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Advanced Microscopy Methods

Molecular Trafficking
Molecular trafficking

Knowledge of the mechanisms regulating molecular trafficking in biological systems is key to the rational design of nanosystems for delivery applications. In fact, fine tuning of molecular trafficking may have significant physiological implications, as it determines the efficiency of biologically active and inert molecules in reaching their target destinations. We apply state-of-the art optical microscopy methods to measure molecular trafficking, including fluorescence recovery after photobleaching (FRAP), single particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) (Fig.1).

FRAP is a perturbation-based ensemble measurement that provides temporal information on the recovery of the concentration of molecules without knowledge of where the fluorescence recovery originates from. On this basis it can be helpful to recover the average dynamic behaviour of a molecule in a whole intracellular compartment or between compartments. SPT, on the other hand, although high in spatial resolution, requires the observation of isolated and large particles for a long time which yields poor statistics. Moreover, the molecule of interest must be purified, properly labelled, and introduced into cells by complex experimental manipulations (e.g. microinjection, electroporation, permeabilization). Besides these experimental drawbacks, the SPT approach is still mandatory if the high-resolution spatial trajectory of the molecule is to be studied . FCS in contrast provides a single-molecule level of information in a selected location with good statistics by averaging the behaviour of many molecules, and has thus emerged as the preferred method for studying the motions of molecules in live samples.

Pair-correlation method
Pair-correlation method

Traditionally FCS is performed as a single point measurement (spFCS). spFCS measures the average time a single fluorescent molecule employs to pass through a certain excitation volume (e.g. the point spread function, PSF, defined by the incident laser beam). If the volume of excitation is known, the local diffusion coefficient and concentration of molecules at the point of excitation can be derived. The limitation of this method of acquisition is that the spatial environment around the excitation volume and thus the route the molecules take prior to crossing the observation volume is not directly observed in the experiment. The combination of the classical single-point FCS approach (local measurement of diffusion) to the recent development of scanning-based approaches (line FCS, Raster Image Correlation Spectroscopy) and data analysis algorithms (pair correlation functions, pCF) has the potential to achieve a more complete description of single molecule behavior in cells. Concerning the pCF algorithm in particular, it shall be noted how it is becoming increasingly used in the study of intracellular transport as it offers a unique tool to probe the directionality of intracellular traffic, by measuring the accessibility of the cellular landscape and its role in determining the diffusive routes adopted by molecules (Fig. 2).

The sensitivity of the pCF method toward detection of barriers means that different structural elements of the cell can be tested in terms of penetrability and mechanisms of regulation imparted on molecular flow. This has been recently demonstrated in a series of studies looking at molecular transport inside live cells (e.g. intranuclear diffusion, nuclear import/export).

On this basis we have decided to activate a platform for the use and development of Fluorescence Correlation Spectroscopy techniques to extract single molecule information in the presence of many molecules, in live, minimally perturbed cells. The interdisciplinary structure of the research team present at CNI conveys the necessary biological background and the expertise in state-of-the-art bioimaging applied to molecular trafficking.

 

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