Fluorescent Probe Assays
Fluorescent Probe Assays - Related Videos
04:02
An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells
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2025
This video demonstrates an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells. Exposure to an efflux pump inhibitor disrupts the transmembrane proton gradient in bacterial cells, upon which a fluorophore-conjugated antibiotic is added. Inhibition of efflux pumps due to the disrupted proton gradient causes accumulation of the antibiotic inside the bacteria. Bacterial cells are lysed, and the fluorescence intensity is measured to confirm antibiotic accumulation.
05:51
A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging
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2023
The present protocol describes a detailed, real-time NIR-II fluorescence imaging operation of a mouse using a NIR-II optics imaging device.
10:59
Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)
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Cited by 9 •
2012
A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.
11:21
Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells
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Cited by 5 •
2010
This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.
07:14
Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes
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Cited by 4 •
2014
We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.
FAQs
The measurable signal changes when a probe binds its biological target or undergoes a chemical change. That event can alter fluorescence intensity, emission wavelength, or the probe’s localization. Because each readout reflects a different consequence of target recognition or reaction, investigators can select intensity, spectral, or spatial measurements to follow molecular events in cells or engineered systems.
Excitation light provides the energy needed to generate fluorescence, while the emitted light supplies the observable signal. Measuring emission intensity can indicate how much signal is present, whereas changes in wavelength can reveal a different fluorescence state. This separation between excitation and emission enables assays to translate probe behavior into quantitative measurements rather than relying only on visual observation.
Probe localization adds spatial information to the assay. A signal can show where a target-associated probe is found, while intensity or wavelength changes report altered fluorescence properties. This combination is especially useful when bioengineers need to examine molecular events within cells, biomaterials, or engineered biological systems instead of measuring an undifferentiated signal from the whole sample.
A practical workflow begins by matching a light-emitting probe to the biological target or chemical change being studied. The assay then exposes the probe-containing sample to excitation light and records the resulting fluorescence. Researchers can analyze intensity, wavelength, or localization with microscopy or fluorimetry, choosing the readout that best represents the event under investigation.
Microscopy and fluorimetry emphasize different kinds of information. Microscopy preserves spatial context, allowing fluorescence to be localized within cells or engineered materials. Fluorimetry provides a fluorescence measurement from the sample for quantitative analysis. Selecting between them depends on whether the experiment prioritizes where a signal occurs, how strong it is, or both.
In bioengineering, fluorescent probe assays support biosensor development, biomaterial characterization, and cell-based analysis. They can also monitor enzyme activity, cellular conditions, and interactions in engineered systems. These applications make the assays useful for evaluating whether an engineered biological function is present and for connecting molecular behavior with observable performance in a designed biological context.