitative translocation tracking in live cells
PLOS ONE. A quantitative method to track protein translocation between intracellular compartments in real time using weighted local variance image analysis.
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CSH Protocols (2008) — Plasma membrane fluorescent labelling
Cold Spring Harbor Protocols. Protocol for labeling the components of the plasma membrane with fluorescent dyes for imaging.
View DOI
Thermo Fisher — EVOS M7000 / CellInsight CX7 UK
Platform pages for multi-channel live-cell inverted imaging and automated high-content compartment analysis.
EVOS M7000 Review
CellInsight CX7 UK
Frequently Asked Questions
What microscope do I need for CellMask and organelle compartment imaging in the UK?
Any inverted fluorescence microscope with sufficient spatial resolution and multi-channel capability will work. For routine compartment segmentation and translocation quantification on a few wells or dishes, the EVOS M7000 with a sensitive camera is practical. For high-throughput multiwell translocation assays, the CellInsight CX5 or CX7 High Content Analysis platform is the better choice.
What does CellMask stain?
CellMask Plasma Membrane Stains label the outer plasma membrane of live cells almost instantly. They are used as a surface reference to define the cell boundary, segment cytoplasm, or measure protein translocation to or from the membrane.
Which organelle dyes are used alongside CellMask in translocation assays?
Common counterstains include Hoechst or DAPI for nuclei, MitoTracker for mitochondria, ER-Tracker for the endoplasmic reticulum, LysoTracker for lysosomes, GolgiTracker or fluorescent lectins for the Golgi, and BODIPY variants for lipid droplets.
How do you measure protein translocation with CellMask?
You image your protein of interest (e.g. GFP-tagged transcription factor or receptor) together with CellMask as a membrane/cytoplasm reference. Image-analysis software then calculates intensity ratios between the membrane, cytoplasm and nucleus over time to quantify translocation.
Can CellInsight CX7 automate compartment-based translocation analysis?
Yes. CellInsight CX7 with HCS Studio can create nuclear, cytoplasmic and membrane segmentation rings, quantify the distribution of one or more fluorescent probes between these compartments, and report population statistics across multiwell plates.
Are CellMask stains toxic for long-term live-cell imaging?
CellMask Plasma Membrane Stains are formulated for short live-cell staining and are generally well tolerated at recommended concentrations for imaging sessions from minutes to a few hours. For multi-day time-lapse, test concentration and photostability; some labs refresh dye or use genetically encoded compartment markers instead.