Improving Accuracy and Precision in Biological - Deep Blue

186 Pages·2009·3.87 MB·English

Improving Accuracy and Precision in Biological Applications of
Fluorescence Lifetime Imaging Microscopy

by

Ching-Wei Chang

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
(Biomedical Engineering)
in The University of Michigan
2009

Doctoral Committee:
Associate Professor Mary-Ann Mycek, Chair
Professor Scott J. Hollister
Professor Nicholas Kotov
Associate Professor Shuichi Takayama
© Ching-Wei Chang 2009
Acknowledgements

First, I would like to thank Dr. Mary-Ann Mycek for providing me plenty of advices
on this work. She gave me correct research directions while still giving me sufficient
freedom to conduct detailed experiments and analyses. I definitely cannot complete this
work without her great support and help.
I also would like to thank Dr. Hollister, Dr. Kotov, and Dr. Takayama for
participating in the dissertation committee and providing me very useful advices.
I want to thank previous Mycek lab members Dr. Wei Zhong and Dr. Dhruv Sud
for their helpful discussions on FLIM techniques and experiments. I also want to thank
the members from our collaborating lab Dr. Sofia Merajver, Dr. Mei Wu, Dr. Zhi-Fen Wu,
Devin Rosenthal, and Elliot Rhee for their work and discussions on the RhoC project.
They all contribute a lot to this work.
In addition, I need to thank Dr. Selim Esedoglu in the Department of Mathematics
for his class and advices on image processing, which provided me the basis to do further
advanced analyses.
Finally, I want to thank my parents. I am very grateful to them for raising me and
providing me support in all these years that let me concentrate on my Ph.D. work without
worrying them.
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Table of contents

Acknowledgements...........................................................................................................ii
List of Figures..................................................................................................................vii
List of Tables...................................................................................................................xv
List of Abbreviations.......................................................................................................xvii
Abstract...........................................................................................................................xx
Chapter 1 Introduction.......................................................................................................1
1.1 Background and motivation.....................................................................................1
1.1.1 Quantitative understanding of living cells.........................................................1
1.1.2 Fluorescence measurement and FLIM.............................................................4
1.1.3 Challenges in live-cell FLIM..............................................................................7
1.2 Hypotheses and specific aims...............................................................................13
1.3 Dissertation overview............................................................................................15
Chapter 2 FLIM instrumentation and analytical procedures............................................17
2.1 Fluorescence lifetime............................................................................................17
2.1.1 Basic theory....................................................................................................17
2.1.2 Key features of lifetime sensing......................................................................19
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2.2 Time-gated fluorescence lifetime imaging microscopy..........................................21
2.2.1 Concepts........................................................................................................21
2.2.2 Instrumentation...............................................................................................22
2.2.3 Data processing.............................................................................................24
2.2.4 System variations...........................................................................................25
2.3 The temporal approach.........................................................................................28
2.3.1 Introduction to optimal gating and curve fitting...............................................28
2.3.2 Optimal gating and parameters used in time-gated integration......................29
2.3.3 Lifetime determination methods.....................................................................32
2.3.4 Monte Carlo simulations.................................................................................36
2.4 The spatial approach.............................................................................................37
2.4.1 Introduction to total variation models..............................................................37
2.4.2 Analytical procedures.....................................................................................40
Chapter 3 Well-controlled FLIM for FRET detection in living cells..................................46
3.1 Introduction to FRET and FLIM-based FRET detection........................................46
3.2 Use of FRET to monitor RhoC interactions...........................................................50
3.3 Choice of fluorophores..........................................................................................57
3.4 Temperature control..............................................................................................60
3.5 CO control............................................................................................................61
2
3.6 Incorporation of both environmental controls........................................................63
3.7 Discussion.............................................................................................................66
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Chapter 4 Temporal approach – Optimal gating and curve fitting to improve the precision
of FLIM............................................................................................................................71
4.1 Simulation results..................................................................................................71
4.1.1 RSD = f(g/τ , τ /τ ) contour plot analysis.........................................................71
1 2 1
4.1.2 minRSD = f(α /α , τ /τ ) analysis.....................................................................74
1 2 2 1
4.1.3 Effects of the number of gates, n, on RSD values..........................................82
4.1.4 dt has higher impact on RSD than g...............................................................84
4.1.5 Analysis of relative mean errors (RMEs)........................................................87
4.1.6 A guideline for the practical use of the temporal approach............................87
4.2 Experimental validation.........................................................................................89
4.2.1 Double-exponential-decay lifetime standards.................................................89
4.2.2 Single-exponential-decay fluorescent beads..................................................92
4.3 Summary and conclusion......................................................................................94
Chapter 5 Spatial approach – Total variation (TV) models for FLIM image denoising....96
5.1 Results of the artificial images, the first version....................................................97
5.1.1 Undenoised images........................................................................................97
5.1.2 Rudin-Osher-Fatemi (ROF) model.................................................................98
5.1.3 Poisson-adapted TV (PoissTV) model.........................................................100
5.1.4 f-weighted TV (FWTV) model.......................................................................103
5.1.5 u-weighted TV (UWTV) model......................................................................105
5.2 Results of the artificial images, the second version............................................107
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5.3 Improvement of FWTV and UWTV – incorporation of G(N) curves....................110
5.3.1 Acquisition of G(N) curves............................................................................110
5.3.2 Effect of G and G(N) curves – results of live-cell images.............................121
5.3.3 Factors affecting lifetime changes with the modified denoising....................124
Chapter 6 Combination of temporal and spatial approaches for further precision
improvement in FLIM....................................................................................................126
6.1 Performance of the combined approach.............................................................126
6.2 Linearity of the two approaches..........................................................................130
Chapter 7 Conclusions and future work........................................................................134
7.1 Conclusions.........................................................................................................134
7.2 Future work.........................................................................................................140
7.3 Potential applications..........................................................................................142
Appendix.......................................................................................................................147
References....................................................................................................................150

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List of Figures

Figure 1-1 Time-gated FLIM concept. The system captures fluorescence intensity image
at a time t after the excitation pulse over the interval g. Lifetime can be created using
G
intensity images captured at several different t . [32].......................................................9
G
Figure 1-2 Two live cells transfected with ECFP (enhanced cyan fluorescent protein) and
emitting different total photon counts due to their different ECFP concentrations. The
intensity image is shown on the left with the total photon counts (TC) labeled on each
cell. The corresponding lifetime map is shown on the right. The variations of the lifetime
values can be clearly seen, especially for the cell with lower TC (the cell in the bottom-
right region). [33].............................................................................................................10
Figure 2-1 Simplified Jablonski diagram. k : Bimolecular quenching constant; [Q]:
q
Quencher concentration (see Section 2.1.2); k : Energy transfer rate constant (see
T
Section 3.1); k: Rate constant for non-radiative processes other than dynamic
j
quenching and FRET. [17]..............................................................................................17
Figure 2-2 Intensity-independent fluorophore lifetimes recovered by FLIM. Despite a
factor of four difference in POPOP concentration and hence signal intensity (left), the 3-
gate protocol lifetime map (right) showed identical mean lifetimes for the two POPOP
samples. The images were of POPOP in ethanol solutions in two quartz capillaries. The
capillary interface was on the diameter, with the circular region as the illumination area.
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The intensity images were background subtracted before calculation of lifetime map. [46]
........................................................................................................................................19
Figure 2-3 The time-gated FLIM setup used for this study. Abbreviations: CCD = charge-
coupled device; HRI = high rate imager; INT = intensifier; TTL I/O = TTL input/output
card; OD = optical discriminator; BS = beam splitter; DC = dichroic mirror; FM =
“flippable” mirror; L1, L2, L3, L4 = quartz lenses; M = mirror. Thick solid lines = light path;
thin solid line = electronic path. [32]................................................................................23
Figure 2-4 A schematic demonstration of the controllable system parameters. g is the
gate width of each gate and dt is the time between starting time points of any two
consecutive gates. D , D , D and D are the integrals of the 1st, 2nd, 3rd, and 4th gates,
0 1 2 3
respectively.....................................................................................................................30
Figure 2-5 The concepts of nonlinear curve fitting used in time-gated fluorescence
lifetime measurements. (W)NLLS minimizes the sum of (weighted) squares of the
residuals (R with i for different gates) and MLE maximizes the likelihood (product of
i
probabilities prob(O, E)), when adapting the model parameters (the lifetimes and pre-
i i
exponential terms) in order to fit the data values. O: Observation values (from data); E:
i i
Expected values (from model); R: Residual = O – E; prob: probability density function;
i i i
D(.): integral under gating as a function..........................................................................34
Figure 2-6 The Monte Carlo simulation procedures for the temporal method.................36
Figure 2-7 The artificial images mimicking the geometry that may be encountered in live-
cell FLIM experiments.....................................................................................................40
Figure 2-8 The Monte Carlo simulation procedures for the spatial method. We first
generate noise-free artificial images with the specified parameters (α , τ , g, dt), and,
1 1
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after Poisson noise addition and denoising in the iterations, RSD and RME values are
calculated for the estimation of accuracy and precision..................................................45
Figure 3-1 Schematic depiction of FRET studies on RhoC and RhoGDIγ. When active,
RhoC is associated with GTP and localizes to the membrane, where it is capable of
binding to its effectors. When inactive, it is associated with GDP and is able to bind with
the inhibitor RhoGDIγ in the cytoplasm. Interactions between RhoGDIγ and RhoC
causes FRET to occur from the donor (CFP) to the acceptor (YFP). Approximate
excitation and emission maxima of CFP and YFP are labeled. The decrease of donor
fluorescence lifetime is monitored to detect FRET..........................................................52
Figure 3-2 ECFP intensity (left) and lifetime (right) values of cells transfected with ECFP
(top) and Cerulean (bottom) fusion plasmids. Group 1 (left box plot in (a) and (b))
consisted of 37 cells transfected with ECFP-RhoGDIγ and EYFP. Group 2 (right box plot
in (a) and (b)) consisted of 60 cells transfected with ECFP-RhoGDIγ + EYFP-RhoC.
Groups 3 and 4 (left and right box plots in both (c) and (d)) consisted of 30 cells
transfected with Cerulean-RhoGDIγ + EYFP and 60 cells transfected with Cerulean-
RhoGDIγ + EYFP-RhoC, respectively. The first quartile, the median, and the third
quartile are shown within the boxes and the 10th/90th percentiles outside the boxes. All
the plots are labeled with p-values of the Student’s t-test, together with the mean
intensity or lifetime value and standard deviation for each group of cells. (a) and (c)
Fluorescence intensities suffered from high variation within the cell population and
revealed no statistically significant difference between groups by the student’s t-test. (b)
and (d) The FRET-induced decreases in lifetime (group 2 vs. group 1; group 4 vs. group
3) were statistically significant (p-value < 0.05)), indicating RhoGDlr-RhoC molecular
interaction. [32]................................................................................................................54
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