single-molecule and super-resolution imaging in living cells a dissertation submitted to the ...

242 Pages·2011·5.49 MB·English

SINGLE-MOLECULE AND SUPER-RESOLUTION IMAGING IN LIVING CELLS

A DISSERTATION

SUBMITTED TO THE DEPARTMENT OF CHEMISTRY

AND THE COMMITTEE ON GRADUATE STUDIES

OF STANFORD UNIVERSITY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Hsiao-lu Lee
December 2011
© 2011 by Hsiao-lu Lee. All Rights Reserved.

Re-distributed by Stanford University under license with the author.

This dissertation is online at: http://purl.stanford.edu/sg023jd4045

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I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
William Moerner, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Michael Fayer
I certify that I have read this dissertation and that, in my opinion, it is fully adequate
in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Robert Pecora
Approved for the Stanford University Committee on Graduate Studies.
Patricia J. Gumport, Vice Provost Graduate Education
This signature page was generated electronically upon submission of this dissertation in
electronic format. An original signed hard copy of the signature page is on file in
University Archives.
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ABSTRACT
Since the first successful detection single molecules over two decades ago,
single-molecule spectroscopy has developed into a burgeoning field with a wealth of
experiments at room temperature and inside living cells. Probing asynchronous and
heterogeneous populations in situ, one molecule at a time, is not only desirable, but
critical for many biological questions. Further, super-resolution imaging based on
sequential imaging of sparse subsets of single molecules, has seen explosive growth
within the last five years. This dissertation describes both the application of live-cell
single-molecule imaging as an answer to important biological questions, and
development and validation of fluorescent probes for targeted super-resolution
imaging.
Chapter 1 is a general introduction to fluorescence, single-molecule
spectroscopy, live-cell imaging, and super-resolution imaging. In this chapter, single-
molecule experiments in living cells are discussed, and the probes and targeting
schemes used for such experiments are summarized and compared. Chapter 2
describes experimental details of single-molecule imaging in live cells. Chapter 3
presents the application of live-cell single-molecule imaging to studying the
interaction of oligoarginine molecular transporters with cell plasma membranes.
Chapter 4 describes the design, development, and validation of a target-specific
photoactivatable fluorogen (a chromophore that is dark until converted to a fluorescent
form using light) for super-resolution imaging in live mammalian and bacterial
systems. Chapter 5.presents the real-time single-molecule imaging and sub-diffraction
localization of native sodium ion channels in live neuronal models (differentiated
PC12 cells). This experiment utilized novel fluorescent saxitoxins created by de novo
synthesis.

Data, figures, tables, and excerpts are used with permission in this dissertation from
the following publications:

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(1) Ondrus, A.*; Lee, H.-L.D.*; Iwanaga, S.; Parson, W.; Andresen, B.; Moerner,
W.E.; DuBois, J. Fluorescent saxitoxins for live cell imaging of single voltage-
gated sodium ion channels beyond the optical diffraction limit. In prep.
(2) Lee, H.-L.D.; Lord, S. J.; Iwanaga, S.; Zhan, K.; Xie, H.; Williams, J. C.; Wang,
H.; Bowman, G. R.; Goley, E. D.; Shapiro, L.; Twieg, R. J.; Rao, J.; Moerner, W.
E. Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using
Photoactivatable Organic Fluorophores. J. Am. Chem. Soc. 2010, 132, 15099-
15101.
(3) Lord, S. J.; Lee, H.-L.D.; Moerner, W. E. Single-Molecule Spectroscopy and
Imaging of Biomolecules in Living Cells. Anal. Chem. 2010, 82, 2192-2203.
(4) Lord, S. J.; Lee, H.-L.D.; Samuel, R.; Weber, R.; Liu, N.; Conley, N. R.;
Thompson, M. A.; Twieg, R. J.; Moerner, W. E. Azido Push–Pull Fluorogens
Photoactivate to Produce Bright Fluorescent Labels. J. Phys. Chem. B 2010, 114,
14157-14167.
(5) Lee, H.-L .; Dubikovskaya, E. A.; Hwang, H.; Semyonov, A. N.; Wang, H.; Jones,
L. R.; Twieg, R. J.; Moerner, W. E.; Wender, P. A. Single-Molecule Motions of
Oligoarginine Transporter Conjugates on the Plasma Membrane of Chinese
Hamster Ovary Cells. J. Am. Chem. Soc. 2008, 130, 9364-9370.
(6) Lord, S. J.; Conley, N. R.; Lee, H. -L. D.; Samuel, R.; Liu, N.; Twieg, R. J.;
Moerner, W. E. A Photoactivatable Push−Pull Fluorophore for Single-Molecule
Imaging in Live Cells. J. Am. Chem. Soc. 2008, 130, 9204-9205.

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ACKNOWLEDGMENTS
I have been very fortunate to have W.E. has a research advisor. Through his
insights, support, and well-timed critique, he has taught me how good science is done
and communicated. Further, the joy and enthusiasm he enjoys for science is extremely
contagious and motivational, which is particularly important for difficult times. I could
not have asked for a better advisor.
The members of the Moerner group have contributed immensely to my
personal and professional development at Stanford. The group has been a source of
friendships as well as good advice and collaboration. Dr. Hanshin Hwang introduced
me to cell culture, a feat considering prior to graduate school all my experiments were
performed in a UHV chamber. Dr. So Yoen Kim whose thoroughness and good cheer
made her not only a great office mate, but also the go-to person for advice during my
first 2 years of graduate school. I have had the pleasure of working with Dr. Sam Lord
and Dr. Nick Conley, to whom I have turned many times for chemistry advice and
experimental discussion. Dr. Julie Biteen was generous and helpful to me in a number
of ways from helping me trouble-shoot bacterial experiments to fitting single point
spread functions. Dr. Steven Lee was helpful in many discussions on sub-diffraction
re-constructions. Whitney Duim helped me through learning many biochemical
methods, and has been a great source of support, and friendship. Matthew Lew has
been kind and generous with his expertise on MATLab and optics. Lana Lau has also
been a great source of support and awesome sounding board for crazy experimental
ideas. I also thank Marissa Lee for helpful fluorophores photophysics discussions.
My time at Stanford was made enjoyable in large part by the many friends that
have become a part of my life. I am grateful for time spent with roommates and
friends: Nicholas J. Ward, Xiao Li, Yale Huang, Tina Xu, Marissa Caldwell, Michael
Stern, Meredith Foster, Lydia Ho, Alisha Weight, and Harry Jao. I am also grateful to
my Wellesley College Professors for continuing to be supportive of me even though I
have graduated. My time at Stanford was also enriched by the warm communities of
dancers in the bay area.
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I would like to thank my family for their unyielding love and encouragement.
My aunt Leena , uncle TC, cousins Arthur and Vicky have made the bay area much
more of a home for me. My sister Raechel has always been there for me in celebration
and in distress. She would spend weekends and nights with me in lab. I would also
like to thank my sister Ivy for holding down the fort in Kaohsiung while Raechel and I
pursue our studies abroad. And most of all, I would like to thank my parents whose
love and sacrifice gave me the opportunity to pursue science; my mother Ching-Mei
has been an unfaltering tower of support in my life, and my father, Jueen, whose
curiosity and intellect has inspired my studies in science. This dissertation is dedicated
to them.

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TABLE OF CONTENT

Abstract .............................................................................................................................. iv
Acknowledgments .............................................................................................................. vi
Table of Content .............................................................................................................. viii
List of Tables ...................................................................................................................... xi
List of Figures .................................................................................................................. xii
1. Introduction and Background .................................................................................... 1
1.1 Fluorescence spectroscopy of molecules .................................................. 3
1.2 Single-molecule experiments in biological cells ....................................... 6
1.2.1 Sample requirements for single-molecule detection ............................................ 6
1.2.2 Additional requirements for imaging in live cells ............................................... 8
1.2.3 Super-resolution in live cells ............................................................................. 19
1.2.4 Single-molecule imaging in cells: a timeline..................................................... 25
1.3 Outlook ..................................................................................................... 28
1.4 Scope of the dissertation .......................................................................... 28
1.5 References ................................................................................................. 29
2. Experimental ............................................................................................................. 44
2.1 Microscopy Instrumentation ................................................................... 46
2.1.1 Microscope Configurations ............................................................................... 46
2.1.2 Detectors ............................................................................................................ 49
2.1.3 Optics ................................................................................................................ 51
2.1.4 Excitation Light Source ..................................................................................... 52
2.2 Cell Culture .............................................................................................. 53
2.2.1 Bacterial Cell Culture ........................................................................................ 53
2.2.2 Mammalian Cell Culture ................................................................................... 64
2.3 Immunofluorescence ................................................................................ 78
2.3.1 Immunofluorescence protocol ........................................................................... 81
2.3.2 Antibody conjugation ........................................................................................ 82
2.4 Total protein isolation from cells/cell lysis ............................................. 84
2.4.1 Total protein isolation from bacterial cells ........................................................ 84
2.4.2 Total protein isolation from mammalian cells ................................................... 85
2.5 Coverslip coating ...................................................................................... 86
2.5.1 Fibronectin surface coating ............................................................................... 86
2.5.2 Collagen surface coating ................................................................................... 86
2.5.3 Polyelectrolyte multilayer surface coating ........................................................ 87
2.6 References ................................................................................................. 87
3. Single-Molecule Motions of Oligoarginine Transporter Conjugates on the
Plasma Membrane of Chinese Hamster Ovary Cells ...................................................... 95
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3.1 Introduction to cell penetrating peptides ............................................... 98
3.2 Experimental .......................................................................................... 107
3.2.1 Fluorescent Conjugates ................................................................................... 107
3.2.2 Epi-illumination for single-molecule imaging ................................................. 108
3.2.3 Cellular sample preparation ............................................................................. 109
3.2.4 Imaging Arginine8-D-V tethered to polyelectrolyte multi-layers ................... 110
3.2.5 Passive diffusion inquiry ................................................................................. 110
3.2.6 Adaptive translocation inquiry ........................................................................ 111
3.2.7 Receptor-mediated endocytosis inquiry .......................................................... 111
3.2.8 Macropinocytosis inquiry ................................................................................ 112
3.2.9 High octaarginine concentration inquiry ......................................................... 113
3.2.10 Effects of addition of oxygen scavenger on imaging ...................................... 114
3.3 Single-molecule fluorescence imaging of positional trajectories ....... 115
3.4 Analysis of single-molecule motion ....................................................... 116
3.5 Results ..................................................................................................... 117
3.5.1 Fluorescently-labeled Arg8 conjugates are readily internalized ...................... 118
3.5.2 Single Arg8-D-V molecules disappear before photobleaching ....................... 120
3.5.3 Length of single-molecule trajectories yields residence time .......................... 122
3.5.4 Diffusion coefficients of single-molecule trajectories ..................................... 124
3.6 Conclusions ............................................................................................. 131
3.7 Acknowledgements ................................................................................ 132
3.8 References ............................................................................................... 133
4. Super-resolution Imaging of Targeted Proteins in Fixed and Living Cells
Using Photoactivatable Organic Fluorophores ............................................................. 139
4.1. Introduction ............................................................................................ 141
4.1.1 Requirements for pointillist super-resolution imaging .................................... 141
4.1.2 Small-molecule photoactivatable fluorophores: the azido-DCDHFs .............. 145
4.1.3 Cellular labeling considerations ...................................................................... 152
4.1.4 Our cellular labeling strategy: HaloTag/HaloEnzyme Enzymatic Targeting . 154
4.2 Experimental .......................................................................................... 157
4.2.1 Plasmids for HaloEnz Fusion Construction ..................................................... 157
4.2.2 Cell Labeling Protocol ..................................................................................... 159
4.2.3 Cell Imaging Protocol ..................................................................................... 161
4.2.4 Photoactivation Quantum Yield Characterization ........................................... 163
4.3 Results ..................................................................................................... 164
4.3.1 Photophysical characterization of Azido DCDHF HaloTag ............................ 164
4.3.2 Specificity and efficacy in in mammalian culture ........................................... 166
4.3.3 Specificity and efficacy in live bacterial samples ............................................ 168
4.4 Acknowledgements ................................................................................ 171
4.5 References ............................................................................................... 171
5. Fluorescent Saxitoxins for Super-Resolution Imaging of Voltage-Gated
Sodium Ion Channels on Live Neuronal Cells .............................................................. 179
5.1 Introduction ............................................................................................ 181
5.1.1 Cell membrane potential and action potential ................................................. 181
5.1.2 Sodium ion channels ....................................................................................... 184
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5.1.3 Saxitoxin .......................................................................................................... 185
5.2 Experimental .......................................................................................... 187
5.2.1 Synthetic design of fluorescent STX ............................................................... 187
5.2.2 Cell culture ...................................................................................................... 188
5.2.3 Electrophysiology experiments ....................................................................... 190
5.2.4 Confocal microscopy on CHO and PC12 cells ................................................ 191
5.2.5 Single-molecule microscopy ........................................................................... 196
5.2.6 Wide-field image and data analysis ................................................................. 197
5.3 Results and discussion ........................................................................... 202
5.3.1 Photo-physical properties of STX-Cy5 and STX-DCDHF ............................. 202
5.3.2 Fluorescent saxitoxins bind specifically to NaV .............................................. 204
5.3.3 NaV block by STX, STX-Cy5 and STX-DCDHF in differentiated PC12 ....... 206
5.3.4 Reversible, specific NaV labeling by STX-Cy5 and STX-DCDHF ................. 209
5.3.5 NaV expression and distribution in NGF-differentiated PC12 ......................... 211
5.3.6 Single-molecule and super-resolution imaging ............................................... 212
5.3.7 Single-molecule tracking of labeled NaVs in NGF-differentiated PC12 ......... 213
5.3.8 Single-molecule imaging of NaV distributions on filopodia ............................ 216
5.3.9 Super-resolution imaging of labeled NaVs in filopodia and neuritic spines .... 218
5.4 Conclusion .............................................................................................. 220
5.5 Acknowledgement .................................................................................. 221
5.6 References ............................................................................................... 221

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