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doi: 10.1186/s44330-025-00048-1. Epub 2025 Nov 3.

Quantitative single-molecule FLIM and PIE-FRET imaging of biomolecular systems

Affiliations

Quantitative single-molecule FLIM and PIE-FRET imaging of biomolecular systems

Irene Silvernail et al. BMC Methods. 2025.

Abstract

Background: The structural dynamics of proteins and nucleic acids are critical for their function in many biological processes but investigating these dynamics is often challenging with traditional techniques. Time-correlated single photon counting (TCSPC) coupled with confocal microscopy is a versatile biophysical tool that enables real-time monitoring of biomolecular dynamics in a variety of systems, across many timescales. Quantitative single-molecule time-resolved fluorescence methods are uniquely positioned to investigate transient interactions and structural changes, yet application in complex biological systems remains limited by technical and analytical challenges. Combining fluorescence lifetime imaging microscopy (FLIM) with pulsed interleaved excitation Förster resonance energy transfer (PIE-FRET) offers a robust approach to overcome these barriers, enabling accurate distance measurements and dynamic studies across diverse sample types.

Methods: We describe practical workflows for implementing FLIM/PIE-FRET for quantitative measurements of nanoscale distances and dynamic processes in various biomolecular systems on a commercial microscope. Benchmark DNA constructs, RNA/DNA hybrids, liposome-encapsulated enzymes, and live Saccharomyces cerevisiae strains were prepared and imaged. Correction factors for FRET efficiency recovery were determined from diffusion-based experiments, and results were validated by direct comparison of intensity- and lifetime-based analyses.

Results: FRET efficiencies from both intensity- and lifetime-based analyses were consistent across systems. DNA standards reproduced expected values, RNA/DNA hybrids reported on substrate dynamics, liposome encapsulation enabled single-enzyme conformational probing, and live-cell imaging revealed transient protein-protein interactions during ribosome biogenesis.

Discussion: This work establishes guidelines for implementing FLIM/PIE-FRET as an accessible method to interrogate nanoscale distances, conformational dynamics, and protein-protein interactions both in vitro and in live cells. The strategies outlined here facilitate broader adoption of quantitative single-molecule time-resolved fluorescence in structural and cell biology.

Supplementary information: The online version contains supplementary material available at 10.1186/s44330-025-00048-1.

Keywords: FLIM; Liposome encapsulation; PIE-FRET; Protein dynamics; RNA/DNA hybrids; Ribosome biogenesis; Single-molecule.

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Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Fig. 1
Fig. 1
A Time-resolved confocal fluorescence microscope schematic. Two diode lasers are driven in pulsed interleaved excitation (PIE) mode and excite the sample through a high numerical aperture objective lens. Fast laser beam scanning is used to image surface-attached molecules. Donor and acceptor molecules are excited sequentially on the nanosecond timescale, the fluorescence is passed through a dichroic mirror and confocal pinhole and detected by single photon counting modules. Each photon is time-tagged by the TCSPC event timer. B Principle of pulsed interleaved excitation – PIE. The red laser pulses in the first time gate, and photons from acceptor molecules are detected only in Channel 1. The excited state decay results from creating a histogram of photon arrival nanotimes. After a 25 ns delay and complete decay of the acceptor excited state, the green laser pulses to excite the donor molecules in the second time gate. Donor emission is detected in Channel 2, and if FRET occurs acceptor emission is detected in Channel 1 during the second time gate (in addition to some spectral crosstalk or leakage that we account for by determining the correction factor, α, Supplementary Information, Section IV) C Schematic of benchmark DNA oligomers for FLIM and PIE-FRET imaging. An acceptor DNA strand containing a 5’ biotin was fluorescently labeled near the 3’ end, indicated by a red circle. The complementary DNA strands were labeled at sites 11, 15, and 23 bases away from the acceptor label, indicated by the green circles. When annealed, lo, mid and hi FRET benchmark samples are produced. The sequences are from an smFRET benchmark study [39] and are listed in Supplementary Table S1
Fig. 2
Fig. 2
FLIM/PIE-FRET single molecule images and traces of benchmark samples. At least 20 individual molecules were probed for each benchmark sample, but we present a detailed analysis for one representative trace from each sample. A formula image, formula image (FRET), and formula image images obtained simultaneously with PIE for each sample (lo, mid, and hi FRET). The brightness of each spot is related to the fluorescence intensity, indicated by the gray scale bar. Higher photon counts are brighter in the image, while lower photon counts are darker. The color of each pixel in the image indicates the Fast fluorescence lifetime at that pixel, which is the average arrival nanotime. Red represents a longer lifetime and blue is a shorter lifetime. B Individual bright spots correspond to single fluorescently labeled annealed benchmark DNA molecules. Single-molecule fluorescence intensity traces for individual duplexes are obtained by clicking on a bright spot and collecting photons from that location. The photon macrotimes were then binned in 50 ms bins to generate the traces. In the traces for individual lo, mid, and hi FRET duplexes, formula image is blue, formula image (FRET) is green, and formula image is magenta. The FRET efficiency (black scatter) below each set of traces was calculated with Eq. 1 using the correction factors in Supplementary Table S5. The photobleach step for the acceptor, which is followed by recovery of the donor fluorescence, is apparent in each trace. The formula image correction factor was calculated with Eq. 2 considering the acceptor photobleach step
Fig. 3
Fig. 3
A Annealed RNA/DNA hybrid MFU substrate was diluted to 15 pM and single molecules were attached to a homebuilt flow cell via biotin-streptavidin linkage, followed by flushing the channel with Image Buffer C (Materials and Methods). An RNA(pink, 15 bases)/DNA(green, 45 bases) hybrid oligo and a complementary DNA strand (green, 45 bases) were all purchased from IDT. The complementary DNA strand was labeled with a donor (Atto550, green circle), and the RNA segment of the hybrid is labeled with an acceptor (Atto647N, red circle). The DNA acceptor strand is biotinylated at the 5’ end for attachment via biotin-streptavidin linkage. At least 20 substrates were probed, but we present detailed analysis for a single trace. We clicked on a single biomolecule (white circle) and collected photons from that location to produce fluorescence intensity traces for the donor (blue, formula image) and acceptor (magenta, formula image) fluorophores attached to the biomolecule. The green trace is the FRET formula image) signal, and the calculated FRET efficiency is below in black. We binned the photon arrival macrotimes for each signal in 100 ms bins. The calculated FRET efficiency below (black scatter, formula image) was corrected for leakage and direct excitation as described in the text. We interpret the loss of the acceptor signal as photobleaching since there is no Nsp15 in this experiment. B Fluorescently labeled Thermus aquaticus MutL enzymes were encapsulated in liposomes and attached to the surface of a homebuilt flow cell. C The liposomes were imaged in PIE mode, and the images were separated based on the excitation laser time gate and detection channel. A fast pattern matching algorithm that separates the photons based on excitation laser time gate and detection channel creates a single RGB image with formula image in green, and formula image in red. Some proteins are donor-only or acceptor-only labeled. Double-labeled (donor-acceptor) may be identified with a yellow color if the photon counts are high enough. D The formula image, formula image (FRET), and formula image images obtained simultaneously. E Three representative traces for different encapsulated MutL enzymes. We clicked on a single encapsulated enzyme (For trace 1, indicated by the white circle in C and D) and collected photons from a single location to produce fluorescence intensity traces for the donor (blue) and acceptor (magenta) attached to the biomolecule. The green trace is the FRET formula image) signal, and the calculated FRET efficiency is below in black. The calculated FRET efficiency was corrected for leakage and direct excitation as described in the text. The calculated gamma factor is indicated on each trace, and a histogram of the calculated corrected FRET efficiency is shown below each trace (gray bars)
Fig. 4
Fig. 4
Live Saccharomyces cerevisiae cells expressing Nop7-GFP/mCherry-Rea1 and Rix7-GFP/Nsa1-mCherry. A FLIM image (DexAem/FRET channel) of a Nop7-GFP/mCherry-Rea1 cell cluster. The gray scale bar correlates the number of photon event counts at each pixel with the image brightness. The color bar indicates the fast lifetime at each pixel, which is the average photon arrival time relative to the laser pulse rate (nanotime) at each pixel. B PIE mode image of the same Nop7-GFP/mCherry-Rea1 expressing cell cluster in A. Nop7-GFP is represented by the green signal and mCherry-Rea1 is represented by the red signal. The yellow signal is colocalization the of red and green signals. The image brightness indicates the number of photon counts at each pixel. C FRET efficiency image for the cell cluster in A and B with pixel-wise calculated FRET efficiency indicated by the pixel color after applying the correction factors as outlined in Supplementary Information, Section VII. The FRET efficiency histogram is inset. D FLIM image (DexAem/FRET channel) of Rix7-GFP/Nsa1-mCherry cluster. E PIE mode image of the same Rix7-GFP/Nsa1-mCherry expressing cell cluster in D. F FRET efficiency image for the cell cluster in D and E with pixel-wise calculated FRET efficiency indicated by the pixel color after applying the correction factors as outlined in Supplementary Information, Section VII. The FRET efficiency histogram is inset

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