Lifetime Resolved Fluorescence Correlation Spectroscopy and Two-Photon Spectroscopy of Amyloid Nanotube Bundles 公开

Guo, Peng (2009)

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Lifetime Resolved Fluorescence Correlation Spectroscopy and Two-Photon Spectroscopy of Amyloid Nanotube Bundles
By Peng Guo

Fluorescence correlation spectroscopy (FCS) has been widely used to investigate molecular dynamics and interactions in biological systems. However, these diffusion based assays currently have a major limitation, which requires that the diffusion coefficients of component species in a sample must be substantially different in order to be resolved. This limitation can be overcome, and the resolution of FCS measurements can be enhanced, by combining FCS measurements with measurements of fluorescence lifetimes. We show that we can dramatically enhance resolution in FCS measurements using global analysis on simultaneously acquired FCS and lifetime data. The method accurately resolves the concentration and diffusion coefficients of multiple sample components, even when their diffusion coefficients are identical, provided that there is a difference in the lifetime of the component species. We show examples of this technique by using both simulations and experiments. It is expected that this method will be of significance for a broad range of researchers studying molecular interactions. In a separate project, a potentially useful amyloid nanotube bundle material from the β-amyloid proteins is studied. We used two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) to investigate the photophysical properties of this material. The emission properties of the bundles are characterized, and their dependence on the moisture level is revealed. The mechanism of the intrinsic fluorescence is discussed to be related to the electron delocalization within the peptides. The spectroscopic and microscopic study of the amyloid nanotube bundles may open up interesting new perspectives in the bio-inspired material science area.

Table of Contents

Table of Contents

Chapter 1 introduction of scope...1
Chapter 2 Lifetime-Resolved Fluorescence Correlation Spectroscopy (LFCS)...2

2.1 Summary...3
2.2 Introduction...4
2.3 Theoretical basis and experimental setup...9

2.3.1 Fluorescence Correlation Spectroscopy...9
2.3.2 Rational of extending FCS technique...14
2.3.3 Fluorescence lifetime analysis...15
2.3.4 Global linkage of FCS and fluorescence lifetime analysis...17

2.4 Materials and Methods...19

2.4.1 Instrumentation...19
2.4.2 Sample preparation...20
2.4.3 Simulation...21
2.4.4 Data analysis...24
2.4.5 Correction of instrument Response Function (IRF) impact in lifetime analysis...26

2.5 Results and Discussion...28

2.5.1 Resolvability analysis...29
2.5.2 Simulation results of two-component system...35
2.5.3 Experimental results...39
2.5.4 Discussion...44 Influence of the diffusion ratio and lifetime ratio...45 Influence of the brightness ratio S...47 When the lifetime and brightness of two components are linked...51 Discussion of experimental condition...52

2.6 Conclusion and outlook...53

Chapter 3 Amyloid nanotube bundles studied by two-photon excited fluorescence and second harmonic generation...54

3.1 Summary...54
3.2 Background...54

3.2.1 Formation of amyloid nanotube bundles...55
3.2.2 Second Harmonic Generation...57

3.3 Materials and Methods...58

3.3.1 Lasers...58
3.3.2 Laser scanning and imaging...58
3.3.3 Detectors and filters...59
3.3.4 TPEF and SHG acquisition...59
3.3.5 Fluorescence lifetime imaging microscopy FLIM...60
3.3.6 Chemicals and other materials...60

3.4 Results and Discussion...61

3.4.1 Optical signals observed in amyloid nanotube bundles...62
3.4.2 Characterization of image-forming signal...63
3.4.3 Fluorescence lifetime study...64
3.4.4 Strong evidence for solvent-assisted fluorescence...68
3.4.5 Application of SHG...71
3.4.6 Spectroscopic study of SHG...72
3.4.7 Imaging contrast between TPEF and SHG...73

3.5 Significance of the finding...74
3.6 Summary and future efforts...75

Chapter 4 Outlook...76

List of Figures

Figure 1.1 search results on fluorescence and molecular interactions...2
Figure 2.1 conceptual basis of FCS and typical time trace of fluorescence intensity...10
Figure 2.2 experimental data of FCS and its fit curve...11
Figure 2.3 schematic of fluorescence lifetime data...16
Figure 2.4 the schematic drawing of the FCS and fluorescence lifetime setup...20
Figure 2.5 comparisons of Koppel noise and our empirical noise generation...22
Figure 2.6 simulated FCS curves and experimental FCS curves...24
Figure 2.7 the tail-fit scheme in the fluorescence lifetime analysis...27
Figure 2.8 the simulation data of two-component fit by one-component model...30
Figure 2.9 the comparison between contour plots of χ2 fit by one component model...31
Figure 2.10 the comparison of χ2 when the minor component concentration is different...34
Figure 2.11 two component LFCS fit of simulation data of a series of titration...36
Figure 2.12 the comparison of two-component FCS with intensity constraint and two-component LFCS analysis...37
Figure 2.13 the experimental data of FCS and lifetime measurements of mixture of R6G and RB when their brightnesses are different...40
Figure 2.14 the results of LFCS analysis on the mixture of R6G and RB...40
Figure 2.15 the experimental data of FCS and lifetime measurements of mixture of R6G and RB when their brightnesses are the same...43
Figure 2.16 the results of LFCS analysis on the mixture of R6G and RB...43
Figure 2.17 the impact of diffusion coefficient and lifetime on the resolvability of LFCS...46
Figure 2.18 the impact of brightness ratio and concentration ratio on the resolvability of LFCS...49
Figure 2.19 the relative error of the minor concentration when the brightness is linked with and without lifetime...51

Figure 3.1 Sulfate-induced axially aligned peptide nanotube macrofilaments...56

Figure 3.2 the SEM images of nanotubes in the absence and presence of sulfate...56

Figure 3.3 optical signals of nanotube bundles under two photon excitation...62

Figure 3.4 the emission spectrum of bundles at excitation λ=780nm and 900nm...63

Figure 3.5 the emission Spectrum of the nanotubes bundles at different excitation λ=780nm, 820nm, 860nm, 900nm...64

Figure 3.6 the lifetime data of SHG contaminated decay and filtered fluorescence decay...65

Figure 3.7 lifetime decay at different excitation λ=780nm, 820nm, 860nm and 900nm of the Aβ(16-22) nanotube bundles...67

Figure 3.8 fluorescence lifetime imaging of nanotube bundles at excitation λ=780nm and 820nm...67

Figure 3.9 the emission spectrum of bundles at excitation λ=780nm...68

Figure 3.10 the emission spectra obtained from both dry and rehydrated nanotube bundles at excitation λ= 780nm, 820nm, 860nm, 900nm...69

Figure 3.11 the emission spectra showing that SHG signal...72

Figure 3.12 the images of nanotube bundles under two-photon excitation at λ=780nm, 820nm and 900nm...73

List of Tables

Table 2.1 the comparison of parameter setting in different fit methods...26

Table 2.2 EGFP sample measured by three different analyses...39

Table 2.3 comparison of resolvability of FCS and LFCS by simulation...48

Table 3.1 lifetime of bundles from 465-495nm emission...65

Table 3.2 lifetime of bundles from 505-700nm emission...66

Table 3.3 lifetime of rhodamine 6G at various excitation conditions...66

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