The second edition of Live Cell Imaging: A Laboratory Manual expands upon and extends the collection of established and evolving methods for studying dynamic changes in living cells and organisms presented in the well-known first edition. There are 16 new chapters and 21 updated chapters in this new edition. They include advances in atomic force microscopy, structured illumination microscopy and other 3-D approaches, as well as imaging in single cells in animals and in plants. New analytical options include live high-throughput/high-content screening in mammalian cells and computational analysis of live cell data. The manual presents hands-on techniques as well as background material, and can serve as a text in advanced courses. The first section covers principles and fundamental issues of detection and imaging; the second provides detailed protocols for imaging live systems.

Section I: Detection and Approaches to Live Cell Imaging

1. Fluorescent Protein Tracking and Detection

M. Rizzo, M. Davidson, and D. Piston

Select a movie: Movie 1_1. African green monkey kidney epithelial cells (CV-1 line) expressing EGFP fused to human α-tubulin Movie 1_2. Pig kidney epithelial cells (LLC-PK1 line) expressing EGFP fused to human α-tubulin and mCherry fused to human histone H2B. Movie 1_3. Fox lung epithelial cells (FoLu line) expressing EGFP fused to human β-actin. Movie 1_4. Fox lung epithelial cells (FoLu line) expressing EGFP fused to human β-actin (second example). Movie 1_5. Fox lung epithelial cells (FoLu line) expressing EGFP fused to microtubule EB3. Movie 1_6. Fox lung epithelial cells (FoLu line) expressing EGFP fused to human α-tubulin. Movie 1_7. Fox lung epithelial cells (FoLu line) expressing EYFP fused to human-light-chain clathrin. Movie 1_8. Fox lung epithelial cells (FoLu line) expressing mCherry fused to human β-actin and mKusabira Orange fused to human mitochondria. Movie 1_9. Fox lung epithelial cells (FoLu line) expressing mKusabira Orange fused to microtubule EB3. Movie 1_10. Fox lung epithelial cells (FoLu line) expressing mKusabira Orange fused to a mitochondrial targeting sequence. Movie 1_11. Fox lung epithelial cells (FoLu line) expressing YPet fluorescent protein fused to microtubule EB3. Movie 1_12. Human cervical carcinoma epithelial cells (HeLa line) expressing EGFP fused to a peroxisomal targeting sequence (SKL). Movie 1_13. Human cervical carcinoma epithelial cells (HeLa line) expressing EYFP fused to a mitochondrial targeting sequence. Movie 1_14. Pig kidney epithelial cells (LLC-PK1 line) expressing mEmerald fluorescent protein fused to microtubule EB3 and mCherry fused to human histone H2B. Movie 1_15. Fox lung epithelial cells (FoLu line) expressing GFP fused to a targeting signal for the Golgi apparatus and DsRed fused to a mitochondrial targeting sequence. Movie 1_16. Human osteosarcoma epithelial cells (U2OS line) expressing mEmerald fluorescent protein fused to an endoplasmic reticulum targeting peptide. Movie 1_17. Opossum kidney epithelial cells (OK line) expressing EGFP fused to human β-actin. Movie 1_18. Opossum kidney epithelial cells (OK line) expressing EGFP fused to an endosome targeting signal. Movie 1_19. Opossum kidney epithelial cells (OK line) expressing EGFP fused to a targeting signal for the Golgi apparatus. Movie 1_20. Opossum kidney epithelial cells (OK line) expressing EGFP fused to LAMP1 (targeting the lysosomes). Movie 1_21. Opossum kidney epithelial cells (OK line) expressing EYFP fused to human β-actin. Movie 1_22. Opossum kidney epithelial cells (OK line) expressing EYFP fused to a targeting signal for the Golgi apparatus.

2. Constructing and Expressing Fluorescent Protein Fusions

D. Spector and B. Goldman

3. Micropatterning Cell-Substrate Adhesions Using Linear Polyacrylamide as the Blocking Agent

W.-h. Guo and Y.-l. Wang

4. CCD Cameras for Fluorescence Imaging of Living Cells

W. Salmon and J. Waters

5. Fluorescence Perturbation Techniques to Study Mobility and Molecular Dynamics of Proteins in Live Cells: FRAP, Photoactivation, Photoconversion, and FLIP

A. Bancaud, S. Huet, G. Rabut, and J. Ellenberg

6. Imaging Protein State in Cells

H. Grecco and P. Bastiaens

7. A Versatile, Multicolor Total Internal Reflection Fluorescence and Spinning-Disk Confocal Microscope System for High-Resolution Live Cell Imaging

W. Shin, R. Fischer, P. Kanchanawong, Y. Kim, J. Lim, K. Myers, Y. Nishimura, S. Plotnikov, I. Thievessen, D. Yarar, B. Sabass, and C. Waterman

8. Confocal Microscopy, Deconvolution, and Structured Illumination Methods

J. Murray

9. Atomic-Force Microscopy for Biological Imaging and Mechanical Testing across Length Scales

M. Plodinec, M. Loparic, and U. Aebi

10. OMX: A New Platform for Multimodal, Multichannel Wide-Field Imaging

I. Dobbie, E. King, R.M. Parton, P. Carlton, J.W. Sedat, J.R. Swedlow, and I. Davis

11. Digital Scanned Laser Light-Sheet Fluorescence Microscopy

P.J. Keller and E.H.K. Stelzer

Select a movie: Movie 11_1. Schematic operation principle of DSLM.

12. First Step for Fluorescence Correlation Spectroscopy for the Live Cell

M. Kinjo, H. Sakata, and S. Mikuni

13. Tracking and Quantitative Analysis of Dynamic Movements of Cells and Particles

K. Rohr, W. Godinez, N. Harder, S. Wörz, J. Mattes, W. Tvaruskó, and R. Eils

14. Imaging Techniques for Measuring Cell Materials Properties

K. Kasza, D. Vader, S. Köster, N. Wang, and D. Weitz

15. Computational Image Analysis of Cellular Dynamics: A Case Study Based on Particle Tracking

K. Jaqaman and G. Danuser

16. Software Tools, Data Structures, and Interfaces for Microscope Imaging

N. Stuurman and J.R. Swedlow

17. High-Throughput Microscopy Using Live Mammalian Cells

S. Terjung, T. Walter, A. Seitz, B. Neumann, R. Pepperkok, and J. Ellenberg

Section II: Imaging of Live Cells and Organisms

18. In Vivo Imaging of Mammalian Cells

J. Swedlow, I. Porter, M. Posch, and S. Swift

19. Live Cell Imaging of Yeast

D. Rines, D. Thomann, J. Dorn, P. Goodwin, and P. Sorger

20. Live Imaging of Caenorhabditis elegans

B. Podbilewicz and Y. Gruenbaum

Select a movie: Movie 20_1. Arrested C. elegans embryos injected with lmn-1 dsRNA. Movie 20_2. Simultaneous imaging of Nomarski (blue) and eff-1promoter::GFP (green) in developing C. elegans embryos reveals timing of cell fusion. Movie 20_3. Expression of AJM-1::GFP in C. elegans epithelial cells during embryonic morphogenesis. Movie 20_4. z-series of two C. elegans vulval toroids expressing egl-17/FGFpromoter::GFP. Movie 20_5. Projection of the z-series and rotation of two C. elegans vulval rings expressing fibroblast growth factor (egl-17). Movie 20_6. FRAP experiment on a C. elegans nucleus expressing GFP::Ce-lamin wild-type protein. Movie 20_7. FRAP experiment on a C. elegans nucleus expressing GFP::Ce-lamin containing an R64P mutation.

21. Live Cell Imaging of Plants

Y. Fang and D. Spector

Select a movie: Movie 21_1. Volume-rendering view of centromere organization in a nucleus of an Arabidopsis leaf epidermal cell. Movie 21_2. Dynamic views of centromere movements through mitosis.

22. Pushing the Limits of Live Cell Imaging in Drosophila

R. Parton, A.M. Vallés, I. Dobbie, and I. Davis

Select a movie: Movie 22_1. Injection into a stage-4 syncytial blastoderm Drosophila embryo (approximately cycle 14–15). Movie 22_2. Bicoid mRNA-transport dynamics followed by bcd-MS2/MCP-GFP labeling of bcd mRNA particles at the anterior of a stage-11 Drosophila egg chamber. Movie 22_3. Multiphoton and confocal imaging deep into a late-stage (12–18 h after laying) Drosophila histone-H2A GFP embryo.

23. Dynamic, Long-Term, In Vivo Imaging of Tumor-Stroma Interactions in Mouse Models of Breast Cancer Using Spinning Disk Confocal Microscopy

A.J. Ewald, Z. Werb, and M. Egeblad

Select a movie: Movie 23_1. Myeloid cells extravasate and infiltrate late carcinoma lesions. Movie 23_2. Myeloid cells migrate in response to wounding on the surface of a normal mammary gland.

24. High-Resolution Multiphoton Imaging of Tumors In Vivo

J. Wyckoff, B. Gligorijevic, D. Entenberg, J. Segall, and J. Condeelis

25. Correlated Live Cell Light and Electron Microscopy Using Tetracysteine Tags and Biarsenicals

G.M. Gaietta, T.J. Deerinck, and M.H. Ellisman

26. Intravital Microscopy of Normal and Diseased Tissues in the Mouse

R.K. Jain, L.L. Munn, and D. Fukumura

Select a movie: Movie 26_1. Tumor blood flow. Movie 26_2. Leukocyte endothelial interaction in tumor vessels. Movie 26_3. Neovasculature in de novo adipose tissue. Movie 26_4. Three-dimensional presentation of tissue-engineered blood vessels.

27. Imaging Lipids in Living Cells

C. Schultz, A.B. Neef, T.W. Gadella, Jr., and J. Goedhart

28. Development of Mammalian Cell Lines with lac Operator-Tagged Chromosomes

Y.G. Strukov, M. Plutz, and A.S. Belmont

29. Imaging Gene Expression in Living Cells

S. Janicki and D. Spector

Select a movie: Movie 29_1. Real-time analysis of gene expression.

30. Studying Mitosis in Cultured Mammalian Cells

P. Wadsworth

Select a movie: Movie 30_1. Mitosis in pig kidney cells expressing GFP-tubulin.

31. Imaging Intermediate Filament Proteins in Living Cells

E.R. Kuczmarski, T. Shimi, and R.D. Goldman

Select a movie: Movie 31_1. A well-spread BHK-21 cell expressing GFP-vimentin. Movie 31_2. Spreading BHK-21 cells showing the motile properties of vimentin particles, the apparent fusion of some particles, and the formation of short filaments or squiggles. Movie 31_3. A PC-12 cell expressing GFP-peripherin in the region of a newly emerging growth cone observed a short time following the addition of nerve growth factor. Movie 31_4. A region of a PtK2 cell transfected with GFP-keratin 14. Movie 31_5. A dividing HeLa cell expressing GFP-lamin A during mitosis.

32. Methods for Expressing and Analyzing GFP-Tubulin and GFP-Microtubule-Associated-Proteins

H.V. Gooodson, J.S. Dzurisin, and P. Wadsworth

Select a movie: Movie 32_1. MT dynamic instability, visualized in the peripheral region of an LLCPK1 cell expressing GFP-tubulin. Movie 32_2. MT growth visualized in an LLCPK1 cell expressing GFP-EB1 to mark the growing MT plus ends. Movie 32_3. MT growth visualized in a COS-7 cell expressing GFP-CLIP-170 to study CLIP-170 plus-end tracking behavior.

33. Imaging of Membrane Systems and Membrane Traffic in Living Cells

E.L. Snapp and P. Lajoie

34. Imaging Live Cells under Mechanical Stress

B.P. Helmke and P.F. Davies

Select a movie: Movie 34_1. Time-lapse movie of deconvolved optical section demonstrating intracellular GFP-vimentin filament movement in 90-sec intervals (green) superimposed on stationary images (red) selected at the start of the no-flow and flow periods. Movie 34_2. Time-lapse movie of deconvolved optical section showing GFP-vimentin filament movement near the apical cell surface

35. Imaging Single Molecules Using Total Internal Reflection Microscopy

S. Reck-Peterson, N. Derr, and N. Stuurman

Select a movie: Movie 35_1. Total internal reflection microscopy imaging of single dynein molecules labeled with TMR (green) moving along axonemal microtubules labeled with Cy5 (red).

36. Cellular Imaging Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

D. Toomre

Select a movie: Movie 36_1. GFP-tubulin-tagged microtubules target the cell cortex of cAR fibroblast cells, often along similar paths. Movie 36_2. YFP-tubulin-tagged microtubules target the cortex of PtK2 epithelial cells. Movie 36_3. YFP-actin shows both retrograde flow and puntae that disappear (likely clathrin-coated pits undergoing endocytosis n PtK2 cells). Movie 36_4. Epifluorescence (red) and TIRFM (green) of the same image plane of vesicular stomatitis virus glycoprotein (VSVG)-YFP-tagged vesicles undergoing exocytosis. Movie 36_5. TIRFM movie of VSVG-YFP vesicles traffic to the cytokinesis furrow of BSC-1 cells from both daughter cells where they subsequently fuse. Movie 36_6. GFP-tagged clathrin light chain expressed in Cos cells show many puntae corresponding to clathrin-coated pits.

37. Visualization and Quantification of Single RNA Molecules in Living Cells

Y. Shav-Tal, S.M. Shenoy, and R.H. Singer