Microscopic view of fluorescently stained cell

Fluorescence imaging of live cells is a powerful approach to the study of dynamic cellular processes and events. Recent advances in fluorescent dye development and imaging technology have resulted in the widespread adoption of using live-cell imaging in many diverse areas, such as developmental and stem cell biology, medical research, drug discovery, and environmental studies.

Whether you are new to cell imaging or an experienced researcher wanting to confirm your knowledge, consider these five proven steps to help ensure that your live-cell images are publication-ready the first time.

With 40 years dedicated to cell imaging research, Invitrogen imaging reagents and antibodies are cited more frequently in published research than any others. Leverage our experience to enable your success and avoid costly wrong turns. We are here to help.

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.


Image gallery for live-cell imaging

Free guide: 5 steps for live-cell imaging

5 steps to live-cell imaging cover Reagent wheel

Live-cell imaging product selection guide

EVOS GFP Light Cube (AMEP4651)
Excitation: 470/22 nm;
Emission: 510/42 nm
Cell structure
Plasma membrane
Nucleus
Cytoskeleton
Endoplasmic reticulum
Lysosomes
Mitochondria
Cell tracking
Cell function
Viability
Oxidative stress detection
Apoptosis (Ap) and autophagy (Au)
Endocytosis (E) and phagocytosis (P)
Antibody internalization
Proliferation
Ion (I) and membrane (M) potential indicators
EVOS RFP Light Cube (AMEP4652)
Excitation: 531/40 nm;
Emission: 593/40 nm
Cell structure
Plasma membrane
Nucleus
Cytoskeleton
Endoplasmic reticulum
Lysosomes
Mitochondria
Cell tracking
Cell function
Viability
Oxidative stress detection
Apoptosis (Ap) and autophagy (Au)
Endocytosis (E) and phagocytosis (P)
Antibody internalization
Proliferation
Ion (I) and membrane (M) potential indicators
EVOS GFP Light Cube (AMEP4651)
Excitation: 470/22 nm;
Emission: 510/42 nm
Cell structure
Plasma membrane
Nucleus
Cytoskeleton
Endoplasmic reticulum
Lysosomes
Mitochondria
Cell tracking
Cell function
Viability
Oxidative stress detection
Apoptosis (Ap) and autophagy (Au)
Endocytosis (E) and phagocytosis (P)
Antibody internalization
Proliferation
Ion (I) and membrane (M) potential indicators
EVOS RFP Light Cube (AMEP4652)
Excitation: 531/40 nm;
Emission: 593/40 nm
Cell structure
Plasma membrane
Nucleus
Cytoskeleton
Endoplasmic reticulum
Lysosomes
Mitochondria
Cell tracking
Cell function
Viability
Oxidative stress detection
Apoptosis (Ap) and autophagy (Au)
Endocytosis (E) and phagocytosis (P)
Antibody internalization
Proliferation
Ion (I) and membrane (M) potential indicators

Follow these 5 steps to capture the best possible live-cell images

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.

Reagent Cell washing Short-term imaging Imaging up to 4 hours Long-term imaging
Gibco PBS, pH 7.4    
Invitrogen Live Cell Imaging Solution  
Gibco FluroBrite 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 reagent compared 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.

HDFn cells loaded with CellTracker Deep Red Dye
 Click image to enlarge

A scratch wound in a culture of HDFn cells loaded with Invitrogen CellTracker Deep Red Dye. (A) The illuminated area was subjected to repeated illumination for 10 hours. Cells in this area show signs of phototoxicity (a loss of viability as cells were not able to grow into the wound). (B) Cells in the non-illuminated area show viable cell growth into the wound.

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 II 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.

Reagent Cell washing Short-term imaging Imaging up to 4 hours Long-term imaging
Gibco PBS, pH 7.4    
Invitrogen Live Cell Imaging Solution  
Gibco FluroBrite 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 reagent compared 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.

HDFn cells loaded with CellTracker Deep Red Dye
 Click image to enlarge

A scratch wound in a culture of HDFn cells loaded with Invitrogen CellTracker Deep Red Dye. (A) The illuminated area was subjected to repeated illumination for 10 hours. Cells in this area show signs of phototoxicity (a loss of viability as cells were not able to grow into the wound). (B) Cells in the non-illuminated area show viable cell growth into the wound.

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 II 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.

  

Assess cell health before imaging

Invitrogen cell health assays have shown excellent results on the Thermo Scientific Varioskan LUX Multimode Microplate Reader equipped with gas module, which can read a 96-well plate in as little as six seconds.

Varioskan LUX Multimode Microplate Reader

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