Dual-color Photometry
Dual-color Photometry - Related Videos
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Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy
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Cited by 12 •
2014
Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.
07:49
Simultaneous Photothrombosis and Fiber Photometry to Induce and Monitor Ischemic Stroke in Behaving Mice
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2025
Using an all-optical approach, ischemic stroke is induced while simultaneously recording neuronal Ca2+ activity using a fluorescent biosensor in the emerging stroke core of an awake, behaving mouse.
10:25
Dual-color Correlative Light and Electron Microscopy for the Visualization of Interactions between Mitochondria and Lysosomes
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Cited by 4 •
2024
This protocol describes a mechanism for using correlative light and electron microscopy to visualize the interaction of mitochondria and lysosomes labeled with mEosEM and APEX2, respectively.
05:16
Mass Photometry to Study Antigen-Antibody Interactions on a Single-Molecule Level
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2025
In this video, we demonstrate the use of mass photometry to study antigen-antibody interactions in a flow chamber. The focused light scatters differently based on the mass of the molecules. A detector collects the scattered light and generates the interference pattern as spots of varied intensities, which correlate with the molecular masses.
09:20
Color Afterimages
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2023
Source: Laboratory of Jonathan Flombaum—Johns Hopkins University Human color vision is impressive. People with normal color vision can tell apart millions of individual hues. Most amazingly, this ability is achieved with fairly simple hardware. Part of the power of human color vision comes from a clever bit of engineering in the human brain. There, color perception relies on what is known as an 'opponent system.' This means that the presence of one kind of stimulus is treated as evidence for...
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Each genetically encoded indicator is associated with a different excitation wavelength, and the emitted fluorescence is directed through dichroic optics and emission filters. These components route light into independent detection channels, reducing the likelihood that one fluorophore’s signal will be interpreted as the other’s. This spectral organization allows two neural measurements to be collected from the same tissue.
The detected fluorescence can reflect contributions from both indicators as well as motion or other optical fluctuations. Ratiometric analysis compares channels, while demixed analysis estimates the contribution of each indicator to the recorded signal. These approaches help separate biologically meaningful changes from recording-related variation, making comparisons between neural signals more interpretable in intact preparations.
A calcium indicator can be paired with an indicator for voltage, a neurotransmitter, or a neuromodulator. This pairing places changes in neuronal activity alongside changes in electrical state or synaptic chemistry. The resulting comparison can reveal whether signals occur together, differ across conditions, or suggest interactions between cellular activity and chemical communication in a circuit.
Single-indicator photometry provides one fluorescence-based measurement, whereas Dual-color Photometry adds a second genetically encoded signal from the same tissue. That second channel enables direct comparison between distinct neural variables rather than requiring separate recordings or indirect inference. In neuroscience, this expanded measurement can improve interpretation of behavior-linked activity and circuit interactions.
The workflow begins by expressing two genetically encoded indicators in the tissue of interest. Separate excitation wavelengths then stimulate the fluorophores, while dichroic optics and emission filters sort the emitted light into independent channels. Researchers can subsequently apply ratiometric or demixed analysis to estimate each indicator’s signal and account for optical fluctuations during recording.
Researchers may choose this approach when they need to relate neuronal activity to another signal in the same tissue during behavior. For example, calcium measurements can be compared with voltage, neurotransmitter, or neuromodulator signals. This design supports investigation of circuit interactions and links cellular activity with synaptic chemistry in intact animals.
Simultaneous measurements can show how distinct neural signals change in relation to one another during behavior. Comparing calcium activity with voltage or chemical-sensor signals may help identify circuit interactions and distinguish neuronal activity from accompanying synaptic or neuromodulatory changes. Accounting for motion and optical fluctuations further supports more reliable interpretation of these behavior-linked recordings.