3-Dimensional particle tracking in a laser scanning fluorescence microscope

VALERIA LEVI AND ENRICO GRATTON

Single Particle Tracking and Single Molecule Energy Transfer

wiley-vch

Lugar: weinheim, germany; Año: 2009;

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Single-particle and single molecules techniques have become essential tools in the fields of Biophysics and Cell Biology [1]. One of the main reasons of the strong impact of these techniques is that they provide crucial information that is averaged out in traditional ensemble methods.  Amongst these new techniques, single particle tracking (SPT) constituted a remarkable new tool to study dynamics of biological processes. Several fluorescence microscopy techniques have been developed to measure the motion of molecules. Two of the methods most widely used are fluorescence recovery after photobleaching (FRAP), developed in the 1970s by Axelrod et al. [2] and fluorescence fluctuation spectroscopy (FCS) established during the same decade by Magde et al. [3]. Figure 1 schematically represents some of the most important characteristics of these techniques. While FRAP averages in time and space the behavior of a large ensemble of molecules, FCS averages the behavior of a small number of molecules within the observation volume. In both cases, the mobility properties determined in these experiments correspond to the average behavior of the observed molecules.  Averaging can be a problem, for example, in the complex environment of the cell where particles can interact with multiple targets resulting in populations with different mobility properties. Moreover, the dynamics of each population may change in time and/or space. In such cases, FRAP and FCS approaches will only give limited dynamical information. SPT was first applied in Biophysics during the 80s-90s (see for example, [4-7]). The number of applications of these techniques has grown significantly since then thanks to advances in microscopy and labeling techniques which significantly improved the accuracy and speed of these methods and opened the possibility of studying more complex processes with better spatial and temporal resolution. These techniques were developed to follow the position of individual particles in time. Provided the spatial and temporal resolution of the method is adequate, these trajectories can be statistically analyzed to extract quantitative information about the mechanism involved in the motion of the particle (for recent reviews, see [8, 9]).   Since the properties of the particles are not averaged as in bulk measurements, SPT is an appealing technique to achieve the ultimate goal of understanding dynamics in cells. In this chapter, we briefly describe the most common techniques used for tracking particles and focus on recent advances in the field of microscopy that resulted in an improvement of these methods. We discuss different strategies followed to obtain information regarding the axial position of the particle in image-based tracking approaches and describe in detail a routine we have designed to achieve 3D tracking with a laser scanning microscope. Finally, we show the application of this technique to the study of chromatin dynamics in interphase cells to exemplify possible uses of this new tracking procedure.