Microscopy has revolutionized our understanding of the world, allowing us to peer into the intricate details of cells, tissues, and even viruses. However, obtaining precise measurements in these minute worlds is quite difficult. Just like fog can make distant objects look different from reality, so does the light treatment of samples in microscopes, which makes them compressed or elongated along the depth axis especially. For decades now, this problem, known as refractive index mismatch (RIM), has troubled researchers.

Recently, there was a breakthrough at Delft University of Technology. In their work published in Optica journal, they present an innovative approach that could be used to determine depth-dependent re-scaling factors, which are very important in correcting distortions caused by RIM. This blog post delves into the complexities of RIM, explores existing approaches to deal with it, and explains how this new method is changing the world of microscopy.

The Challenge of Refractive Index Mismatch

Imagine looking out of a window onto a teeming city street; you get an unobstructed view if the glass on the window is squeaky clean. However, you may see it distorted if there is a layer of droplets or any difference in refractive index between the air and the glass. The bending of light as it passes from one material to another results in an illusion that objects are closer or farther away than they are.

This principle holds for microscopy as well. When light travels from the objective lens into a biological sample, the image may be distorted because of differences in refractive indices between immersion liquids (e.g., water or oil) surrounding the lens and the sample itself. This distortion manifests as a compression of the depth axis, making structures appear flatter than they are.

Existing Methods: A One-Size-Fits-All Approach

Considerable effort has been put into researching RIM (Refractive Index Mismatch) and its consequences on quantifying depth. Several approaches have been proposed to address this issue over time. In most cases, such methods involve applying scaling factors to measured values of depth. However, one major drawback associated with these techniques is that they assume constant scaling throughout all depths within samples.

Although the distortion caused by RIM is non-uniform in reality, it is not so for light rays entering the sample at larger depths as well as for those near the surface. Research has recognized that there is a depth-dependent variation of this type of distortion, but no method was available to account for it directly when calculating.

A Depth-Aware Solution: The New Analytical Theory

Dr. Jacob Hoogenboom and his colleagues from the Delft University of Technology reconsidered the problem. They presented an innovative analytical theory that reflects variations in re-scaling factors according to depth. The idea underlying their work is “leading constructive interference band” at the objective lens pupil part under RIM.

Essentially, the objective lens behaves like a filter, allowing light rays to go through and be involved in making pictures. In other words, wavelengths within a particular range in the spectrum will interfere constructively in what may be termed a leading constructive interference band. The researchers could, therefore, determine how this band changes depth-wise due to RIM within the specimen and calculate a re-scaling factor dependent on depth.

Testing the Theory: Experiments and Simulations

The researchers had to go on validating their theoretical framework. They did this using two methods, namely wave-optics calculations and experimental measurements.

The wave-optics calculation calculates how light behaves in a microscope under different conditions, like the interaction of light with an objective lens or sample. The calculations were made at various depths in the sample so that the comparison between theoretical model predictions and predicted the researchers could make depth-dependent re-scaling factor.

For the experiments, there was a smartly designed configuration where two substrates were slowly brought into proximity of each other. In between, they used liquid with specific refractive index values to represent some biological specimens. This experiment provided them with an experimental re-scaling factor through which they measured apparent distances between substrates and true ones at varying depths.

The results were positive. The new theory’s calculated depth-dependent re-scaling factor matched very closely those obtained from both wave-optics simulations and experimental measurements. This validation supported their approach as a correct and useful one.

The Benefits of Depth-Dependent Re-scaling

This advance offers better alternatives to the current depth-independent methods. Researchers can use a re-scaling factor that is dependent on depth to obtain highly accurate measurements in their samples. This becomes particularly important when studying minute protein and organelle structures whose function cannot be understood without precise measurements.

The improvements go beyond accuracy. The new technique eliminates the need for corrections of distortions related to depth, thereby allowing researchers to save time and resources. As a result, researchers can carry out more studies in less time, thus enhancing our understanding of biological processes. The inclusion of depth-dependent re-scaling also allows us to investigate previously un-analyzed samples that were inhibited by strong RIM.

Turning Theoretical Concepts into Practical Tools for Scientists

In this regard, the Delft University group has come up with two valuable tools:

  • Online Web Applet: This easy-to-use online tool helps visualize how deep a microscope can re-scale. It is composed of parameters that can be filled with the numerical aperture (NA) of the objective, refractive indices of immersion medium and sample, and wavelength of light used to generate a graph showing depth dependence of re-scaling factor for a given sample. What it does show is an optical distortion.
  •  Software Toolkit: Besides the web app, the researchers have also made available Python software, which can be downloaded. This software allows researchers to directly apply the depth-dependent re-scaling factor to their microscopy data. Researchers may include this in their analysis workflows so they can automatically correct for distortions that may result from differences in depth, thus saving them time and helping ensure accurate measurements.

These are user-friendly tools that eliminate cumbersome calculations and make this novel approach usable by all kinds of scholars.

The Ripple Effect: Beyond Depth Measurement

This latest way of doing things is not limited to fixing problems of depth in microscopy only. It creates room for the numerous advancements in connected fields such as:

  • Improved Drug Discovery: It is important to know protein structures with precision in order to develop drugs. This new method helps researchers accurately describe protein targets and thus create targeted therapies that work better.
  • Advanced Biomanufacturing: The field of biomanufacturing relies on manipulating biological structures at a minute level. With better accuracy in measuring these structures, the precision and control of biomanufacturing processes can be significantly improved.
  • Neuroscience Breakthroughs: Unravelling neurological disorders depends on understanding complex connections within the brain. More accurate mapping of neural networks can result from using this new method, hence opening up possibilities for further discoveries in neuroscience.

These are just some instances where a re-scaling approach depending on depth could revolutionize different scientific areas.

Conclusion: A Clearer Perspective on the Microworld.

This new scaling factor, which takes into account the depth-dependent nature of the light traveling through a material, made by Delft University of Technology researchers, is a significant advance in microscopy. This technique allows for more precise and dependable measurements within the microscopic realm by taking into account the intricacies involved in light–sample interactions. Moreover, this method can be easily accessed online with other user-friendly tools and software that make it possible for researchers in various fields to unravel the mysteries hidden in tiny life phenomena. In summary, as scientists delve deeper into exploiting this new approach, they are bound to reveal more fascinating facts and developments ahead.

Article Source: Reference Paper | Reference Article

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Anchal is a consulting scientific writing intern at CBIRT with a passion for bioinformatics and its miracles. She is pursuing an MTech in Bioinformatics from Delhi Technological University, Delhi. Through engaging prose, she invites readers to explore the captivating world of bioinformatics, showcasing its groundbreaking contributions to understanding the mysteries of life. Besides science, she enjoys reading and painting.


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