Introduction

The invention relates to a novel optical microscope which provides the possibility to perform inertia free axial scanning of the detected focus plane. The velocity and depth of the axial scanning can be modulated. When the axial scanning is slow, it is possible to acquire an image for each focused plane to obtain a spatial z- stack of the sample. When the axial scanning is fast, it is possible to collect the optical signals from all the scanned planes and project them on a single image.

Technical features

We present a novel optical configuration to perform an inertia free axial scanning of the sample in order to acquire a z-stack of the sample, or to perform extended depth imaging, at single cell resolution, with a variable extension of the depth of field. The system is based on a simple widefield configuration, where we introduced a liquid tunable lens (TL) in the detection path in order to perform dynamic phase modulation. The liquid tunable lens (commercially available) is a lens with variable focal length that can be modulated by a driving external voltage (temporal bandwidth up to 1 kHz). By varying the focal length of the tunable lens, we modify the optical transfer function of the detection pathway, and thus we scan the in-focus plane of the objective in the axial direction of the sample. In such a way we can reconstruct the 3D architecture of the sample and combine it with the fast wide volume time lapse 3D imaging to monitor the functional activity of the sample at single cell resolution.

Possible Applications

• Functional time-lapse imaging of 3D cell laden constructs;
• Wide volume calcium imaging with single cell resolution in 3D samples;
• Fast z-axis scanning;
• Extended depth field microscopy;
• High throughput screening of 3D samples.

Advantages

• Dynamic phase modulation of the optical detection pathway;
• Detection of fluorescence signal fluctuations by a time lapse imaging within a modulable extended depth of field;
• Inertia-free axial scanning;
• Tunable high velocity axial scanning.