When viewing biological samples with a microscope, the light beam is disturbed if the lens of the objective is in a different medium than the sample. For example, when looking at a watery sample with a lens sur­rounded by air, the light rays bend more sharply in the air around the lens than in the water. This distur­bance leads to the measured depth in the sample being smaller than the actual depth. As a result, the sample appears flattened. “This problem has been known for a long time, and since the 80s, theories have been developed to determine a corrective factor for determining the depth. However, all these theories assumed that this factor was constant, regardless of the depth in the sample. This happened despite the fact that the later Nobel laureate Stefan Hell pointed out in the 90s that this scaling could be depth-dependent”, explains Jacob Hoogen­boom from the Delft University of Technology.

Sergey Loginov, a former postdoc at Delft University of Techno­logy, has shown with calculations and a mathematical model that the sample indeed appears more strongly flattened closer to the lens than farther away. PhD candidate Daan Boltje and postdoc Ernest van der Wee sub­sequently confirmed in the lab that the corrective factor is depth-dependent. Van der Wee: “We have compiled our results into a web tool and software provided with the article. With these tools, anyone can determine the precise corrective factor for their experiment.”

“Partly thanks to our calculation tool, we can now very precisely cut out a protein and its surroundings from a biological system to determine the structure with electron microscopy. This type of micro­scopy is very complex, time-consuming, and incredibly expensive. Ensuring that you are looking at the right structure is therefore very important”, says Boltje. “With our more precise depth deter­mination, we need to spend much less time and money on samples that have missed the biological target. Ultimately, we can study more relevant proteins and bio­logical structures. And determining the precise structure of a protein in a biological system is crucial for understanding and ultimately combating abnormali­ties and diseases.”

In the web tool, you can fill in the relevant details of your experiment, such as the refrac­tive indices, the aperture angle of the objective, and the wavelength of the light used. The tool then displays the curve for the depth-dependent scaling factor. You can also export this data for your own use. Addi­tionally, you can plot the result in combination with the result of each of the existing theories. (Source: TU Delft)

Reference: S. V. Loginov et al.: Depth-dependent scaling of axial distances in light microscopy, Optica 11, 553 (2024); DOI: 10.1364/OPTICA.520595

Link: Dept. of Imaging Physics, Delft University of Technology, Delft, The Netherlands