Introduction

Super-resolution (SR) fluorescence microscopy, is the preferred method for analysing living biological samples; however, it is limited to depths of 10-15 pm. The need to view biological activities in thicker (above 15pm), non-transparent, and non- homogeneous samples (e.g., mice brains, tumours, etc.) has called for new methods to be developed. The greater the depth at which the analysis is carried out, the more complex becomes the reconstruction of the corresponding image. Depletion microscopy by stimulated emission (STED) was the first SR technique that experimentally showed the use of the concept of Reversible Saturable Optical Linear Fluorescence Transitions (RESOLFT) for fast acquisition of images, allowing visualization of biological dynamics at 60-80 nm depths.

Technical features

STED microscopy thou successful, has limitations when used at high depths in biological samples. This is because it requires a mirror to be place above the sample (making it unsuitable for thick samples) and requires a minimum of two images to be taken sequentially from different angles, thus making it unsuitable for fast imaging applications. This invention addresses these challenges by providing a lightening method which uses an excitation beam followed by a depletion beam to acquire a fluorescence signal, to analyse a biological sample in a depletion microscopy system. A method with increased optical resolution, acquiring images in all three spatial dimensions, suitable for thick and non-homogenous sample imagining, all done in a simple and economical manner.

Possible Applications

• Stimulated emission depletion microscopy (STED);
• Biological and in vivo sample analysis.

Advantages

• Increased optical resolution of biological samples;
• Allows for fast imagining;
• Suitable for thick, non-transparent and non-homogenous samples;
• Acquired images in all three spatial dimensions;
• Simple and economical to use.