What is fluorescence live-cell imaging?

Fluorescence imaging of live cells is used to observe dynamic cellular processes and track cellular biomolecules and structures over time. Live-cell fluorescence imaging allows researchers to examine cells in their native state so that cellular function is preserved, enabling the study of cellular processes including cytoskeletal rearrangement, apoptosis, cell migration, endocytosis, phagocytosis, and organelle dynamics to obtain more relevant findings in drug discovery, cancer, developmental biology, environmental science, and other medical research fields.

Use this guide to build live-cell imaging experiments, pick reagents, and generate publication quality data.

Build live-cell experiments for your imaging platform with an incubator or microscope

How to choose reagents for live-cell imaging

Live-cell imaging reagents include both targeted fluorescent proteins and small membrane permeant fluorescent dyes. To enable live-cell imaging experiments, many reagents are designed for time-lapse over several hours or days, while others are optimal for end-point assays in which cells are imaged and analyzed immediately after staining. Staining concentration, incubation time, and the appropriate time window and imaging interval/frequency should be determined empirically to minimize cytotoxicity and preserve cellular function.

The reagents found within the live-cell imaging guide below are compatible with automated high-content and incubator-based fluorescence imaging systems such as: Thermo Fisher Scientific’s  EVOS M7000 and M5000 Cell Imaging Systems with EVOS Onstage Incubator, CellInsight HCS Platforms, PerkinElmer’s MuviCyte™ live-cell imaging system*, Leica’s Thunder and Mica*, and Sartorius’ Incucyte® Live-Cell Analysis System™*.

 EVOS DAPI Light CubeEVOS GFP Light Cube
Excitation: 470/22 nm;
Emission: 510/42 nm
EVOS RFP Light Cube Excitation: 531/40 nm; Emission: 593/40 nm EVOS Red Light Cube Excitation: 585/29 nm; Emission: 624/40 nm EVOS Cy5 Light Cube Excitation: 628/40 nm; Emission: 693/40 nm Live-cell imaging time frame
Incucyte Green FilterIncucyte Orange FilterIncucyte Red FilterIncucyte NIR Filter
Antibody internalization 

pHrodo Green iFL antibody labeling reagents

 

pHrodo Red iFL antibody labeling reagents

 pHrodo Deep Red antibody labeling reagentsLong-term (hours to overnight)
Apoptosis CellEvent Caspase-3/7 Green Detection Reagent

 

CellEvent Caspase-3/7 Red Detection Reagent Long-term (overnight to 48-72 hours)
Autophagy Premo Autophagy Sensors (p62 and LC3B) GFPPremo Autophagy Sensors (p62 and LC3B) RFP  Long-term (overnight to 48 hours)
Cell trackingCellTracker dyesShort- to long-term (72 hours/~3 or more generations)
Cytoskeleton (actin) CellMask Green Actin Tracking StainCellMask Orange Actin Tracking Stain CellMask Deep Red Actin Tracking StainShort- to long-term (24+ hours)
Cytoskeleton (tubulin) Tubulin Tracker Green  Tubulin Tracker Deep RedShort-term
Endosomes/endocytosis pHrodo and Alexa Fluor dextransShort- to long-term (minutes to hours; overnight or longer for some applications)
Ligand internalization Alexa Fluor, BODIPY, and pHrodo LDL conjugatesShort- to long-term (minutes to overnight or longer)
Alexa Fluor and pHrodo EGF and transferrin conjugates
Endoplasmic reticulum ER-Tracker Green

ER-Tracker Red

End-point
Hypoxia Image-iT Green Hypoxia Reagent   End-point 
Calcium Fluo-4 Calcium Imaging KitRhod-3 Calcium Imaging Kit  Short-term (minutes to hours)
LysosomesLysoTracker and LysoSensor dyesShort-term (minutes to hours)
Membranes (plasma and internal) Vybrant cell labeling solutionsLong-term
Mitochondria structure MitoTracker dyesLong-term
Mitochondria function  TMRM  Short-term (minutes to hours)
TMRE
NucleusHCS NuclearMask Blue StainSYTO 9 Green Fluorescent Nucleic Acid Stain HCS NuclearMask Red StainHCS NuclearMask Deep Red StainEnd-point
Hoechst 33342   NucRed Live 647 ReadyProbes Reagent
NucBlue Live ReadyProbes Reagent   DRAQ5 Fluorescent Probe Solution
Plasma membrane CellMask Green Plasma Membrane StainCellMask Orange Plasma Membrane Stain CellMask Deep Red Plasma Membrane StainShort-term (10-90 minutes, can image longer but will internalize)
Reactive oxygen species CellROX Green ReagentCellROX Orange Reagent CellROX Deep Red ReagentLong-term (24 hours)
 H2DCFDA dyes   Short-term (~2 hours)
ThiolTracker VioletMitoSOX Green Superoxide Indicator   End-point
Phagocytosis pHrodo Green BioParticles ConjugatespHrodo Red BioParticles Conjugates pHrodo Deep Red BioParticles ConjugatesShort- to long-term (minutes to hours or longer)  
ViabilityCalcein Blue, AMCalcein, AM, cell-permeant dye   Short-term (minutes to hours) 
 SYTOX Green Nucleic Acid StainSYTOX Orange Nucleic Acid Stain SYTOX Deep Red Nucleic Acid Stain
 LIVE/DEAD Viability/Cytotoxicity Assay Kit (Green/Deep Red)  LIVE/DEAD Viability/Cytotoxicity Assay Kit (Green/Deep Red)
Fluorescent protein–based cell structure reagents CellLight GFP BacMam 2.0CellLight RFP BacMam 2.0  Long-term (overnight to days/2-3 generations)

How to maintain environmental control during live-cell imaging

Fluorescence imaging platforms combined with incubators provides a controlled and stable environment that allows long-term imaging of cells in their natural state. The EVOS Onstage Incubator for the EVOS M7000 and M5000 Cell Imaging Systems and the Invitrogen HCA Onstage Incubator for automated high-content screening and analysis on the CellInsight HCS/HCA platforms enable time-lapse and kinetic imaging and analysis of live cells within an environmental chamber. When performing long-term live-cell imaging within an incubated environment, reagents should be used in traditional cell culture medium or FluoroBrite DMEM (Cat. No. A1896701) buffered with bicarbonate to maintain physiological pH in a 5% CO2 environment.

Example: apoptosis in live-cell imaging experiments

Cellular apoptosis is a tightly controlled programmed cell death process used to remove excess, damaged, and unneeded cells or tissues. Apoptosis can be detected through caspase activation, DNA fragmentation, disruption in active mitochondria, and plasma membrane changes. The CellEvent Caspase 3/7 reagents are fluorogenic caspase substrates that detect caspase activation to assay for apoptosis in live cells. These reagents do not require wash steps and can be used for real-time and time-lapse live-cell imaging of apoptosis for 48 hours or longer.

CellEvent Caspase 3/7 workflow

Figure 1. Apoptosis live-cell endpoint assay workflow using the CellEvent Caspase-3/7 Green Detection Reagent (Cat. No. C10432). Seed cells and follow with treatment.  Add diluted CellEvent Caspase-3/7 Green Detection Reagent to cells. CellEvent Caspase 3/7 dyes may be imaged directly on cells in complete media without wash. Measure fluorescence starting as early as 30 minutes to as long as 72 hours.

Download Caspase 3/7 Protocol

Cells stained with CellEvent Caspase 3/7 Green as indicated with apoptotic cells being bright green and live cells being dark, and imaged with Incucyte® ZOOM Live-Cell Analysis

Figure 2. Example of Live Cell Imaging of apoptotic cells stained with CellEvent Caspase 3/7 Green. HuVEC cells treated with a cocktail stained with CellEvent Caspase 3/7 Green Assay (Cat. No. C10432)  and imaged with Incucyte® ZOOM Live-Cell Analysis System at 0, 24, 48, and 72 hours after treatment. (Image from: Sambi M, Samuel V, Qorri B, Haq S, Burov SV, Markvicheva E, Harless W, Szewczuk MR. A Triple Combination of Metformin, Acetylsalicylic Acid, and Oseltamivir Phosphate Impacts Tumour Spheroid Viability and Upends Chemoresistance in Triple-Negative Breast Cancer. Drug Des Devel Ther. 2020;14:1995-2019 PMID: 32546966)

Example: mitochondrial activity in live-cell imaging experiments

Live-cell imaging reagents include both cell structure reagents used to identify cellular components and cell function reagents to analyze cellular functions and processes. For instance, MitoTracker dyes accumulate in active mitochondria in live cells and covalently attach to mitochondria to allow evaluation of mitochondrial localization and abundance. In contrast, TMRM and TMRE are dynamic mitochondria membrane potential indicator dyes that fluctuate in and out of the mitochondria based on membrane potential.

Cells stained with MitoTracker Red as indicated with cells with active mitochondria being bright red, the dead cells are stained in green, the nucleus in blue, and imaged with Incucyte® ZOOM Live-Cell Analysis

Figure 3. Monitoring active mitochondria in live cells. Fibroblast lines from patients were cultured and stained with MitoTracker Red (Cat. No. M22426), Image-iT DEAD Green dyes (Cat. No. I10291), Hoechst (Cat. No. R37165) and imaged on an Incucyte® ZOOM Live-Cell Analysis System. (Image from Smith GA, Jansson J, Rocha EM, Osborn T, Hllett PJ, Isacson O. Fibroblast Biomarkers of Sporadic Parkinson's Disease and LRRK2 Kinase Inhibition. Mol Neurobiol. 2016 Oct;53(8):5161-77. PMCID: PMC5012155. )


Tips to detect changes in live-cell experiments

Step 1: Plan

Design your experiment with careful consideration of the tools and resources needed for each step.

Advantages

  • Observe dynamic cellular processes as they happen
  • Study and image several processes and functions simultaneously using multiplexed assays
  • Study cellular structures in their native environment, resulting in more realistic results closer to in vivo scenarios
  • Track cellular biomolecules and structures over time
  • Observe interactions between cells
  • Cellular enzymes and other cytosolic biomolecules remain in the cell

Considerations

  • Must have a specific way to label your target with minimal toxicity – whether it is a molecule, a cellular function, or a cellular state
  • Living cells are generally not permeable to large detection molecules such as antibodies
  • Moving objects can be more difficult to keep in focus
  • Certain techniques can be harmful to living cells
  • Cells must be kept in their natural physiological state

Step 2: Culture

Maintain or grow your cells in optimum conditions.

Keeping cells alive and healthy during various experimental manipulations, detection, and imaging is no small task. The choice of medium is particularly important for time-lapse imaging and experiments where cells are exposed to ambient conditions for longer periods. For reliable results with live cells, it is essential that the cells be healthy and kept in an environment as close as possible to physiological temperature, pH, oxygen level, and other conditions.

Learn more about cell culture

Product highlights

These media and wash buffers are created specifically for live-cell imaging and detection. Employing them in your experiments can help you improve image clarity, reduce background fluorescence, and optimize cell viability.

Tips

You can improve image clarity, reduce background fluorescence, and optimize cell viability by using media and wash buffers created specifically for live-cell imaging and detection. See product selection guide

Step 3: Label

Target cell structures, cell functions, and proteins of interest with selective dyes and stains.

The appropriate fluorophore (targeted fluorescent protein or small membrane-permeant reagent) should be used to monitor your target cellular structure or process. Additional fluorophores can be used to monitor multiple cellular structures and processes, but the excitation and emission spectra should be checked using the Fluorescence SpectraViewer to ensure minimal spectra overlap. It is critical to avoid using too much fluorescent label because excessive fluorescent labeling can result in:

  • Nonspecific staining with increased background signals
  • Physiological artifacts and structural perturbations
  • Cytotoxicity
  • Spectral overlap

Note that:

  • Live-cell structure reagents—help identify cellular components
  • Live-cell function reagents—help identify cellular functions and processes

Product highlights

  • Invitrogen CellLight reagents have proven to be the easiest to use for labeling specific structures in live cells. Targeted fluorescent proteins are introduced using the Invitrogen BacMam transduction system; no molecular biology techniques are required. Simply add the reagent to your cells, incubate overnight, and you’re ready to image in the morning.
  • Invitrogen CellTracker reagents are a diverse reagent class used for labeling mammalian cells to view changes in morphology or location. These nontoxic fluorescent dyes are designed to freely pass through cell membranes into cells, where they are transformed into cell-impermeant reaction products. Incubating cells with a CellTracker reagent for 30 minutes will provide at least 72 hours of fluorescent signal (typically three to six generations).
  • Invitrogen pHrodo indicators are fluorogenic dyes that dramatically increase in fluorescence as the pH of their surroundings becomes more acidic. When conjugated to dextrans, proteins, or other particles, pHrodo dyes can be used as highly specific sensors of endocytic and phagocytic internalization and lysosomal sequestration in live cells, offering a superior alternative to conjugates of other fluorescent dyes such as fluorescein and tetramethylrhodamine.

Tips

  • Consider using a longer wavelength fluorescent reagent if extended light exposure is required. This will require lower excitation power, which can correlate to lower phototoxicity and healthier cells.
  • Staining must be optimized for the particular assay readout, spectral compatibility, and signal-to-background ratio.
  • Removing the labeling solution and rinsing with fresh medium will reduce background fluorescence.

Step 4: Optimize

Minimize background and maintain photostability of fluorescence signals.

Signal-to-background ratio can be optimized by using reagents that reduce extracellular fluorescence and increase fluorophore photostability. It is important to image in media that have been specifically designed for maintaining cell health while reducing or eliminating background fluorescence in live-cell imaging experiments (see Table 1). The addition of a background suppressor compatible with live cells can also help reduce extracellular background fluorescence and eliminate the need for a wash step. Antifade mounting media for live cells can be applied to samples to reduce photobleaching of fluorophores, preventing signal loss with multiple or long exposures.

Table 1. Imaging media comparison.

ReagentCell washingShort-term imagingImaging up to 4 hoursLong-term imaging
Gibco PBS, pH 7.4  
Gibco FluoroBrite DMEM

Product highlights

Invitrogen BackDrop Background Suppressor imaging results

Live HeLa cells labeled with Tubulin Tracker Green dye and Tubulin Tracker Deep Red dye. Both labels show high off-cell background when the probe is left in the staining solution (left). Addition of BackDrop Background Suppressor greatly reduces extracellular background while leaving intracellular labeling unaffected (right), thus enabling a no-wash protocol for high-contrast imaging of tubulin in live cells.

Invitrogen ProLong Live Reagent confocal imaging results

The overall signal protection offered by ProLong Live reagentcompared to untreated samples is calculated based on the scan number where treated and untreated samples reach the EC50 value. The addition of ProLong Live reagent permitted 100% more captures with Invitrogen CellLight Mitochondria-RFP reagent.

Invitrogen ProLong Live Reagent fluorescence imaging

After 120 exposures using a standard time-lapse imaging protocol, samples treated with ProLong Live reagent are >20% brighter than untreated cells, enabling more data collection time.

Tips

  • If no further culture is planned, a background suppressor can be used to optimize the signal by reducing the haze and increasing the contrast.
  • The use of an antifade reagent has been shown to increase fluorophore photostability and decrease the effect of phototoxicity in a variety of sample types.

Step 5: Image

Live-cell imaging of dynamic processes requires active observation over time

Illumination and detection

To minimize phototoxicity, choose imaging systems that give you the greatest control of light sources. Try to minimize light intensity, exposure time, wavelength range, and amount of excitation energy for illuminating your cells while still generating a good signal with low background. Use the illumination that gives you the highest signal with the lowest level of fluorophore excitation. In some cases (particularly when you wish to image over a long period of time), it is advisable to sacrifice resolution by using shorter exposure times or lower magnification in exchange for healthier cells.

Live-cell imaging over longer periods of time can be challenging because the target may move out of focus during the course of the experiment. Many microscopes have autofocusing features that can help keep your target in focus longer and reduce focal drift. Additionally, maintaining cells at a constant temperature and keeping the volume of solution in the vessel constant will help with focal drift.

Environmental control

Many cells cannot tolerate deviations from their optimal temperature, osmolarity, pH, and humidity. Requirements vary depending on what experimental question you are asking. For example, experiments investigating cell growth and division may have a different set of requirements than experiments involving receptor activation and calcium accumulation. Some robust immortalized cell lines will tolerate being imaged or monitored for short periods of time without any environmental control. Conversely, for long-term imaging and detection studies, good results with both immortalized cells and primary cells typically require tightly controlled environmental parameters.

Catastrophic blebbing of the cell membrane

The top cell shows catastrophic blebbing of the cell membrane caused by excessive light exposure. Blebbing is a term used to describe membrane perturbation caused by toxicity. By contrast, the bottom cell remains relatively healthy and is not displaying aberrant morphology.

Product highlights

Countess II FL Automated Cell Counter

To avoid the pitfall of proceeding to the next step in your experiment with unhealthy cells, a quick check for cell health can be done on the Countess III FL Automated Cell Counter when used in conjunction with a variety of fluorescent reagents to detect cell viability, apoptosis, cytotoxicity, and transfection efficiency. The reusable slide option reduces consumption cost.

Invitrogen EVOS M5000 Imaging System with EVOS Onstage Incubator

Designed specifically for Invitrogen EVOS imaging systems, the Invitrogen EVOS Onstage Incubator is an environmental chamber that enables precise control of temperature, humidity, and three gases for time-lapse imaging of live cells under both physiological and nonphysiological conditions.

CellInsight CX7 LZR High-Content Screening Platform with HCA Onstage Incubator

The Invitrogen HCA Onstage Incubator for Thermo Scientific CellInsight HCA platforms allows precise control of temperature, humidity, and CO2 levels so that you may observe and measure biological activity and changes over time. Data gathered from longer-term imaging studies are the basis of quantitative analysis studies, especially when combined with Thermo Scientific HCS Studio Software for increased statistical power.

Tips

  • For short-term imaging experiments, use a large volume of imaging medium to prevent changes in osmolarity and oxygen resulting from evaporation of the medium.
  • To focus on a sample, start with a low magnification. This will minimize the time the sample is exposed to light.
  • Avoid using autofocus for every image taken during time-lapse imaging. Autofocus can increase the amount of light energy hitting the sample by as much as 10 times.
  • For longer time-course imaging or imaging of sensitive cells, an onstage incubator (OSI) may be added to the imaging equipment to allow precious control of temperature, humidity, and CO2 levels.

Step 1: Plan

Design your experiment with careful consideration of the tools and resources needed for each step.

Advantages

  • Observe dynamic cellular processes as they happen
  • Study and image several processes and functions simultaneously using multiplexed assays
  • Study cellular structures in their native environment, resulting in more realistic results closer to in vivo scenarios
  • Track cellular biomolecules and structures over time
  • Observe interactions between cells
  • Cellular enzymes and other cytosolic biomolecules remain in the cell

Considerations

  • Must have a specific way to label your target with minimal toxicity – whether it is a molecule, a cellular function, or a cellular state
  • Living cells are generally not permeable to large detection molecules such as antibodies
  • Moving objects can be more difficult to keep in focus
  • Certain techniques can be harmful to living cells
  • Cells must be kept in their natural physiological state

Step 2: Culture

Maintain or grow your cells in optimum conditions.

Keeping cells alive and healthy during various experimental manipulations, detection, and imaging is no small task. The choice of medium is particularly important for time-lapse imaging and experiments where cells are exposed to ambient conditions for longer periods. For reliable results with live cells, it is essential that the cells be healthy and kept in an environment as close as possible to physiological temperature, pH, oxygen level, and other conditions.

Learn more about cell culture

Product highlights

These media and wash buffers are created specifically for live-cell imaging and detection. Employing them in your experiments can help you improve image clarity, reduce background fluorescence, and optimize cell viability.

Tips

You can improve image clarity, reduce background fluorescence, and optimize cell viability by using media and wash buffers created specifically for live-cell imaging and detection. See product selection guide

Step 3: Label

Target cell structures, cell functions, and proteins of interest with selective dyes and stains.

The appropriate fluorophore (targeted fluorescent protein or small membrane-permeant reagent) should be used to monitor your target cellular structure or process. Additional fluorophores can be used to monitor multiple cellular structures and processes, but the excitation and emission spectra should be checked using the Fluorescence SpectraViewer to ensure minimal spectra overlap. It is critical to avoid using too much fluorescent label because excessive fluorescent labeling can result in:

  • Nonspecific staining with increased background signals
  • Physiological artifacts and structural perturbations
  • Cytotoxicity
  • Spectral overlap

Note that:

  • Live-cell structure reagents—help identify cellular components
  • Live-cell function reagents—help identify cellular functions and processes

Product highlights

  • Invitrogen CellLight reagents have proven to be the easiest to use for labeling specific structures in live cells. Targeted fluorescent proteins are introduced using the Invitrogen BacMam transduction system; no molecular biology techniques are required. Simply add the reagent to your cells, incubate overnight, and you’re ready to image in the morning.
  • Invitrogen CellTracker reagents are a diverse reagent class used for labeling mammalian cells to view changes in morphology or location. These nontoxic fluorescent dyes are designed to freely pass through cell membranes into cells, where they are transformed into cell-impermeant reaction products. Incubating cells with a CellTracker reagent for 30 minutes will provide at least 72 hours of fluorescent signal (typically three to six generations).
  • Invitrogen pHrodo indicators are fluorogenic dyes that dramatically increase in fluorescence as the pH of their surroundings becomes more acidic. When conjugated to dextrans, proteins, or other particles, pHrodo dyes can be used as highly specific sensors of endocytic and phagocytic internalization and lysosomal sequestration in live cells, offering a superior alternative to conjugates of other fluorescent dyes such as fluorescein and tetramethylrhodamine.

Tips

  • Consider using a longer wavelength fluorescent reagent if extended light exposure is required. This will require lower excitation power, which can correlate to lower phototoxicity and healthier cells.
  • Staining must be optimized for the particular assay readout, spectral compatibility, and signal-to-background ratio.
  • Removing the labeling solution and rinsing with fresh medium will reduce background fluorescence.

Step 4: Optimize

Minimize background and maintain photostability of fluorescence signals.

Signal-to-background ratio can be optimized by using reagents that reduce extracellular fluorescence and increase fluorophore photostability. It is important to image in media that have been specifically designed for maintaining cell health while reducing or eliminating background fluorescence in live-cell imaging experiments (see Table 1). The addition of a background suppressor compatible with live cells can also help reduce extracellular background fluorescence and eliminate the need for a wash step. Antifade mounting media for live cells can be applied to samples to reduce photobleaching of fluorophores, preventing signal loss with multiple or long exposures.

Table 1. Imaging media comparison.

ReagentCell washingShort-term imagingImaging up to 4 hoursLong-term imaging
Gibco PBS, pH 7.4  
Gibco FluoroBrite DMEM

Product highlights

Invitrogen BackDrop Background Suppressor imaging results

Live HeLa cells labeled with Tubulin Tracker Green dye and Tubulin Tracker Deep Red dye. Both labels show high off-cell background when the probe is left in the staining solution (left). Addition of BackDrop Background Suppressor greatly reduces extracellular background while leaving intracellular labeling unaffected (right), thus enabling a no-wash protocol for high-contrast imaging of tubulin in live cells.

Invitrogen ProLong Live Reagent confocal imaging results

The overall signal protection offered by ProLong Live reagentcompared to untreated samples is calculated based on the scan number where treated and untreated samples reach the EC50 value. The addition of ProLong Live reagent permitted 100% more captures with Invitrogen CellLight Mitochondria-RFP reagent.

Invitrogen ProLong Live Reagent fluorescence imaging

After 120 exposures using a standard time-lapse imaging protocol, samples treated with ProLong Live reagent are >20% brighter than untreated cells, enabling more data collection time.

Tips

  • If no further culture is planned, a background suppressor can be used to optimize the signal by reducing the haze and increasing the contrast.
  • The use of an antifade reagent has been shown to increase fluorophore photostability and decrease the effect of phototoxicity in a variety of sample types.

Step 5: Image

Live-cell imaging of dynamic processes requires active observation over time

Illumination and detection

To minimize phototoxicity, choose imaging systems that give you the greatest control of light sources. Try to minimize light intensity, exposure time, wavelength range, and amount of excitation energy for illuminating your cells while still generating a good signal with low background. Use the illumination that gives you the highest signal with the lowest level of fluorophore excitation. In some cases (particularly when you wish to image over a long period of time), it is advisable to sacrifice resolution by using shorter exposure times or lower magnification in exchange for healthier cells.

Live-cell imaging over longer periods of time can be challenging because the target may move out of focus during the course of the experiment. Many microscopes have autofocusing features that can help keep your target in focus longer and reduce focal drift. Additionally, maintaining cells at a constant temperature and keeping the volume of solution in the vessel constant will help with focal drift.

Environmental control

Many cells cannot tolerate deviations from their optimal temperature, osmolarity, pH, and humidity. Requirements vary depending on what experimental question you are asking. For example, experiments investigating cell growth and division may have a different set of requirements than experiments involving receptor activation and calcium accumulation. Some robust immortalized cell lines will tolerate being imaged or monitored for short periods of time without any environmental control. Conversely, for long-term imaging and detection studies, good results with both immortalized cells and primary cells typically require tightly controlled environmental parameters.

Catastrophic blebbing of the cell membrane

The top cell shows catastrophic blebbing of the cell membrane caused by excessive light exposure. Blebbing is a term used to describe membrane perturbation caused by toxicity. By contrast, the bottom cell remains relatively healthy and is not displaying aberrant morphology.

Product highlights

Countess II FL Automated Cell Counter

To avoid the pitfall of proceeding to the next step in your experiment with unhealthy cells, a quick check for cell health can be done on the Countess III FL Automated Cell Counter when used in conjunction with a variety of fluorescent reagents to detect cell viability, apoptosis, cytotoxicity, and transfection efficiency. The reusable slide option reduces consumption cost.

Invitrogen EVOS M5000 Imaging System with EVOS Onstage Incubator

Designed specifically for Invitrogen EVOS imaging systems, the Invitrogen EVOS Onstage Incubator is an environmental chamber that enables precise control of temperature, humidity, and three gases for time-lapse imaging of live cells under both physiological and nonphysiological conditions.

CellInsight CX7 LZR High-Content Screening Platform with HCA Onstage Incubator

The Invitrogen HCA Onstage Incubator for Thermo Scientific CellInsight HCA platforms allows precise control of temperature, humidity, and CO2 levels so that you may observe and measure biological activity and changes over time. Data gathered from longer-term imaging studies are the basis of quantitative analysis studies, especially when combined with Thermo Scientific HCS Studio Software for increased statistical power.

Tips

  • For short-term imaging experiments, use a large volume of imaging medium to prevent changes in osmolarity and oxygen resulting from evaporation of the medium.
  • To focus on a sample, start with a low magnification. This will minimize the time the sample is exposed to light.
  • Avoid using autofocus for every image taken during time-lapse imaging. Autofocus can increase the amount of light energy hitting the sample by as much as 10 times.
  • For longer time-course imaging or imaging of sensitive cells, an onstage incubator (OSI) may be added to the imaging equipment to allow precious control of temperature, humidity, and CO2 levels.

Featured webinar

Best practices: 5 steps to live-cell imaging

Whether you are new to live-cell imaging or you are an experienced researcher, this webinar will show you how to obtain publication-quality live-cell images every time while avoiding the frustration of wasted time and resources.

Examples of live-cell images and videos

Video gallery for live-cell imaging

Live-cell imaging resources

For additional information and data, please see the following posters:

Poster: New generation sensors for caspase activation and mitochondrial superoxide in live cell microscopy
Poster: New and improved cellular health evaluation of 2D and 3D cellular models using microplate reader assays
Poster: Hypoxia measurements in live and fixed cells using fluorescence microscopy and high-content imaging
Poster: Evaluation of Cellular Senescence through Fluorescence Characterization

*Incucyte® Live-Cell Analysis System is trademarked or registered trademarks of Essen BioScience. Incucyte, Essen BioScience, and all names of Essen BioScience products are registered trademarks and the property of Essen BioScience unless otherwise specified. Essen BioScience is a Sartorius Company. MuviCyte™ live-cell imaging system is trademarked or registered trademarked of PerkinElmer. THUNDER Imager Live Cell & 3D Assay and MICA Microhub Microscope are sold by Leica Microsystems CMS GmbH.

 

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