Ratiometric Pericam
Ratiometric Pericam - Related Videos
13:05
Ratiometric Imaging of Extracellular pH in Dental Biofilms
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Cited by 24 •
2016
A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.
14:39
Measuring Phagosome pH by Ratiometric Fluorescence Microscopy
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Cited by 26 •
2015
Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.
12:42
Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator
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Cited by 13 •
2017
This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.
02:39
Imaging the Mitochondrial Redox State in Primary Neurons Using a Ratiometric Indicator
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2025
Source: Katsalifis, A., et al. Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator. J. Vis. Exp. (2021)This video demonstrates the imaging of mitochondrial redox state in primary neurons using a ratiometric fluorescent indicator. By applying two excitation wavelengths under a fluorescence microscope, the fluorescence intensity ratio is measured to assess mitochondrial oxidation. The protocol includes N-methyl-D-aspartate (NMDA) stimulation...
13:38
Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET
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Cited by 21 •
2012
FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.
FAQs
Ca2+ binding changes the conformation of the sensor formed by circularly permuted green fluorescent protein, calmodulin, and the M13 peptide. This structural rearrangement changes how the fluorescent protein responds to excitation, producing an excitation-dependent shift in fluorescence. The resulting change provides a molecular connection between intracellular calcium binding and the measured optical signal.
The two-wavelength measurement allows researchers to calculate a fluorescence ratio rather than rely on absolute intensity alone. This ratio reduces errors associated with differences in probe concentration, illumination, or cell movement. As a result, changes in the signal more reliably represent intracellular Ca2+ dynamics, even when imaging conditions introduce intensity variation.
A single-intensity measurement can change because of calcium levels or because the amount of probe, illumination, or position of the cell changes. Ratiometric Pericam compares fluorescence intensities produced by two excitation wavelengths, helping separate calcium-dependent signal changes from these technical influences. This makes the measurement more robust for monitoring calcium dynamics in living biological samples.
Researchers image the sensor in living cells or tissues while acquiring fluorescence signals at two excitation wavelengths. They then compare the intensities by calculating their ratio and follow how that value changes over time. Transient ratio changes can be interpreted as changes in intracellular Ca2+, allowing real-time calcium dynamics to be related to cellular activity.
The indicator is useful when researchers need to observe intracellular Ca2+ changes in real time within living cells or tissues. Its measurements can help connect transient calcium signals with neuronal activity, secretion, and cell regulation. The approach therefore supports studies that examine calcium dynamics alongside biological processes rather than measuring calcium as an isolated endpoint.
Ratiometric Pericam can help researchers examine how transient intracellular Ca2+ changes relate to neuronal activity, secretion, and broader cell regulation. By recording these changes over time in living cells or tissues, the method provides a way to associate calcium signaling patterns with ongoing biological events. Its value lies in linking dynamic signals to cellular function.