Live cell imaging

The health of the cells needs to be maintained, especially in long term imaging over hours or days to prevent changes in metabolic function that could alter the process being observed. To do this, physiological pH, oxygenation, temperature and osmolarity are controlled. Since most cells and tissues do not normally encounter light exposure, and UV light is known to cause DNA damageDNA damage, experimenters should minimize the quantity of illumination the cells receive to avoid phototoxicity. Also the excitation of fluorescent proteins and dye molecules releases free radicals that interact with cellular components. Illumination can be limited by using the lowest amounts of a fluorescent probe, using phenol red-free medium and efficient microscope design with an optimal light path and optimized detectors. Controls need to be performed in each experiment to check that the imaging process has not affected cellular function.

Stem cells are imaged in their native environment in model organisms or explants from model organisms such as mouse, zebrafish, fruit fly (DrosophilaDrosophila) and nematode worms (Caenorhabditis elegans). Traditional methods involve labeling individual cells or groups of cells followed by fixation of tissue at certain time points. Live cell imaging allows the behavior of cells to be directly observed, such as following individual cell fate choices over time, which can provide valuable information about the process of self-renewal and tissue maintenance.

Tracking of RNARNA in live cells is a way to study cellular processes that occur during development or due to changes to environment or various stimuli. MS2 labeling of RNA is a method that tags an RNA of interest with a stem-loop RNA sequence derived from the bacteriophage MS2 genome which binds to bacteriophage coat protein. The coat protein is fused to a fluorescent protein so that it can be visualized and it binds to the stem-loop tagged RNA. CRISPR/Cas tools have been used to insert an MS2 cassette to tag stem cell transcription factor Esrrb in mouse embryonic stem cells.

The discovery of green fluorescent protein (GFP) naturally occurring in jellyfish by Osamu Shimomura in 1962 revolutionized live cell imaging. After the GFP gene was cloned in 1992, mutants were created to produce different color variants and improve fluorescence signal and photostability. In 2008 the Nobel PrizeNobel Prize in Chemistry was awarded to Shimomura, Martin Chalfie and Roger Tsien for their work on the genetically encoded fluorescent proteins. GFP and other fluorescent proteins are introduced into the cell as a gene fused with a gene that codes for a protein of interest. Fusion proteins can keep their functional properties while having fluorescence properties that allow them to be analysed with live imaging.

Stem cells are imaged in their native environment in model organisms or explants from model organisms such as mouse, zebrafish, fruit fly (Drosophila) and nematode worms (Caenorhabditis elegansCaenorhabditis elegans). Traditional methods involve labeling individual cells or groups of cells followed by fixation of tissue at certain time points. Live cell imaging allows the behavior of cells to be directly observed, such as following individual cell fate choices over time, which can provide valuable information about the process of self-renewal and tissue maintenance.

Live cell imaging allows the observation of dynamic changes. These techniques are less prone to experimental artifacts as compared to imaging fixed cells. Live cell imaging is often used to examine structural components of the cell, to study dynamic processes and to see where different molecules are localized within the cell. It is possible to use live cell imaging to monitor cellular integrity, endocytosis, exocytosis, protein trafficking, signal transduction and enzyme activity. The movement of molecules in response to environmental cues can be studied with live cell imaging. Some imaging systems can follow cells within live animals, such as tracing neural stem cell and progenitor cellsprogenitor cells in the mouse brain. Tissue repair is an area of research that has benefited from live cell imaging approaches.

The discovery of green fluorescent protein (GFP) naturally occurring in jellyfish by Osamu ShimomuraOsamu Shimomura in 1962 revolutionized live cell imaging. After the GFP gene was cloned in 1992, mutants were created to produce different color variants and improve fluorescence signal and photostability. In 2008 the Nobel Prize in Chemistry was awarded to Shimomura, Martin Chalfie and Roger Tsien for their work on the genetically encoded fluorescent proteins. GFP and other fluorescent proteins are introduced into the cell as a gene fused with a gene that codes for a protein of interest. Fusion proteins can keep their functional properties while having fluorescence properties that allow them to be analysed with live imaging.

The discovery of green fluorescent proteingreen fluorescent protein (GFP) naturally occurring in jellyfish by Osamu Shimomura in 1962 revolutionized live cell imaging. After the GFP gene was cloned in 1992, mutants were created to produce different color variants and improve fluorescence signal and photostability. In 2008 the Nobel Prize in Chemistry was awarded to Shimomura, Martin Chalfie and Roger Tsien for their work on the genetically encoded fluorescent proteins. GFP and other fluorescent proteins are introduced into the cell as a gene fused with a gene that codes for a protein of interest. Fusion proteins can keep their functional properties while having fluorescence properties that allow them to be analysed with live imaging.

The discovery of green fluorescent protein (GFP) naturally occurring in jellyfish by Osamu Shimomura in 1962 revolutionized live cell imaging. After the GFP gene was cloned in 1992, mutants were created to produce different color variants and improve fluorescence signal and photostability. In 2008 the Nobel Prize in ChemistryChemistry was awarded to Shimomura, Martin Chalfie and Roger Tsien for their work on the genetically encoded fluorescent proteins. GFP and other fluorescent proteins are introduced into the cell as a gene fused with a gene that codes for a protein of interest. Fusion proteins can keep their functional properties while having fluorescence properties that allow them to be analysed with live imaging.

The discovery of green fluorescent protein (GFP) naturally occurring in jellyfish by Osamu Shimomura in 1962 revolutionized live cell imaging. After the GFP gene was cloned in 1992, mutants were created to produce different color variants and improve fluorescence signal and photostability. In 2008 the Nobel Prize in Chemistry was awarded to Shimomura, Martin ChalfieMartin Chalfie and Roger Tsien for their work on the genetically encoded fluorescent proteins. GFP and other fluorescent proteins are introduced into the cell as a gene fused with a gene that codes for a protein of interest. Fusion proteins can keep their functional properties while having fluorescence properties that allow them to be analysed with live imaging.

Tracking of RNA in live cells is a way to study cellular processes that occur during development or due to changes to environment or various stimuli. MS2 labeling of RNA is a method that tags an RNA of interest with a stem-loop RNA sequence derived from the bacteriophage MS2bacteriophage MS2 genome which binds to bacteriophage coat protein. The coat protein is fused to a fluorescent protein so that it can be visualized and it binds to the stem-loop tagged RNA. CRISPR/Cas tools have been used to insert an MS2 cassette to tag stem cell transcription factor Esrrb in mouse embryonic stem cells.

Organic fluorescent molecules are coupled to biomolecules lielike peptides, proteins, oligonucleotides or cell components by chemical reaction or by intermediate attachment with a peptide or antibody specific to the biomolecule. Alexa Fluors are a family of organic dyes which are sulfonated rhodamine derivatives commercially available from Molecular Probes. Cyanine, Cy2, Cy3, Cy5, Cy7 are another family of stable dyes that are commercially available.

Tracking of RNA in live cells is a way to study cellular processes that occur during development or due to changes to environment or various stimuli. MS2 labeling of RNA is a method that tags an RNA of interest with a stem-loop RNA sequence derived from the bacteriophage MS2 genome which binds to bacteriophage coat protein. The coat protein is fused to a fluorescent protein so that it can be visualized and it binds to the stem-loop tagged RNA. CRISPR/Cas tools have been used to insert an MS2 cassette to tag stem cell transcription factor Esrrb in mouse embryonic stem cells.

Live cell imaging allows the observation of dynamic changes. These techniques are less prone to experimental artifacts as compared to imaging fixed cells. Live cell imaging is often used to examine structural components of the cell, to study dynamic processes and to see where different molecules are localized within the cell. It is possible to use live cell imaging to monitor cellular integrity, endocytosis, exocytosis, protein trafficking, signal transduction and enzyme activity. The movement of molecules in response to environmental cues can be studied with live cell imaging. Some imaging systems can follow cells within live animals, such as tracing neural stem cell and progenitor cells in the mouse brain. Tissue repair is an area of research that has benefited from live cell imaging approaches.

Stem cells are imaged in their native environment in model organisms or explants from model organisms such as mouse, zebrafish, fruit fly (Drosophila) and nematode worms (Caenorhabditis elegans). Traditional methods involve labeling individual cells or groups of cells followed by fixation of tissue at certain time points. Live cell imaging allows the behavior of cells to be directly observed, such as following individual cell fate choices over time, which can provide valuable information about the process of self-renewal and tissue maintenance.

Using lineage analysis of fixed samples in the brain it was not clear whether neural stem cells (NSCs) could self-renew indefinitely or whether they get depleted. In adult zebrafish brain, multiphoton imaging was performed to follow NSCs over a period of one month. The imaging revealed that the majority of NSCs sustain their numbers by asymmetric divisions where one daughter cell remains a stem cell and one daughter cell commits to differentiation. The live imaging showed that some NSCs differentiate directly into neural progenitors, which supports the hypothesis that NSCs can be depleted over time.

Live imaging of zebrafish tissue and fetal liver in mouse has allowed the observation of endothelial niche cells seeming to provide a niche environment by remodelling to surround hematopoietic stem cells (HSCs) which have newly arrived to the area. The HSCs were observed to remain inside these structures.

Live cell imaging allows the observation of dynamic changes. These techniques are less prone to experimental artifacts as compared to imaging fixed cells. Live cell imaging is often used to examine structural components of the cell, to study dynamic processes and to see where different molecules are localized within the cell. It is possible to use live cell imaging to monitor cellular integrity, endocytosis, exocytosis, protein trafficking, signal transduction and enzyme activity. The movement of molecules in response to environmental cues can be studied with live cell imaging. Some imaging systems can follow cells within live animals, such as tracing neural stem cell and progenitor cells in the mouse brain.

Live cell microscopy approaches include transmission microscopy (bright field, dark field, phase contrast, differential interference contrast), epifluorescence, confocal, spinning disk, multiphoton microscopy, light sheet microscopy, ion imaging, Forster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), fluorescence recovery after photo-bleaching (FRAP), fluorescence correlation spectroscopy (FCS), single particle tracking (SPT) and SPT photoactivated localisation microscopy (sptPALM).

The health of the cells needs to be maintained, especially in long term imaging over hours or days to prevent changes in metabolic function that could alter the process being observed. To do this, physiological pH, oxygenation, temperature and osmolarity are controlled. Since most cells and tissues do not normally encounter light exposure, and UV light is known to cause DNA damage, experimenters should minimize the quantity of illumination the cells receive to avoid phototoxicity. Also the excitation of fluorescent proteins and dye molecules releases free radicals that interact with cellular components. Illumination can be limited by using the lowest amounts of a fluorescent probe, using phenol red-free medium and efficient microscope design with an optimal light path and optimized detectors. Controls need to be performed in each experiment to check that the imaging process has not affected cellular function.

Fluorescent probes are designed to penetrate the cell without causing damage, which can be done with esterification of dyes, synthetic vesicles and mechanical techniques like microinjection and electroporation.

The discovery of green fluorescent protein (GFP) naturally occurring in jellyfish by Osamu Shimomura in 1962 revolutionized live cell imaging. After the GFP gene was cloned in 1992, mutants were created to produce different color variants and improve fluorescence signal and photostability. In 2008 the Nobel Prize in Chemistry was awarded to Shimomura, Martin Chalfie and Roger Tsien for their work on the genetically encoded fluorescent proteins. GFP and other fluorescent proteins are introduced into the cell as a gene fused with a gene that codes for a protein of interest. Fusion proteins can keep their functional properties while having fluorescence properties that allow them to be analysed with live imaging.

GFP and its derivatives lose fluorescence over time under illumination and also can aggregate. Subsequent developments in fluorescent labeling molecules include photoactivatable, photoconvertible or photo-switchable fluorescent proteins such as PA-GFP, mEOS or Dronpa.

Organic fluorescent molecules are coupled to biomolecules lie peptides, proteins, oligonucleotides or cell components by chemical reaction or by intermediate attachment with a peptide or antibody specific to the biomolecule. Alexa Fluors are a family of organic dyes which are sulfonated rhodamine derivatives commercially available from Molecular Probes. Cyanine, Cy2, Cy3, Cy5, Cy7 are another family of stable dyes that are commercially available.

Direct labeling of proteins entails the incorporation of labels, often at the N-terminus or Cys/Lys side chains of a peptide whereas indirect labeling is the use of peptide tags or protein tags conjugated to organic fluorophores.