Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level

Abstract

We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06± 0.37 ns (Cy5) and 3.89± 0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short offstates t off of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield Y ISC of ~0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.

Optical Probing of Single Fluorescent Molecules and Proteins

“…Recently, Tinnefeld et al reported a powerful method to monitor fluctuations in the excited lifetime of single molecules by recording fluorescence lifetime trajectories with millisecond time resolution. [59] In agreement with the observations of Macklin et al, [13] a broad distribution of the fluorescence decay times for molecules dispersed in space was found and similarly attributed to the presence of a dielectric interface, which modifies the radiative component of the excited-state lifetime. We have done similar experiments with DiD molecules embedded in thin PMMA layers and have recorded temporal and spatial lifetime trajectories.…”

Optical Probing of Single Fluorescent Molecules and Proteins

“…Recently, Tinnefeld et al reported a powerful method to monitor fluctuations in the excited lifetime of single molecules by recording fluorescence lifetime trajectories with millisecond time resolution. [59] In agreement with the observations of Macklin et al, [13] a broad distribution of the fluorescence decay times for molecules dispersed in space was found and similarly attributed to the presence of a dielectric interface, which modifies the radiative component of the excited-state lifetime. We have done similar experiments with DiD molecules embedded in thin PMMA layers and have recorded temporal and spatial lifetime trajectories.…”

Room-Temperature Solution Fluorescence Excitation Correlation Spectroscopy

“…Fourier transformation of this data with respect to wedge position x yields an excitation wavelength-resolved fluorescence lifetime trace as shown in Figure b, which shows a decay with a time constant of 1.25 ns without noticeable excitation wavelength dependence. The obtained lifetime is close to those of indodicarbocyanine dyes (∼1 ns), which have a similar backbone structure. , The integration of the data over the microtime t axis provides a fluorescence excitation spectrum, which reproduces the linear absorption spectrum (Figure b, top), demonstrating that an excitation spectrum of the solution sample can be obtained by Fourier transform spectroscopy using broadband femtosecond pulses. In the case of TIPS-Pn (Figure c), the fluorescence interferogram shows a distinct beating pattern.…”

“…The obtained lifetime is close to those of indodicarbocyanine dyes (∼1 ns), which have a similar backbone structure. 38,39 The integration of the data over the microtime t axis provides a fluorescence excitation spectrum, which reproduces the linear absorption spectrum (Figure 2b, top), demonstrating that an excitation spectrum of the solution sample can be obtained by Fourier transform spectroscopy using broadband femtosecond pulses. In the case of TIPS-Pn (Figure 2c), the fluorescence interferogram shows a distinct beating pattern.…”

Monitoring the Coating of Single DNA Origami Nanostructures with a Molecular Fluorescence Lifetime Sensor

“…Single‐molecule fluorescence lifetime imaging microscopy (FLIM) scans were acquired on a custom‐built confocal microscope with a time‐correlated single photon counting unit (TCSPC). [ 43 ] Acquired FLIM scans were analyzed by picking individual nanostructures and extracting the spot‐integrated fluorescence lifetime information. Distributions of the obtained fluorescence lifetime values were then fitted by Gaussian distribution functions, to determine the mean of each individual distribution.…”

Optical Probing of Single Fluorescent Molecules and Proteins

“…Recently, Tinnefeld et al reported a powerful method to monitor fluctuations in the excited lifetime of single molecules by recording fluorescence lifetime trajectories with millisecond time resolution. [59] In agreement with the observations of Macklin et al, [13] a broad distribution of the fluorescence decay times for molecules dispersed in space was found and similarly attributed to the presence of a dielectric interface, which modifies the radiative component of the excited-state lifetime. We have done similar experiments with DiD molecules embedded in thin PMMA layers and have recorded temporal and spatial lifetime trajectories.…”

Room-Temperature Solution Fluorescence Excitation Correlation Spectroscopy

“…Fourier transformation of this data with respect to wedge position x yields an excitation wavelength-resolved fluorescence lifetime trace as shown in Figure b, which shows a decay with a time constant of 1.25 ns without noticeable excitation wavelength dependence. The obtained lifetime is close to those of indodicarbocyanine dyes (∼1 ns), which have a similar backbone structure. , The integration of the data over the microtime t axis provides a fluorescence excitation spectrum, which reproduces the linear absorption spectrum (Figure b, top), demonstrating that an excitation spectrum of the solution sample can be obtained by Fourier transform spectroscopy using broadband femtosecond pulses. In the case of TIPS-Pn (Figure c), the fluorescence interferogram shows a distinct beating pattern.…”

“…The obtained lifetime is close to those of indodicarbocyanine dyes (∼1 ns), which have a similar backbone structure. 38,39 The integration of the data over the microtime t axis provides a fluorescence excitation spectrum, which reproduces the linear absorption spectrum (Figure 2b, top), demonstrating that an excitation spectrum of the solution sample can be obtained by Fourier transform spectroscopy using broadband femtosecond pulses. In the case of TIPS-Pn (Figure 2c), the fluorescence interferogram shows a distinct beating pattern.…”

Monitoring the Coating of Single DNA Origami Nanostructures with a Molecular Fluorescence Lifetime Sensor

“…Single‐molecule fluorescence lifetime imaging microscopy (FLIM) scans were acquired on a custom‐built confocal microscope with a time‐correlated single photon counting unit (TCSPC). [ 43 ] Acquired FLIM scans were analyzed by picking individual nanostructures and extracting the spot‐integrated fluorescence lifetime information. Distributions of the obtained fluorescence lifetime values were then fitted by Gaussian distribution functions, to determine the mean of each individual distribution.…”

Optical Probing of Single Fluorescent Molecules and Proteins

“…Recently, Tinnefeld et al reported a powerful method to monitor fluctuations in the excited lifetime of single molecules by recording fluorescence lifetime trajectories with millisecond time resolution. [59] In agreement with the observations of Macklin et al, [13] a broad distribution of the fluorescence decay times for molecules dispersed in space was found and similarly attributed to the presence of a dielectric interface, which modifies the radiative component of the excited-state lifetime. We have done similar experiments with DiD molecules embedded in thin PMMA layers and have recorded temporal and spatial lifetime trajectories.…”

Room-Temperature Solution Fluorescence Excitation Correlation Spectroscopy

“…Fourier transformation of this data with respect to wedge position x yields an excitation wavelength-resolved fluorescence lifetime trace as shown in Figure b, which shows a decay with a time constant of 1.25 ns without noticeable excitation wavelength dependence. The obtained lifetime is close to those of indodicarbocyanine dyes (∼1 ns), which have a similar backbone structure. , The integration of the data over the microtime t axis provides a fluorescence excitation spectrum, which reproduces the linear absorption spectrum (Figure b, top), demonstrating that an excitation spectrum of the solution sample can be obtained by Fourier transform spectroscopy using broadband femtosecond pulses. In the case of TIPS-Pn (Figure c), the fluorescence interferogram shows a distinct beating pattern.…”

“…The obtained lifetime is close to those of indodicarbocyanine dyes (∼1 ns), which have a similar backbone structure. 38,39 The integration of the data over the microtime t axis provides a fluorescence excitation spectrum, which reproduces the linear absorption spectrum (Figure 2b, top), demonstrating that an excitation spectrum of the solution sample can be obtained by Fourier transform spectroscopy using broadband femtosecond pulses. In the case of TIPS-Pn (Figure 2c), the fluorescence interferogram shows a distinct beating pattern.…”

Monitoring the Coating of Single DNA Origami Nanostructures with a Molecular Fluorescence Lifetime Sensor

“…Single‐molecule fluorescence lifetime imaging microscopy (FLIM) scans were acquired on a custom‐built confocal microscope with a time‐correlated single photon counting unit (TCSPC). [ 43 ] Acquired FLIM scans were analyzed by picking individual nanostructures and extracting the spot‐integrated fluorescence lifetime information. Distributions of the obtained fluorescence lifetime values were then fitted by Gaussian distribution functions, to determine the mean of each individual distribution.…”