Abstract
We introduce and use Wavelet Imaging on Multiple Scales (WIMS) as an improvement to fluorescence correlation spectroscopy to measure physical processes and features that occur across multiple length scales. In this study, wavelet transforms of cell images are used to characterize molecular dynamics at the cellular and subcellular levels (i.e. focal adhesions). We show the usefulness of the technique by applying WIMS to an image time series of a migrating osteosarcoma cell expressing fluorescently labelled adhesion proteins, which allows us to characterize different components of the cell ranging from optical resolution scale through to focal adhesion and whole cell size scales. Using WIMS we measured focal adhesion numbers, orientation and cell boundary velocities for retraction and protrusion. We also determine the internal dynamics of individual focal adhesions undergoing assembly, disassembly or elongation. Thus confirming as previously shown, WIMS reveals that the number of adhesions and the area of the protruding region of the cell are strongly correlated, establishing a correlation between protrusion size and adhesion dynamics. We also apply this technique to characterize the behavior of adhesions, actin and myosin in Chinese hamster ovary cells expressing a mutant form of myosin IIB (1935D) that displays decreased filament stability and impairs front-back cell polarity. We find separate populations of actin and myosin at each adhesion pole for both the mutant and wild type form. However, we find these populations move rapidly inwards toward one another in the mutant case in contrast to the cells that express wild type myosin IIB where those populations remain stationary. Results obtained with these two systems demonstrate how WIMS has the potential to reveal novel correlations between chosen parameters that belong to different scales.
Highlights
Fluorescence fluctuation techniques [1, 2] have been extended to the fluorescence imaging domain, such as image correlation spectroscopy, which analyzes the intensity fluctuations in space and time from a fluorescence image time series
At fine scales Wavelet Imaging on Multiple Scales (WIMS) is a technique that can analyze diverse behavior of multiple clusters existing in the same focal adhesion to coarse scales where it can reveal large scale details such as the relationship between the number of adhesions and the cell boundaries
We can integrate the information acquired across different scales to discover relationships such as that between the cell boundary velocity and the adhesion dynamics (Panels A,B,D in S2 Fig)
Summary
Fluorescence fluctuation techniques [1, 2] have been extended to the fluorescence imaging domain, such as image correlation spectroscopy, which analyzes the intensity fluctuations in space and time from a fluorescence image time series. Spatio-temporal image correlation spectroscopy was introduced to analyze both space and time fluctuations and measure the velocity and diffusion coefficients of fluorescently labeled molecules across multiple regions of cells [10]. In an effort to work around the changing morphology, Mohl et al [20] transformed each image of an irregularly shaped cell to fit into an unvarying circle This mapping was used to look at the fluorescent protein distribution and dynamics, the results were not transformed back into the original reference frame. Other focal adhesion detection methods use thresholding (such as a high pass filter) to find adhesions in an image, tracking their individual movement in time while collecting information on their size, shape and lifetime [21, 22]. We decided to use a wavelet since the goal was to detect adhesions of finite length, which is better suited to a wavelet basis in detecting and localizing them
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
