Fluorescence Lifetime correlation spectroscopy(FLCS)

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Contents
01 Introduction

02 Methods

03 Result

04 Conclusion

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Introduction
Kapusta, P., Wahl, M., Benda, A. et
al. Fluorescence Lifetime Correlation
Fluorescence Lifetime Spectroscopy. J Fluoresc 17, 43–48 (2007).
https://doi.org/10.1007/s10895-006-0145-1

The lifetime is the time it takes for the number of

excited molecules to decay to 1/e or 36.8% of the
original population ( n*(t)/n*(0) = e-t/tau)

D(i) = wa · a(i) + wb · b(i) 3

Introduction
Two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS)

Fluorescence correlation spectroscopy(FCS)

  : the fluorescence intensity at time t

F(t)
<F(t)> = (1/T)dt : the time average
σF(t) = F(t) – F(t) : the fluctuations
τ : the correlation (or lag) time.

G(0) = 1/N = 1/VC

Showing the probability that the signal at

different time still belongs to the same
molecular event.

Diffusion or internal dynamics makes fluctuation of intensity.

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Introduction
Two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS)

Fluorescence lifetime correlation spectroscopy(FCS)

FLCS : improving quality of FCS data by removing noise and distortion

- lifetime-scale
- FCS-scale

uses differences in
fluorescence intensity decays to obtain separate FCS
autocorrelation functions (ACFs) of individual
fluorophore populations in a mixture.

TCSPC channel number : j

Fluorescence decay curve : Ij
Decay components : M
Decay pattern linear combination : pj(k)
Multiplied number : w(k)
Filter function : fj(k)

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Paper

Resolving the rapid conformational isomerization

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Introduction & Method

Arpino JAJ, Rizkallah PJ, Jones DD (2012) Crystal Structure of Enhanced Green Fluorescent Protein to 1.35 Å Resolution
Reveals Alternative Conformations for Glu222. PLOS ONE 7(10): e47132. https://doi.org/10.1371/journal.pone.0047132 7
Data

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Data

Excitation & Emission spectra of the two states

are highly overlapping and inseparable at
room temperature
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Data

p-hydroxybenzylideneimidazolone(HBDI-)

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Result

kf = k1→2 + k2 →1 ks = k1→d + kd→1 - Transition rates are faster than interconversion(Dark state)

Kf >> ks - Photoisomerization or protonation affects the bright

state(No relation with pH and excitation laser power)

- State 2 can be reached protonated D through

intermediate state 1. And it is similar I state in wt-GFP

- The occupancy values are good aggrement with crystal

data

- Transition between D and fluorescent state is associated

with protonation dynamics of HBDI-.

- High pH conditions, dark state transition is related

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Paper

Two-dimensional fluorescence lifetime correlation spectroscopy

is used to explore the conformational dynamics

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Data

 I(t) =

Fluorescence lifetime distribution : α(τ)

Pair of emission delay time (t’,

t’’) are separated by certain
interval(ΔT).
2D inverse Laplace
transform(ILT) is applied to 2D
map.
Diagonal : lifetime components
Off-diagonal : correlation
among lifetime components
divided by ΔT.

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Data

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Data

Native state(N) : sp1(70 ps)

Unfolded state(U) : sp4(3.3 ns)
Folding intermediate state(I1, I2, I3) : sp2(280 ps), sp3(300 ps, 1.7 ns) 15
Data

This schem assuming that intermediate states like that Dark state lifetime is shorter
However, it is possible to involve off-pathway or parallel that time resolution
pathway.

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Conclusion

- FLCS shows that the conformational transitions within each ensemble

on the microsecond or shorter timescale, wheras between different
ensembles take longer than a millisecond.
- These ensembles is separated by a substantial potential barrier
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