When using a microscope to view biological samples, it is crucial to consider the medium in which the lens of the objective is placed. The light beam can be disturbed if the lens is in a different medium than the sample, causing the light rays to bend differently. This disturbance can result in the sample appearing flattened, with the measured depth being smaller than the actual depth.
The Evolution of Corrective Theories
For decades, researchers have been aware of this issue and have developed theories to determine a corrective factor for depth measurement. However, these theories previously assumed that the corrective factor was constant, regardless of the depth of the sample. It was not until Nobel laureate Stefan Hell suggested in the 90s that this factor could be depth-dependent that researchers began to explore this concept further.
Recent research by Sergey Loginov, Daan Boltje, and Ernest van der Wee at Delft University of Technology has shed light on the importance of depth-dependent correction in microscopy. Through calculations and a mathematical model, Loginov demonstrated that samples appear more flattened closer to the lens than farther away. Boltje and van der Wee confirmed this finding in the lab, highlighting the significance of considering depth-dependent factors in microscopy.
By developing a web tool and software to determine the precise corrective factor for experiments, the researchers have made it easier for scientists to accurately analyze biological structures using electron microscopy. This advancement has the potential to save time and resources by ensuring that researchers are focusing on the right structures in their samples.
According to Daan Boltje, this newfound precision in depth determination allows researchers to study more relevant proteins and biological structures, ultimately aiding in the understanding and treatment of abnormalities and diseases. By accurately determining the structure of proteins within biological systems, researchers can make significant strides in combating various health issues.
The web tool provided by the researchers allows users to input relevant details of their experiments, such as refractive indices, aperture angle of the objective, and the wavelength of light used. The tool then generates a curve for the depth-dependent scaling factor, which can be exported for further analysis. Furthermore, users can compare the results with existing theories to gain a comprehensive understanding of depth correction in microscopy.
The discovery of depth-dependent correction in microscopy has revolutionized the way researchers analyze biological samples. By taking into account the varying factors that affect depth perception, scientists can now conduct more accurate and efficient studies, leading to advancements in various fields of research.