Image segmentation is a key aspect of image-based high-content screening (HCS) assays and is the first step of automated image acquisition and analysis. Image segmentation involves separating objects of interest (cells) from background or other features not relevant to the analysis and allows determination of items of interest in their appropriate spatial context such as translocation of biomarkers or changes in biomarker levels within particular cellular compartments.  Key approaches for image segmentation involve using fluorescent stains selective for specific parts of the cell—nucleus, cytoplasm, plasma membrane, or other organelles—to delineate the whole cell. Life Technologies Molecular Probes® fluorescent stains and dyes are validated for high-content analysis and provide you with the tools you need for accurate cell analysis.

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Nuclear Image Segmentation

For many software algorithms, the cell identification process begins with the detection of fluorescently stained nuclei then extrapolating to build a mask that marks the probable position of the cytoplasmic region. The versatile HCS NuclearMask™ stains, which survive standard formaldehyde-based fixation and detergent-based permeabilization methods, can be applied to live and fixed cells. HCS NuclearMask™ reagents are available in a wide range of fluorescent colors from blue to deep red for multiplexing flexibility. Sufficient quantities are provided to stain ten 96-well plates using an assay volume of 100 μL per well.


Figure 1. Nuclear image segmentation of live and formaldehyde-fixed HeLa cells stained with HCS NuclearMask™ Deep Red stain. Live HeLa cells were incubated with HCS NuclearMask™ Deep Red stain for 30 minutes at 37°C and 5% CO2 and imaged on a Thermo Scientific Cellomics® ArrayScan® VTI platform (left panel). The cells were fixed and imaging was repeated on the same field of view.

Nuclear Image Segmentation

Product Cat. no.
Emission Color*
Ex/Em† Live‡ Fixed§ Fixable** Key attributes
HCS NuclearMask™ Blue stain H10325 340/361
Developed for image-based high content screening (HCS) assays to give distinct nuclear labeling with minimal cytoplasmic background.  Measures DNA content and nuclear morophology.
HCS NuclearMask™ Red stain H10326

622/645

HCS NuclearMask™ Deep Red stain H10294 638/686
* Gray represents fluorescence emission that is beyond the limit of human vision. † Ex/Em = Fluorescence excitation and emission maxima in nm. ‡ Stains live cells. § Stains fixed cells. ** Staining pattern is fixable. NA = Not applicable.

Cytoplasmic Image Segmentation

HCS CellMask™ stains can also be used to measure quantitative differences in cell size resulting from drug treatment. The ability to measure cytoskeletal disruption is an important aspect of cytotoxicity screening in drug discovery and development. While fluorescent conjugates of phalloidin and anti-tubulin antibodies are popular probes used for measuring cytoskeletaldisruption, a mask of the entire cell also has great utility in both cellular demarcation and cell size determination. Cellular demarcation with HCS CellMask™ stains enables automated image analysis software to determine where the cytoskeleton of one cell ends and another begins, a parameter that cannot be measured accurately with a nuclear counterstain alone (Figure 2).

Cytoplasm Segmentation Stains
Figure 2. The effects of cytochalasin D treatment on HeLa cell size as measured by HCS CellMask™ Blue stain. HeLa cells were treated with DMSO vehicle (left) or 10 µM cytochalasin D (right) for 3 hr before fixation and permeabilization. Samples were then labeled with HCS CellMask™ Blue stain, Alexa Fluor® 488 dye–conjugated phalloidin to visualize filamentous actin (red), mouse anti–α-tubulin IgG (detected with Alexa Fluor® 555 goat anti-mouse IgG, green), and TO-PRO®-3 iodide to counterstain nuclei (magenta). The bar graph represents quantitative measurements of cell size as determined by HCS CellMask™ Blue stain to show the effects of cytochalasin D treatment.

Cytoplasmic Image Segmentation

Product Cat. no.
Emission Color*
Ex/Em† Live‡ Fixed§ Fixable** Key attributes
HCS CellMask™ Blue stain H32720 346/442  
 

Label cytoplasm and nucleus via nucleic acid binding; no wash steps are required, and imaging can be performed in stain solution; validated as a counterstain and a segmentation tool for HCS applications

HCS CellMask™ Green stain H32714 493/516  
 
HCS CellMask™ Orange stain H32713

556/572

 
 
HCS CellMask™ Red stain H32712 558/612  
 
HCS CellMask™ Deep Red stain H32721 650/665  
 
* Gray represents fluorescence emission that is beyond the limit of human vision. † Ex/Em = Fluorescence excitation and emission maxima in nm. ‡ Stains live cells. § Stains fixed cells. ** Staining pattern is fixable. NA = Not applicable.

Plasma Membrane Image Segmentation

Binding events at the cell surface, endocytosis, receptor internalization and membrane-nuclear translocation are all key events that require the ability to accurately segment and identify the cell’s boundary.  In addition identifying individual cells in clusters or clumps cannot be performed easily using whole cell or nuclear image segmentation approaches.  For this reason we developed our CellMask™ Plasma Membrane stains. These are lipophilic dyes that can be used to accurately label and identify the plasma membrane in live cells for segmentation (Figure 3).  Orange- or red-fluorescent colors are available.

Please note: Due to their lipophilic nature, these stains can tolerate formaldehyde fixation but will be washed out with detergent permeabilization.

  Figure 3.  Plasma membrane image segmentation of live cells using CellMask™ Deep Red plasma membrane stain. HaCaT cells (an immortalized human keratinocyte cell line) transfected with a mitochondrion-targeted eGFP (pseudocolored green) construct were incubated with 1 µM Hoechst 33342 (pseudocolored blue) and then 8 µg⁄ml CellMask™ Deep Red plasma membrane stain (pseudocolored red). Cells were imaged with a Zeiss Cell Observer HS microscope. The image stack was deconvolved with the Zeiss AxioVision 3D Deconvolution module with the point spread function acquired using a 200 nm TetraSpeck™ fluorescent microsphere. Image submitted by Christian Junker, University of Saarland, Department of Biophysics, Homburg, Germany.

Plasma Membrane Image Segmentation

Product Cat. no.
Emission Color*
Ex/Em† Live‡ Fixed§ Fixable** Key attributes
CellMask™ Orange plasma membrane stain C10045 554/567
 
Developed for image-based high content screening (HCS) assays to products not yet validated for HCS applications.  Will survive formaldehyde fixation with some signal loss but will be extracted by detergents and other permeabilization agents.
CellMask™ Deep Red plasma membrane stain C10046 649/666
 
* Gray represents fluorescence emission that is beyond the limit of human vision. † Ex/Em = Fluorescence excitation and emission maxima in nm. ‡ Stains live cells. § Stains fixed cells. ** Staining pattern is fixable. NA = Not applicable.

Organelle Image Segmentation

Organelle markers in HCS assays are used both as structural landmarks (e.g., in co-localization analysis) and as functional indicators (e.g., organelle integrity). We offer a wide range of choices for live-cell or fixed-cell imaging applications.