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Figure 17: Image deconvolution. Left: Original fluorescence image. Right: Image after deconvolution, showing increased detail. Credit: Author.

The imaging system may also include elements such as apertures and filters that select certain portions of light from the sample, for example to see only light that has been scattered off the sample, or only light of a certain color or wavelength. As in the case of the illumination system, this type of filtering can be extremely useful to single out certain features of interest that would remain hidden when imaging all the light from the sample.Overall, both the illumination and the imaging system play a key role in how well a light microscope performs. To get the best out of light microscopy in your application, it is essential to have a good understanding of how a basic light microscope works, and what variations exist today.

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Some of the most fundamental processes in nature occur at the microscopic scale, far beyond the limits of what we can see by eye, which motivates the development of technology that allows us to see beyond this limit. As early as the 4th century AD, people had discovered the basic concept of an optical lens, and by the 13th century, they were already using glass lenses to improve their eyesight and to magnify objects such as plants and insects to better understand them.1 With time, these simple magnifying glasses developed into advanced optical systems, known as light microscopes, which allow us to see and understand the microscopic world beyond the limits of our perception. Today, light microscopy is a core technique in many areas of science and technology, including life sciences, biology, materials sciences, nanotechnology, industrial inspection, forensics and many more. In this article, we will first explore the basic working principle of light microscopy. Building on this, we will discuss some more advanced forms of light microscopy that are commonly used today and compare their strengths and weaknesses for different applications.

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What's the difference between light microscopy vs electron microscopy? Light microscopy typically uses wavelengths of light in the visible spectrum, which inherently limits it spatial resolution due to the Rayleigh criterion to approximately half the wavelength used (approximately 200 nm at best). However, even when using objectives with high NA and advanced image processing, this fundamental limit cannot be overcome. Instead, observing smaller structures requires the use of electromagnetic radiation of shorter wavelength. This is the underlying principle of electron microscopy, where electrons are used to illuminate the sample instead of visible light. Electrons have an associated wavelength which is much shorter than visible light, which allows magnifications of up to 10,000,000 x to be achieved, such that even single atoms can be resolved.

Strong background suppression from non-birefringent areas of a sample, allows measurement of sample thickness and birefringence

Allows individual fluorophores and particular areas of interest in a sample to be singled out, can overcome the resolution limit

What is light microscopy? Light microscopy is used to make small structures and samples visible by providing a magnified image of how they interact with visible light, e.g., their absorption, reflection and scattering. This is useful to understand what the sample looks like and what it is made of, but also allows us to see processes of the microscopic world, such as how substances diffuse across a cell membrane.

What is light microscopy?Parts of a microscope and how a light microscope worksSimple and compound microscopesTypes of light microscopy-     Bright field microscopy-     Dark field microscopy-     Phase contrast microscopy-     Differential interference contrast microscopy-     Polarized light microscopy-     Fluorescence microscopy-     Immunofluorescence microscopy-     Confocal microscopy-     Two-photon microscopy-     Light sheet microscopy-     Total internal reflection fluorescence microscopy-     Expansion microscopy

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Figure 7: Phase contrast microscopy of a human embryonic stem cell colony. Credit Sabrina Lin, Prue Talbot, Stem Cell Center University of California, Riverside.

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Figure 16: Sample preparation for expansion microscopy. A cell is first stained and then linked to a polymer gel matrix. The cell structure itself is then dissolved (digested), allowing the stained parts to expand isotropically with the gel, allowing the stained structure to be imaged with more detail.

Deconvolution in light microscopyWhat's the difference between light microscopy vs electron microscopy?Summary and conclusionLight microscopy techniques comparison table

Figure 9: Polarization microscopy. Photomicrograph of olivine adcumulate, formed by the accumulation of crystals with different birefringence. Variations of thickness and refractive index across the sample result in different colors. Credit: R. Hill, CSIRO.

Figure 8: Differential interference contrast microscopy. Left: Schematic setup for DICM. Right: Live adult Caenorhabditis elegans (C. elegans) nematode imaged by DICM. Credit: Bob Goldstein, Cell Image Library. Reproduced under a Creative Commons Attribution 3.0 Unported license (CC BY 3.0).

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