ArticleBiophysical journal2005
Pulsed interleaved excitation.
Article in Biophysical journal, 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 235 papers.
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Who cites it
235 citing papers in PubMed.
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- Time-Resolved Single-Molecule FRET Reveals Length-Dependent Nucleosome Decompaction by Poly(ADP-ribose).bioRxiv : the preprint server for biology · 2026Article
- Fluorescence Correlation Spectroscopy to Examine Protein-Lipid Interactions in Membranes.Methods in molecular biology (Clifton, N.J.) · 2026Article
- High-Throughput Fabrication of Zero-Mode Waveguide Nanoaperture Arrays with Sol-Gel Nanoimprint Lithography for Enhanced Single Molecule Fluorescence Detection.Small (Weinheim an der Bergstrasse, Germany) · 2026Article
- Bound or unbound: mapping and monitoring receptor oligomerization by time-resolved fluorescence live-cell imaging.Frontiers in molecular biosciences · 2026Article
- Differential conformational expansion of NUP98-HOXA9 oncoprotein from nanosized assemblies to macrophases.Nature communications · 2025Article
- Building a dynamic model for EGFR-family dimerization using quantitative, live-cell fluorescence methods.Biophysical journal · 2025Review
- Single-fibril Förster resonance energy transfer imaging and deep learning reveal concentration dependence of amyloid β 42 aggregation pathways.PNAS nexus · 2025Article
- Accuracy of distance distributions and dynamics from single-molecule FRET.Biophysical journal · 2025Article
175 more citing papers are in PubMed but not listed here.
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Authors and funding
4 authors.
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No grant is acknowledged in the PubMed record.
Abstract
In this article, we demonstrate the new method of pulsed interleaved excitation (PIE), which can be used to extend the capabilities of multiple-color fluorescence imaging, fluorescence cross-correlation spectroscopy (FCCS), and single-pair fluorescence resonance energy transfer (spFRET) measurements. In PIE, multiple excitation sources are interleaved such that the fluorescence emission generated from one pulse is complete before the next excitation pulse arrives. Hence, the excitation source for each detected photon is known. Typical repetition rates used for PIE are between approximately 1 and 50 MHz. PIE has many applications in various fluorescence methods. Using PIE, dual-color measurements can be performed with a single detector. In fluorescence imaging with multicolor detection, spectral cross talk can be removed, improving the contrast of the image. Using PIE with FCCS, we can eliminate spectral cross talk, making the method sensitive to weaker interactions. FCCS measurements with complexes that undergo FRET can be analyzed quantitatively. Under specific conditions, the FRET efficiency can be determined directly from the amplitude of the measured correlation functions without any calibration factors. We also show the application of PIE to spFRET measurements, where complexes that have low FRET efficiency can be distinguished from those that do not have an active acceptor.
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