Measuring Time-Lapse Experiments: An Overview

 Kota Miura (Centre for Molecular and Cellular Imaging, EMBL), 29.June.2006

 

EMBO Practical Course 2006

“Microinjection and Detection of Probes”

 

Abstract

 

Time series of digital images, usually called ‘a stack’, contains temporal dynamics of position and intensity. By analyzing these dynamics, we can extract numerical parameter which then enables us to characterize the biological system. There are three types of dynamics. (i) Position does not change but intensity changes over time. (ii) Position changes but the intensity does not change. (iii) Both Position and Intensity change over time. Since (iii) is a combination of (i) and (ii), I will explain the basics of the measurement of type (i) and (ii). An example of type (i) is the measurement of cargo transport dynamics in vesicle trafficking (Hirschberg et al., 1998). Transition of protein localization from ER to Golgi then to the plasma membrane was measured over time by measuring the signal intensity in each statically positioned compartment. This type of technique has evolved to various sophisticated methods based on the same principle such as FRAP technique. Type (ii) corresponds to the measurement of movement, or object tracking, and an example is the single particle tracking of membrane surface proteins (Murase et al., 2004).

 

Notes

 

Single Particle Tracking (SPT)

 

(Saxton and Jacobson, 1997)   A review on SPT, also discusses about mean-square-displacement plot and interpretations.

(Kusumi et al., 1993)    Excellent application of SPT on constrained diffusion.

(Qian et al., 1991) Theoretical Comparison of SPT and FRAP

(Miura, 2005) A review on tracking techniques in cell biology.

 

Active Contour (SNAKES) Demo (Link)

 

FRAP reviews

Reviews on FRAP (Phair et al., 2004; Sprague and McNally, 2005). Another review is a bit older, but good for overviewing classic literatures (Reits and Neefjes, 2001)[1].

 

Models for FRAP analysis

 

Diffusion: Axelrod et. al.’s paper is a frequently cited classic paper on FRAP (Axelrod et al., 1976). They measured pure diffusion. Closed solution for Axelrod’s model was proposed later and still used by many researchers (Soumpasis, 1983).

Several empirical formula for fitting diffusion-FRAP can be found in other literatures (Ellenberg et al., 1997; Yguerabide et al., 1982).

 

Reaction: Jacquez ‘s book is good for learning the compartmental analysis used for modelling reaction-dominant FRAP recovery  (Jacquez, 1972) The book is also informative and excellent for modelling biochemical dynamics in general. Recent advances in biochemistry incorporate interaction with immobile (non-diffusive) entity, which radically changes the interpretation of parameter acquired by fitting exponential equations (Bulinski et al., 2001; Sprague et al., 2004)

 

Advanced Models for FRAP

 

Diffusion-Reaction: Formula considering both diffusion and reaction were recently proposed (Sprague et al., 2004). This paper is interesting not only for this diffusion-reaction approach but also for derivation of pure-diffusion, effective diffusion and reaction dominant FRAP.

 

Considerations on Membrane Architecture: Mobility of proteins is generally constrained by the complex architecture of intracellular space, the shape of organelle. Such steric effects has been omitted from FRAP analysis for the estimation mobility parameters e.g. diffusion coefficient.  Recent literature includes this effect for the FRAP analysis by reconstructing the ER membrane geometry by 3D rendering and simulating the movement of protein along that geometry (Sbalzarini et al., 2006; Sbalzarini et al., 2005). 

 

 

Cytoplasmic Architecture

 

Diffusion within cytoplasm is not a simple pure-diffusion. Cytoskeletons, organelle and supramolecular complexes become obstacles to the movement of proteins. In a very small scale, the vacant spaces between these structures allow the molecule to move around without encountering these structures. In this vacant space, the cytoplasmic viscosity is said to be similar to water, or 2-3 folds higher than water. Measurement of small scale diffusion needs special techniques. On the other hand, we also can measure the movement of molecules in a larger scale. In this case, diffusion encounters steric hindrances and bindng/reaction with other molecules. Diffusion coefficient that includes this slowing factor is thus an apparent diffusion. More specifically when the molecule mobility is slowed down due to binding/reactions, this type pf diffusion is called effective diffusion.

 

To know more about microscopic diffusion and macroscopic diffusion inside cell, refer to Luby-Phelps papers (Luby-Phelps, 1994; Luby-Phelps, 2000).

 

 

ImageJ website

Free and powerful software for quantitative image analysis (Link).

 

EAMNET (European Advanced Microscopy Network) website

The website (Link) is maintained by Stefan Terjung (ALMF, EMBL). Download page links to many useful Macros for analyzing image-stacks.

 

 

References

 

 

Axelrod, D., D.E. Koppel, J. Schlessinger, E. Elson, and W.W. Webb. 1976. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 16:1055-69.

Bulinski, J.C., D.J. Odde, B.J. Howell, T.D. Salmon, and C.M. Waterman-Storer. 2001. Rapid dynamics of the microtubule binding of ensconsin in vivo. J Cell Sci. 114:3885-97.

Ellenberg, J., E.D. Siggia, J.E. Moreira, C.L. Smith, J.F. Presley, H.J. Worman, and J. Lippincott-Schwartz. 1997. Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J Cell Biol. 138:1193-206.

Hirschberg, K., C.M. Miller, J. Ellenberg, J.F. Presley, E.D. Siggia, R.D. Phair, and J. Lippincott-Schwartz. 1998. Kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport intermediates in living cells. J Cell Biol. 143:1485-503.

Jacquez, J.A. 1972. Compartmental analysis in biology and medicine. Elsevier.

Kusumi, A., Y. Sako, and M. Yamamoto. 1993. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys J. 65:2021-40.

Luby-Phelps, K. 1994. Physical properties of cytoplasm. Curr Opin Cell Biol. 6:3-9.

Luby-Phelps, K. 2000. Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int Rev Cytol. 192:189-221.

Miura, K. 2005. Tracking Movement in Cell Biology. In Advances in Biochemical Engineering/Biotechnology. Vol. 95. J. Rietdorf, editor. Springer Verlag, Heidelberg. 267.

Murase, K., T. Fujiwara, Y. Umemura, K. Suzuki, R. Iino, H. Yamashita, M. Saito, H. Murakoshi, K. Ritchie, and A. Kusumi. 2004. Ultrafine membrane compartments for molecular diffusion as revealed by single molecule techniques. Biophys J. 86:4075-93.

Phair, R.D., S.A. Gorski, and T. Misteli. 2004. Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy. Methods Enzymol. 375:393-414.

Qian, H., M.P. Sheetz, and E.L. Elson. 1991. Single particle tracking. Analysis of diffusion and flow in two-dimensional systems. Biophys J. 60:910-21.

Reits, E.A., and J.J. Neefjes. 2001. From fixed to FRAP: measuring protein mobility and activity in living cells. Nat Cell Biol. 3:E145-7.

Saxton, M.J., and K. Jacobson. 1997. Single-particle tracking: applications to membrane dynamics. Annu Rev Biophys Biomol Struct. 26:373-99.

Sbalzarini, I.F., A. Hayer, A. Helenius, and P. Koumoutsakos. 2006. Simulations of (an)isotropic diffusion on curved biological surfaces. Biophys J. 90:878-85.

Sbalzarini, I.F., A. Mezzacasa, A. Helenius, and P. Koumoutsakos. 2005. Effects of organelle shape on fluorescence recovery after photobleaching. Biophys J. 89:1482-92.

Soumpasis, D.M. 1983. Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys J. 41:95-7.

Sprague, B.L., and J.G. McNally. 2005. FRAP analysis of binding: proper and fitting. Trends Cell Biol. 15:84-91.

Sprague, B.L., R.L. Pego, D.A. Stavreva, and J.G. McNally. 2004. Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys J. 86:3473-95.

Yguerabide, J., J.A. Schmidt, and E.E. Yguerabide. 1982. Lateral mobility in membranes as detected by fluorescence recovery after photobleaching. Biophys J. 40:69-75.