Introduction

An optical microscopy method that enables the three-dimensional location of nanometric objects with high precision and over wide and tunable axial ranges is provided.

Technical features

The present method is based on splitting the detection arm of a microscope into 2 different pathways in combination with a high-speed varifocal lens. The 2 pathways pass through the varifocal lens at different positions, one perfectly aligned (AP) and one slightly decentered with respect to the lens optical axis (DP), and the corresponding images are collected using 2 different cameras or 2 regions within a single camera. From the AP pathway, an extended DOF image is obtained that is used to determine the x-y position of the species. By comparing the AP versus the DP images corresponding to the same species, a quantification of the z position of the different species can also be carried out.

Possible Applications

• Neuroscience: study of neuronal network dynamics at physiological conditions;
• Virology: analysis of virus trafficking;
• Molecular biology: characterization of any intracellular diffusion process in vitro;
• Flow analysis in fluid mechanics.

Advantages

• Tracking over large volumes;
• Tunable precision, down to the nanometer scale;
• High temporal resolution;
• Ease of implementation in commercial microscopes.