PHYS 598 BP :: Physics Illinois :: University of Illinois at Urbana-Champaign

Lab

Handouts and Answer Keys

References

1

Handout:
Ensemble Fluorescence
Appendices

Lab 1 Answer Key

Organic Fluorophores
[Structural Effects on Fluorescence Emission, Valeur Chapter 4]
[Time Resolve Fluorescence Techniques, Valeur Chapter 10]
[Frequency Domain Lifetime Measurements, Lakowicz Chapter 5]

FRET and Anisotropy
[Fluorescence Properties and Photophysics of the Sulfoindocyanine
Cy3 Linked Covalently to DNA]
[Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids]

2

Handout: Brightfield and Fluorescence Microscopy

Lab 2 Answer Key

General Microscopy and Specific Topics
  [Basic Concepts in Microscopy]
  [Introduction to Optical Microscopy, Digital Imaging, and Photomicrography]
  [Interactive Tutorials] (under Microscopy Basics)
  [Zeiss Axiovert 200M Microscope with Apotome] (IGB)

Image Acquisition
  [Zen 2012]
  [ZenLite software download]

3D Visualization and Deconvolution
  [Autoquant Deconvolution Package]
  [Biophotonics Deconvolution]

IGB Map
[Map]

3

Handout: 
smFRET
TIRF Alignment

Lab 3 Answer Key

Background
Single-Molecule Techniques: A Laboratory Manual. Cold Spring Harbor Laboratory Press ISBN 978-087969775-4, 507 pp (2008), Chapter 2,  Joo, C. and T. Ha, "Single-Molecule FRET with Total Internal Reflection Microscopy."
Roy, R., S. Hohng and T. Ha., "A practical guide to single-molecule FRET.", Nat. Methods 5, 507-516 (2008)

G-quadruplex dynamics
Lee J.Y., Okumus B, Kim D.S., and Ha T. “Extreme conformational diversity in human telomeric DNA”, Proc Natl Acad Sci. 102(52), 18938–43 (2005).

Hidden Markov Modeling
Rabiner, LR. 1989. A Tutorial on Hidden Markov-Models And Selected Applications In Speech Recognition. Proc. IEEE 77 (2): 257-286.  
Liu, Y; Park, J; Dahmen, KA; Chemla, YR; Ha, T. 2010. A Comparative Study of Multivariate and Univariate Hidden Markov Modelings in Time-Binned Single-Molecule FRET Data Analysis. J. Phys. Chem. B 114 (16): 5386-5403. 

4

Handout: 
FIONA Lab

FIONA Lab Answer Key

Background
[FIONA: Application to Molecular Motors]
[Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization]
[Precise Nanometer Localization Analysis for Individual Fluorescent Probes]
[Kinesin Walks Hand-Over-Hand]

Matlab Codes
[FIONA Tutorial]
[Photon Counting]
[Stepping Analysis]

Camera Performance Sheets
[Sub-zero]
[Scorpion]

5

Handout: STORM

STORM Lab Answer Key

PALM/STORM principles
B. Huang, M. Bates, X. Zhuang, "Super-resolution Fluorescence Microscopy", Annual Review Biochemistry 78, 993-1016 (2009)
X. Zhuang, "Nano-imaging with STORM", Nature Photonics 3, 365-367 (2009)
B. Huang, W. Wang, M. Bates, X. Zhuang, "Three-dimensional Super-resolution Imaging by Stochastic Optical Reconstruction Microscopy", Science 319, 810-813 (2008)

[Removing free dyes after labeling antibodies]

[Excitation with two different wavelengths]

[z-axis drift correction]

Handout: Light Sheet Microscopy

Light Sheet Lab Answer Key

References
• Jan Huisken et al., Science 305, 1007 (2004)
Illumination Microscopy Optical Sectioning Deep Inside Live Embryos by Selective Plane
• Jan Huisken and Didier Y. R. Stainier OPTICS LETTERS / Vol. 32, No. 17 (2007)
Even fluorescence excitation by multidirectional selective plane  illumination microscopy (mSPIM)
• IGB light sheet instrument webpage
• LSM Z1 brochure
• LSM Sample Preparation
• IMARIS 3D analysis software information – from IGB core website