An Introduction to Microscopy and Imaging Techniques

PART 1. MICROSCOPY 101

Adapted from the TRI Skin Course, 2025 

Dr Jessica Turner  Staff Scientist

Dr Philippa Cranwell Technical Content Creator

Figure 1: Illustration of a flea, 1664. Image taken from Robert Hooke’s Micrographia

Figure 2:  End of a broken hair created by brushing in a repeated grooming experiment, viewed by SEM at TRI

The marvels of the microscopic world have fascinated scientists and non-scientists alike for centuries, ever since the first amazing pictures of the stinging hairs of a nettle and the anatomy of a flea were published by Robert Hooke in 1665 (Figure 1). Since then, microscopy and imaging techniques have become indispensable tools for scientists in all fields of research, and are a key technology in the field of cosmetic science research and claims substantiation (Figure 2).

There are two main branches of microscopy: optical and electron. This mini-series will discuss both, outlining key differences between the two, as well as providing key use-cases within cosmetic science and examples of the outputs that can be generated. 

Optical vs Electron Microscopy

Optical microscopy and electron microscopy are extremely useful analytical techniques, with both allowing magnification, visualization, and analysis of specimens, which may be biological in origin, such as cells or bacteria, or synthetic, for example polymers. However, the application of each microscopy is very different due to differences in the scale at which they operate (Figure 3). 

Figure 3: Comparison of the difference in scale at which optical and electron microscopy operate. Image taken from Science Learning Hub, Pokapū Akoranga Pūtaiao.

Before we discuss the fundamental differences between optical and electron microscopy in more detail, there are two terms that need to be defined:

• Magnification: the ability of the microscope to enlarge the apparent size of an object. 

• Resolution: the ability of the microscope to show two nearby points as separate, meaning fine details are observed instead of a blurry image.

Optical microscopes use visible light of wavelength 400 – 700 nm and a series of lenses to visualize specimens, Table 1.

While up to 1,500 times magnification can be achieved, resolution is limited to 200 nm, therefore samples that are smaller than 200 nm will not give a clear enough image for accurate analysis. Optical microscopy is suitable for live samples and sample preparation is relatively simple, often relying upon mounting a sample upon a glass slide (which usually only takes a few minutes). 

Two main types of light microscopy can be selected, including transmission light microscopy, and reflectance light microscopy.  For transmission light microscopy, the sample must be transparent so light can pass through. If contrast is required, or certain aspects of a sample need to be visualized, stains such as carmine, which stains animal starch (or glycogen) red, or fluorophores (chemical molecules that can absorb light at one wavelength and re-emit the light at a longer wavelength) can be used.  Reflectance light microscopy, also termed micro- or macro-imaging, does not require transparent samples.  Instead, these techniques require good contrast between the objects of interest and the background, and good illumination. Electron microscopes, in contrast, use a beam of electrons to visualize the sample, Table 1.

A common example of this technique is scanning electron microscopy, or SEM, in which a beam of electrons is scanned across the sample, giving information about the sample’s surface. Transmission Electron Microscopy (TEM), in which a beam of electrons is passed through the sample, is also possible, but is less commonly used within cosmetic science. The use of electron microscopy allows for magnification of over 1,000,000 times, and in some cases observation of individual atoms is possible. It also gives higher resolution; in fact, resolution of < 1 nm can be achieved. However, this extremely high resolution comes at a cost: sample preparation can be time-consuming, and samples need to be dead as they are placed under vacuum conditions and often cooled to cryogenic temperatures. In addition, staining requires coating samples with heavy metals. Furthermore, all electron microscope images are black and white; it is not possible to see real colors under the electron microscope, so color is often added during analysis. An advantage of electron microscopy, however, is that it can be combined with other analytical techniques, enabling identification of the chemical elements present within a sample.

Table 1: Summary of the differences between optical and electron microscopy. 

Optical vs electron: which should I choose?

The type of microscopy that you should choose depends upon several aspects, including the sample type, what information is desired from the sample, and what measurements will be undertaken. Key questions for consideration may include:

• Sample collection: is it invasive or non-invasive?

• Sample preparation: do tissues need to be alive/bio-active; do cells need to be ‘fixed’ at a certain time-point?

• Area of focus: is the sample area deep or on the surface?

• Resolution: are tissues or cells being imaged, or is it smaller molecules like proteins or lipids?

• Level of information required: is just visualization needed for comparison, or is qualitative or qualitative analysis of the chemical elements present in the sample needed?

The team at TRI are able to advise and guide you towards the best microscopy for your requirements. Get in contact with us today to arrange a chat with one of our experts.

We would like to thank Matthew Almond, Emeritus Professor at the University of Reading, for his help and guidance when preparing these articles.