An Introduction to the Light Microscope, Light Microscopy Techniques and Applications – Technology Networks

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.

What is light microscopy?

Parts of a microscope and how a light microscope works

Early microscopes used an illumination system comprising sunlight that was collected and reflected onto the sample by a mirror. Today, most microscopes use artificial light sources such as light bulbs, light-emitting diodes (LEDs) or lasers to make more reliable and controllable illumination systems, which can be tailored to a given application. In these systems, light from the source is typically collected using a condenser lens and then shaped and optically filtered before being focused onto the sample. Shaping the light is essential to achieve high resolution and contrast, and often includes controlling the sample area that is illuminated and the angles at which light impinges on it. Optical filtering of the illumination light, using optical filters that modify its spectrum and polarization, can be used to highlight certain features of a sample, to improve the visibility of weak signatures or to observe a samples fluorescence.

The imaging system collects illuminating light that has interacted with the sample and produces a magnified image that can be viewed (Figure 1). This is achieved using two main groups of optical elements: first, an objective lens that collects as much light from the sample as possible and second, an eyepiece lens which relays the collected light to the observers eye or a camera system. 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.

Simple and compound microscopes

A single lens can be used as a magnifying glass which increases the apparent size of an object when it is held close to the lens. Looking through the magnifying glass at the object, we see a magnified and virtual image of the object. This effect is used in simple microscopes, which consist of a single lens that images a sample held clamped into a frame and illuminated from below, as is shown in Figure 2. This type of microscope can achieve a magnification of typically 2-6 x, which is sufficient to study relatively large samples. However, achieving higher magnification and better image quality requires the use of more optical elements, which led to the development of the compound microscope (Figure 3).

In a compound microscope, the sample is illuminated from the bottom to observe transmitted light, or from the top to observe reflected light. Light from the sample is collected by an optical system consisting of two main lens groups: the objective and the eyepiece, whose individual powers multiply to enable much higher magnifications than those achieved by a simple microscope. The objective collects light from the sample and typically has a magnification of 40-100 x. Some compound microscopes feature multiple objective lenses on a rotating turret known as a nose piece, allowing the user to choose between different magnifications. The image from the objective is picked up by the eyepiece, which magnifies the image again and relays it to the users eye, with typical eyepieces having a magnification of 10 x. Therefore, the total magnification of a compound microscope, which is the product of the objective magnification and the eyepiece magnification, typically lies in the range of 400-1000 x.

r = 0.61 (/NA)

In standard compound microscopes (Figure 4a), the sample (often on a glass slide) is held on a stage that can be moved manually or electronically for higher precision, and the illumination system is in the lower part of the microscope, while the imaging system is above the sample. However, the microscope body can usually also be adapted to particular uses. For example, stereo microscopes (Figure 4b) feature two eyepieces at a slight angle to each other, allowing the user to see a slightly three-dimensional image. In many biology applications, an inverted microscope design (Figure 4c) is used, where both the illumination system and the imaging optics are below the sample stage to facilitate placing e.g., containers of cell cultures onto it. Finally, comparison microscopes (Figure 4d) were often used in forensics, for example to compare fingerprints or bullets by eye before the advent of digital microscopy, which allowed images to be saved and compared.

Types of light microscopy

In the following, we will present a selection of different light microscopy techniques available today, discuss their main operating principles and the strengths and weaknesses of each technique.

Bright field microscopy (BFM) is the simplest form of light microscopy, where the sample is illuminated from above or below, and light transmitted through or reflected from it is collected to form an image that can be viewed. Contrast and color in the image are formed because absorption and reflection vary over the area of the sample. BFM was the first type of light microscopy developed and uses a relatively simple optical setup, which allowed early scientists to study microorganisms and cells in transmission. Today, it is still very useful for the same purposes, and is also widely used to study other partially transparent samples such as thin materials in transmission mode (Figure 5), or microelectronics and other small structures in reflection mode. However, the magnification of BFM is limited to 1300 x and it is not suitable for imaging highly transparent samples.

Figure 5: Bright field microscopy. Left: Transmission mode - flakes of graphite (dark grey) and graphene (lightest grey) as seen in a bright field microscope. Here, the difference in brightness seen on the image is proportional to the thickness of the graphite layer. Right: Reflection mode - flakes of graphene and graphite on a SiO2 surface. Small surface contaminants are also visible. Credit: Author.

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

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).

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 10: Fluorescence microscopy. Left: Working principle - illumination light is filtered by a short-pass excitation filter and reflected towards the sample by a dichroic mirror. Fluorescence from the sample passes the dichroic mirror and is additionally filtered by an emission filter to remove residual excitation light in the image. Right: Fluorescence image of molecules hosted in an organic crystal (crystal outline shown dashed yellow). The background is not completely dark due to fluorescence from other molecules and the crystal material. Credit: Author.

Figure 11: Immunofluorescence microscopy. Two interphase cells with immunofluorescence labeling of actin filaments (purple), microtubules (yellow), and nuclei (green). Credit: Torsten Wittmann, NIGMS Image Gallery.

A disadvantage of TPM is that the probability of two-photon absorption is much lower than single-photon absorption and thus requires high-intensity illumination such as pulsed lasers to achieve a practical fluorescence signal intensity.

Figure 13: Two-photon microscopy. Thin optical section of pollen, showing fluorescence mostly form the outer layers. Credit: Michael Cammer, Cell Image Library.

Total internal reflection fluorescence (TIRF) is a fluorescence microscopy technique that allows 2D fluorescence images to be made of an extremely thin (approximately 100 nm thick) sample slice.10 This is achieved by exciting the fluorescence of the sample by evanescent fields of the illuminating light, which occur when it undergoes total internal reflection at a boundary between two materials of different refractive index (n). Evanescent fields have the same wavelength as the illuminating light but are tightly bound to the interface. In TIRF microscopy, the excitation light typically undergoes total internal reflection at the interface between a glass slide (n = 1.52) and the aqueous medium (n = 1.35) the sample is dispersed in. The intensity of the evanescent field falls off exponentially with distance from the interface, such that only fluorophores close the interface are observed in the final image. This also leads to a strong suppression of fluorescence background from areas outside the slice, which allows weak fluorescence signals to be picked up, for example when localizing single molecules. This makes TIRF extremely useful to observe the weak signal of fluorescent proteins (Figure 15) involved in intercellular interactions, but also requires the sample to be dispersed in an aqueous medium, which may limit the types of samples that can be measured.

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 microscopy

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

Light microscopy vs electron microscopy

Summary and conclusion

Light microscopy techniques comparison table

Technique

Advantages

Limitations

Typical applications

Bright field microscopy

Relatively simple setup with few optical elements

Low contrast, fully transparent objects cannot be imaged directly and may require staining

Imaging colored or stained samples15 and partially transparent materials16

Dark field microscopy

Reveals small structures and surface roughness, allows imaging of unstained samples

High illumination power required can damage the sample, only scattering image features seen

Imaging particles in cells,17 surface inspection18

Phase contrast microscopy

Enables imaging of transparent samples

Complex optical setup, high illumination power required can damage the sample, generally darker images

Tracking cell motion,19 imaging larvae20

Differential interference contrast microscopy

Higher resolution than PCM

Complex optical setup, high illumination power required can damage the sample, generally darker images

High resolution imaging of live, unstained cells21 and nanoparticles22

Polarized light microscopy

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

Requires a birefringent sample

Imaging collagen,23 revealing grain boundaries in crystals24

Fluorescence microscopy

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

Requires a fluorescent sample and a sensitive detector, photobleaching can diminish signal

Imaging cell components, single molecules, proteins25

Immunofluorescence microscopy

Visualize specific biomolecules using antibody targeting

Extensive sample preparation, requires a fluorescent sample, photobleaching

Identifying and tracking cells26 and proteins27

Confocal microscopy

Low background signal, possible to create 3D images

Slow imaging speed, requires a complicated optical system

3D cell imaging, imaging samples with weak fluorescence signals, surface profiling28.

Two-photon microscopy

Deep sample penetration, low background signal, less photobleaching

Slow imaging speed, requires a complicated optical system and high-power illumination

Neuroscience,29 deep tissue imaging30

Light sheet microscopy

Images only an extremely thin slice of the sample, can create 3D images by rotating the sample

Slow imaging speed, requires a complicated optical system

3D imaging of cells and organisms8

Total internal reflection fluorescence microscopy

Strong background suppression, extremely fine vertical sectioning

Imaging limited to thin area of sample, requires a complicated optical system, sample needs to be in aqueous medium

Single molecule imaging,31 imaging molecular trafficking32

Expansion microscopy

Increases effective resolution of standard fluorescence microscopy

Requires chemical processing of the sample, not suitable for live samples

High resolution imaging of biological samples11

B

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An Introduction to the Light Microscope, Light Microscopy Techniques and Applications - Technology Networks