Research team solves decades-old problem in microscopy

When observing biological samples through a microscope, if the medium surrounding the objective lens differs from the medium of the sample, light beams can become distorted. For instance, when using an air-surrounded lens to view a water-based sample, the light rays in the air surrounding the lens bend more than they do in the water. This distortion can lead to depth measurements appearing shallower than they actually are, making the sample look flatter than it is.

This issue has persisted for a long time. Since the 1980s, various theoretical approaches have been proposed to correct the depth measurement, assuming a constant correction factor regardless of the sample's depth. However, as noted by Jacob Hoogenboom, Associate Professor at Delft University of Technology, Nobel laureate Stefan Hell pointed out in the 1990s that this ratio could depend on the depth, but the theoretical approaches still assumed a constant correction factor.

Sergey Loginov, a former postdoctoral researcher at Delft University of Technology, has now demonstrated through computational and mathematical models that samples closer to the lens appear flatter than those further away. PhD student Daan Boltje and postdoctoral researcher Ernest van der Wee later confirmed experimentally that the correction factor varies with depth.

This breakthrough was published in the journal Optica. The researchers have provided a web tool and software alongside the article to allow anyone to determine an accurate correction factor for their own experiments. In this web tool, users can enter relevant experimental details such as refractive index, objective lens numerical aperture, and the wavelength of the light used. The tool then displays a scaling factor curve in relation to depth. Users can also export these data for further analysis and even combine the results with existing theoretical outcomes.

Researcher Daan Boltje mentioned that with these more precise depth measurements, less time and money would be spent on samples that miss the biological target. This could eventually lead to more accurate studies of proteins and biological structures. Understanding and eventually preventing anomalies and diseases requires detailed knowledge of the precise structure of proteins within biological systems.

This new computational tool could greatly benefit researchers using microscopy by providing a more accurate correction factor for depth measurements, enabling more reliable results and facilitating a better understanding of complex biological structures.

Sources: Phys.org, Optica.