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+ | Measuring Time-Lapse Experiments: | ||
+ | |||
+ | 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 | ||
+ | |||
+ | < | ||
+ | |||
+ | { 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: | ||
+ | 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: | ||
+ | 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: | ||
+ | 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, | ||
+ | |||
+ | **Considerations on Membrane Architecture: | ||
+ | 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, | ||
+ | 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/ | ||
+ | includes this slowing factor is thus an *apparent diffusion*. More | ||
+ | specifically when the molecule mobility is slowed down due to | ||
+ | binding/ | ||
+ | diffusion*. | ||
+ | |||
+ | To know more about microscopic diffusion and macroscopic diffusion | ||
+ | inside cell, refer to Luby-Phelps papers (Luby-Phelps, | ||
+ | Luby-Phelps, | ||
+ | |||
+ | { ImageJ website} | ||
+ | |||
+ | Free and powerful software for quantitative image analysis. | ||
+ | |||
+ | http:// | ||
+ | |||
+ | { EAMNET (European Advanced Microscopy Network) website} | ||
+ | |||
+ | http:// | ||
+ | |||
+ | The website is maintained by Stefan Terjung (ALMF, EMBL). Download page | ||
+ | links to many useful Macros for analyzing image-stacks. | ||
+ | |||
+ | { References} | ||
+ | |||
+ | [^1]: Good for overviewing FRAP; but I don’t agree with statement such | ||
+ | as below; Quote: “*When motion due to active transport or | ||
+ | unidirectional flow can be discounted, protein mobility in a cell is | ||
+ | due to brownian motion.*”, | ||
+ | by Brownian motion *and *the structural environment, | ||
+ | FRAP curve fitting difficult. | ||
+ | </ | ||
+ |
playground/markdown.1337000975.txt.gz · Last modified: 2016/05/24 12:46 (external edit)