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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.
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
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.
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.
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 |
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:
Tips
|
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 | ![]() | ![]() | ![]() | ![]() |
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
|
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.
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.
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
|
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.
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.
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 |
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:
Tips
|
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 | ![]() | ![]() | ![]() | ![]() |
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
|
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.
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.
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
|
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.
For Research Use Only. Not for use in diagnostic procedures.