Ratiometric Indicators
Ratiometric Indicators - Related Videos
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...
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.
07:47
Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator
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2021
This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.
02:39
Indicators
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2020
Certain organic substances change color in dilute solution when the hydronium ion concentration reaches a particular value. For example, phenolphthalein is a colorless substance in any aqueous solution with a hydronium ion concentration greater than 5.0 × 10−9 M (pH < 8.3). In more basic solutions where the hydronium ion concentration is less than 5.0 × 10−9 M (pH > 8.3), it is red or pink. Substances such as phenolphthalein, which can be used to determine the pH of a solution, are called...
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.
FAQs
A ratio can reduce the influence of factors that affect signal intensity without representing a biological change. Variations in probe concentration, illumination, sample thickness, or focal conditions may alter both measured channels, while their relationship can remain more informative. This makes the calculated value less dependent on optical variability and better suited for interpreting intracellular signals in neural tissue.
The biological analyte changes how the indicator responds to light, either by altering excitation behavior or by shifting emission behavior. Researchers measure the resulting responses at two wavelengths and compare them rather than relying on one intensity value. Changes in the ratio can then serve as an estimate of intracellular conditions, including calcium or pH.
Ratiometric measurements can help separate genuine changes in neural signaling from intensity changes caused by movement, uneven labeling, or changing focal conditions. These effects may make a single fluorescence channel appear to change even when intracellular physiology has not. Comparing channels provides a way to assess whether an apparent response is more consistent with biology or with altered optical conditions.
The workflow begins by measuring the indicator’s optical responses in two wavelength channels during the experiment. Researchers then calculate the ratio between those signals and examine how it changes over time or across the sample. The resulting pattern is interpreted as an estimate of an intracellular condition, such as calcium or pH, while considering optical factors that could affect the measurement.
They are particularly useful when researchers need to monitor intracellular conditions in complex tissue preparations, where illumination, labeling, thickness, movement, or focal conditions may vary. In neuroscience, the approach supports imaging of calcium or pH and helps relate those measurements to neural signaling and cellular physiology. It is therefore relevant to experiments examining activity across nonuniform neural samples.
Changes in the calculated optical ratio can provide estimates of intracellular calcium or pH, depending on the indicator used. These measurements help researchers examine cellular physiology and neural signaling while reducing confusion from changes in imaging conditions. In activity studies, the approach can support interpretation of spatial or temporal signal changes as possible indicators of neuronal activity rather than purely optical artifacts.