• Universitat de València

• Angel Tolosa Ruiz
• Nicolo Incardona
• Emilio Sánchez Ortiga

The present invention comprises an accessory, attachable to a camera port of a host microscope, and enables a stack of focal images to be obtained at different depths.

A conventional optical microscope, working with 3D samples, provides the sharp image of one section of the sample, the one above the focal plane targeted by the microscope objective. The rest of the sample sections provide a very blurred image. Only by shifting the stage it is possible to focus on sections of the sample at other depths. To capture a stack of 3D cross-sectional images of samples, an axial mechanical scanning process is necessary in which, at each step, the stage is mechanically moved. This process requires, for each step, waiting some time for the system to stabilise before finally capturing the image with the pixel sensor. This whole process can be too slow for moving samples. Even in the case of static samples, but immersed in a liquid medium, the mechanical scanning causes vibrations that can modify the characteristics of the sample.

To avoid these problems, there are different proposals for optical accessories that allow the capture of image stacks without moving the stage. However, none of these solutions preserves the characteristics of the host microscope in terms of magnification, resolution, depth of field and aberration correction. A simple, robust and portable accessory for obtaining a focal image stack is therefore necessary. This accessory must be attachable to any microscope, maintaining its characteristics intact and providing fast focusing capacity without causing vibrations, inertia or changes in the scale of the focal stack images.

Researchers at the Universitat de València have developed a novel accessory for obtaining focal image stacks that can be attached to a host microscope, which provides a simple, robust and portable solution to the problem of capturing focal image stacks of 3D samples free of aberrations, with constant lateral magnification and avoiding mechanical vibrations. From this stack, a 3D image of the sample can be obtained using computational techniques.

The invention is of interest in a multitude of applications, especially those in which the vibrations introduced by mechanical systems are critical because they seriously alter the characteristics of the 3D sample. The following applications, among others, can be highlighted:

• Microfluidic analysis: avoiding vibrations during the 3D registration process.
• Assisted reproduction: analysis of 3D images of embryos.
• Biomedicine: analysis of the evolution of cancer cells; or bioprinting and tissue  engineering applied to regenerative medicine.
• Biology and microbiology: study of biofilms generated by bacteria.
• Surgery: extending the focus range of surgical microscopes.
• Forensic science: analysis of marks in solid samples.
• Industrial: quality control and machine vision analysis.
• Microelectronics: analysis of the elements present in motherboard circuits and their connectors.

This invention has the following advantages: stability; constancy of scale for the entire 3D image; accuracy and reliability of measurements; versatility; simplicity and handiness; constancy of the native properties of the host microscope; width of the axial focusing range, speed; immediate cost reduction and cost reduction in the medium and long term.

• Patent granted