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
Live HeLa cells labeled with Tubulin Tracker Deep Red and NucBlue Live ReadyProbes Reagent, enabling visualization of the microtubule cytoskeleton and nuclei in live cells, respectively. High resolution images were acquired using a confocal system.
HeLa cells labeled using Invitrogen Tubulin Tracker Green dye and Invitrogen MitoTracker Red CMXRos dye show superb multiplexing capability and uniformity of staining compared with overexpressed fluorescent protein fusions. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 20X Super Apochromat objective using GFP and Texas Red EVOS light cubes.
HeLa cells labeled using NucBlue Live ReadyProbes Reagent, Tubulin Tracker Green dye, and Invitrogen LysoTracker Deep Red dye show superb multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Super Apochromat Oil objective using DAPI, GFP, and Cy5 EVOS light cubes.
U2OS cells labeled using Invitrogen LysoTracker Blue DND22 dye, Invitrogen MitoTracker Green FM dye, and Tubulin Tracker Deep Red dye show superb multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Super Apochromat Oil objective using DAPI, GFP, and Cy5 EVOS light cubes.
HeLa cells labeled using NucBlue Live ReadyProbes Reagent, Tubulin Tracker Green, and CellLight Mitochondria-RFP show superb multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Super Apochromat Oil objective> using DAPI, GFP, and RFP EVOS light cubes.
HeLa cells labeled using NucBlue Live ReadyProbes Reagent, ER-Tracker Green, and Tubulin Tracker Deep Red show superb multiplexing capability and uniformity of staining compared with overexpressed fluorescent protein fusions. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 20X Super Apochromat objective using DAPI, GFP, and Cy5 EVOS light cubes.
U2OS cells labeled using NucBlue Live ReadyProbes Reagent, CellLight Actin-GFP, CellLight Mitochondria-RFP, and Tubulin Tracker Deep Red show superb multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Super Apochromat Oil objective> using DAPI, GFP, RFP, and Cy5 EVOS light cubes.
HeLa cells grown in MEM with 10% FBS were transduced with CellLight Mitochondria-RFP and CellLight Golgi-RFP. After 24 hours, nuclei were counter stained with NucBlue Live ReadyProbes Reagent. Image was captured on an EVOS FL Auto 2 Imaging System using a 60X Apo objective and DAPI, GFP and RFP LED light cubes.
Four-plex image of COS7 live cell. Tubulin is labeled with Invitrogen Tubulin Tracker Green dye, mitochondria are labeled with Invitrogen MitoTracker Orange dye, plasma membrane is labeled with Invitrogen CellMask Deep Red dye, and nucleus is stained blue with Hoechst 33342 reagent. Image was taken with a Zeiss ELYRA S.1 microscope using Structured Illumination Microscopy (SIM). Image is provided to Thermo Fisher Scientific courtesy of M. Sauer and L. Behringer-Pließ, Biocenter University Wuerzburg.
Four-plex image of U2OS live cells. Tubulin is labeled with Tubulin Tracker Green dye, mitochondria are labeled with MitoTracker Orange dye, plasma membrane is labeled with CellMask Deep Red dye, and nuclei are stained blue with Hoechst 33342 reagent. Image was taken with an ELYRA S.1 microscope using Structured Illumination Microscopy (SIM). Image is provided to Thermo Fisher Scientific courtesy of M. Sauer and L. Behringer-Pließ, Biocenter University Wuerzburg.
HeLa cells were transduced with Invitrogen CellLight Mitochondria-RFP red dye and CellLight Talin-GFP green dye for 24 hours, then labeled with Invitrogen NucBlue Live ReadyProbes Reagent-Hoechst 33342 for 15 minutes. For photobleach protection, cells were incubated with Invitrogen ProLong Live Antifade Reagent for 90 minutes before imaging on an Invitrogen EVOS cell imaging system.
Three-plex image of COS7 live cell. Tubulin is labeled with Tubulin Tracker Green dye, plasma membrane is labeled with CellMask Orange dye, and nucleus is stained blue with Hoechst 33342 reagent . Image was taken using an ELYRA S.1 microscope using Structured Illumination Microscopy (SIM). Image is provided to Thermo Fisher Scientific courtesy of M. Sauer and L. Behringer-Pließ, Biocenter University Wuerzburg.
HeLa cells were transduced with CellLight Mitochondria-RFP red dye and CellLight Talin-GFP green dye for 24 hours, then labeled with NucBlue Live ReadyProbes Reagent-Hoechst 33342 for 15 minutes. For photobleach protection, cells were incubated with ProLong Live Antifade Reagent for 90 minutes before imaging on an EVOS cell imaging system.
Free guide: 5 steps for live-cell imaging
Live-cell imaging product selection guide
Cultureware, media, buffers
Extracellular matrices
Transfection
Growth factors
EVOS DAPI Light Cube (AMEP4650)
Excitation: 357/44 nm;
Emission: 447/60 nm
| Cell structure | |
|---|---|
| Plasma membrane | |
| Nucleus | |
| Cytoskeleton | |
| Cell tracking | |
| Cell function | |
| Viability | |
| Oxidative stress detection | |
| 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 | |
EVOS Red Light Cube (AMEP4655)
Excitation: 585/29 nm;
Emission: 624/40 nm
| Cell structure | |
|---|---|
| Nucleus | |
| Endoplasmic reticulum | |
| Mitochondria | |
| Cell tracking | |
| Cell function | |
| Viability | |
| Oxidative stress detection | |
| Endocytosis (E) and phagocytosis (P) | |
| Antibody internalization | |
| Proliferation | |
| Ion (I) and membrane (M) potential indicators | |
EVOS Cy5 Light Cube (AMEP4656)
Excitation: 628/40 nm;
Emission: 693/40 nm
| Cell structure | |
|---|---|
| Plasma membrane | |
| Nucleus | |
| Cytoskeleton | |
| Lysosomes | |
| Mitochondria | |
| Cell tracking | |
| Cell function | |
| Oxidative stress detection | |
| Endocytosis (E) and phagocytosis (P) | |
| Proliferation | |
| Antibody internalization | |
Media and solutions
Background suppressor
Mountant and antifade
Cultureware, media, buffers
Extracellular matrices
Transfection
Growth factors
EVOS DAPI Light Cube (AMEP4650)
Excitation: 357/44 nm;
Emission: 447/60 nm
| Cell structure | |
|---|---|
| Plasma membrane | |
| Nucleus | |
| Cytoskeleton | |
| Cell tracking | |
| Cell function | |
| Viability | |
| Oxidative stress detection | |
| 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 | |
EVOS Red Light Cube (AMEP4655)
Excitation: 585/29 nm;
Emission: 624/40 nm
| Cell structure | |
|---|---|
| Nucleus | |
| Endoplasmic reticulum | |
| Mitochondria | |
| Cell tracking | |
| Cell function | |
| Viability | |
| Oxidative stress detection | |
| Endocytosis (E) and phagocytosis (P) | |
| Antibody internalization | |
| Proliferation | |
| Ion (I) and membrane (M) potential indicators | |
EVOS Cy5 Light Cube (AMEP4656)
Excitation: 628/40 nm;
Emission: 693/40 nm
| Cell structure | |
|---|---|
| Plasma membrane | |
| Nucleus | |
| Cytoskeleton | |
| Lysosomes | |
| Mitochondria | |
| Cell tracking | |
| Cell function | |
| Oxidative stress detection | |
| Endocytosis (E) and phagocytosis (P) | |
| Proliferation | |
| Antibody internalization | |
Media and solutions
Background suppressor
Mountant and antifade
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.
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.
- Thermo Scientific Nunc cell culture vessels with Nunclon Delta surface treatment have been validated with Gibco media to confirm consistent cell growth across multiple cell lines. It’s a proven combination for happy cells and happy scientists.
- Gibco extracellular matrices, scaffolds, and proteins provide in vivo–like morphology and physiologically relevant environments for more realistic cell biology and better intercellular interactions.
- Invitrogen Countess II FL Automated Cell Counter is an affordable and automated tool for checking the health of your cells quickly and objectively. It has reusable or disposable slide options.
TipsYou 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
|
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 is used when observing high background signal or weak fluorescence in the blue, green, or red channels.
- Invitrogen ProLong Live Antifade Reagent is used to increase fluorophore photostability.
- Gibco PBS, pH 7.4 is ideal for cell washing and short-term imaging where prolonged incubation is not required.
- Invitrogen Live Cell Imaging Solution is an optically clear solution used for imaging, dye loading, and wash steps. It helps keep cells healthy for up to 4 hours.
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.
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.
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
|
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.
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.
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
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.
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.
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
|
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.
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.
- Thermo Scientific Nunc cell culture vessels with Nunclon Delta surface treatment have been validated with Gibco media to confirm consistent cell growth across multiple cell lines. It’s a proven combination for happy cells and happy scientists.
- Gibco extracellular matrices, scaffolds, and proteins provide in vivo–like morphology and physiologically relevant environments for more realistic cell biology and better intercellular interactions.
- Invitrogen Countess II FL Automated Cell Counter is an affordable and automated tool for checking the health of your cells quickly and objectively. It has reusable or disposable slide options.
TipsYou 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
|
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 is used when observing high background signal or weak fluorescence in the blue, green, or red channels.
- Invitrogen ProLong Live Antifade Reagent is used to increase fluorophore photostability.
- Gibco PBS, pH 7.4 is ideal for cell washing and short-term imaging where prolonged incubation is not required.
- Invitrogen Live Cell Imaging Solution is an optically clear solution used for imaging, dye loading, and wash steps. It helps keep cells healthy for up to 4 hours.
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.
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.
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
|
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.
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.
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
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.
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.
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
|
Assess cell health using microplate reader assays
Invitrogen microplate reader assays to assess cell health 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.
Color your way through 30 fantastic illustrations inspired by actual cell images submitted by researchers around the world. Don’t wait! Download the free coloring book that helps inspire your creative scientific expression.
Resources
5 Steps Resources
Support
Application notes
Scientific posters
For Research Use Only. Not for use in diagnostic procedures.
