Intracellular Fluorescence
Intracellular Fluorescence - Related Videos
11:17
Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes
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Cited by 44 •
2014
Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.
11:54
Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator
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Cited by 9 •
2017
Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.
08:14
Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles
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Cited by 1 •
2015
This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.
08:55
Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy
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Cited by 5 •
2017
This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on Förster resonance energy transfer, and fluorescence lifetime imaging microscopy.
07:18
Intracellular Refolding Assay
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Cited by 9 •
2012
In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.
FAQs
Fluorophores first absorb light at a particular excitation wavelength and then emit light at a longer wavelength. This shift enables fluorescence microscopy to record the emitted signal and identify where the probe is located. The resulting image can also indicate relative signal intensity within different cellular regions, supporting analysis of molecular distribution and biological activity.
Probe selection determines which cellular feature or event becomes visible, while labeling places the fluorescent signal at the relevant target. Imaging conditions then influence specificity, background signal, and photobleaching, the loss of fluorescence during observation. Balancing these factors helps preserve a detectable, target-associated signal rather than obscuring it with unwanted fluorescence.
Living and fixed cells support different observational goals. Fixed cells can reveal cellular structures and organelle organization, whereas living cells permit observation of dynamic events such as signaling or transport. This distinction guides experimental design: researchers choose the cell state according to whether spatial organization or ongoing biological activity is the main outcome.
A practical workflow begins by selecting a probe suited to the structure, molecule, or event of interest. Researchers then label the cells, establish imaging conditions, and use fluorescence microscopy to record emitted light. Reviewing probe location and signal intensity helps connect the image with protein localization, organelle organization, gene expression, or another measured cellular feature.
Intracellular fluorescence can address questions about where proteins reside, how organelles are organized, whether gene expression is occurring, and how ion concentrations change. It also supports observation of signaling and transport, particularly when researchers need to follow processes rather than only map static cellular features. The selected probe determines which question the image can address.
In disease research and drug development, the technique provides a way to examine cellular organization, molecular localization, and biological activity inside cells. Researchers can use changes in fluorescence location or intensity to investigate effects on signaling, transport, proteins, organelles, gene expression, or ion concentrations. Its value depends on maintaining specificity and limiting background and photobleaching.