Techniques in Optical Microscopy
Welcome to this week's exciting POSES! This week, we will be sharing the techniques in optical microscopy method.
As you might probably already be aware of, optical microscopy is widely utilised in the industry, as well as in schools, hospitals, and laboratories (amongst others).
But did you know that there are multiple ways to obtain microscopic images of specimens using the same optical microscope?
Read on to find out more in Part 1 of this exciting series on Optical Microscopical Techniques!
If you have any suggestions for topics to read about, please leave them in the comments section!
Techniques in Optical Microscopy – Part 1
1.????Brightfield microscopy
Brightfield microscopy is probably the most common & earliest form of obtaining images of specimens when viewed with an optical microscope. As its name implies, brightfield microscopy implies that the background of the field of view (FOV) is brightly illuminated [1]. This is traditionally obtained when a light source is directly illuminating the specimen from beneath [transmitted light (diascopic) illumination], or from above and is reflected (by the specimen) into the microscope’s objective lens [incident light (episcopic) illumination]. Figure 1 below illustrates the optical train in each of these 2 modes of optical microscopy:
As brightfield microscopy represents the most elementary form of optical microscopical imaging [1] without imposing a need for additional components (and thus expenses), it is routinely employed in schools and hospitals, and is the preferred mode of optical microscopy for beginners in the field. This does not however imply that brightfield microscopy is a poor microscopical technique – in fact, brightfield microscopy is employed in >95% of all preliminary microscopical procedures as a means of quality inspection and diagnosis. If the specimen contrast and resolution is poor, this may be enhanced through the use of suitable stains/dyes [1], for artificial colouration of key structural features in the specimen (as depicted in Figure 2 below):
Nonetheless, if one desires to obtain clear photomicrographs[1] of living cells which are optically transparent without the need for staining, brightfield microscopy may not be the most suitable technique to use in this context. This would necessitate us to consider other more specialized techniques, some of which are described in the following sections.
2. Darkfield microscopy
As with brightfield microscopy, darkfield (or darkground) microscopy refers to an optical microscopical setup where the background of the field of view appears dark (black) while the object is brightly illuminated [3]. Darkfield microscopy is often used for imaging living cells and transparent specimens, where the image contrast is noticeably poor when observed under brightfield microscopy. Figure 3 below depicts an image of a unicellular protist (Paramecium sp.) obtained under brightfield & darkfield microscopy respectively:
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In most instances when the numerical aperture (N.A.) of the objective lens being used is below 1.0, darkfield microscopy can be easily achieved through the use of a filter as shown in Figure 4A below. This filter is often positioned just below the substage condenser (most microscope condenser mounts include a filter holder which accommodates a typical 32mm diameter filter) or above the base illuminator (in a potentially swing-out filter holder). For higher-powered objectives where the N.A. of the objective lens is >1.0 (such as the oil-immersion 100X objective lens), special darkfield condensers might need to be used, as illustrated in Figure 4B below:
The optical train of a transmitted light darkfield microscope is illustrated in Figure 5A below. This contrasts with reflected light (episcopic) darkfield microscopy [4] (the optical train of which is shown in Figure 5B), where the darkfield filter is placed in the incident light path and special darkfield objective lenses (as depicted in Figure 6A) may need to be used. Traditionally, a darkfield filter used in episcopic darkfield microscopy may be in the form of a filter slider, or a filter cube mounted in the turret of a fluorescence microscope adapted for episcopic darkfield as well (as shown in Figure 6B below):
So that’s it for now – we hope that you have found the read both informative & enjoyable Bookmark the POSES website to learn more about optical microscopy techniques in our future articles in this series!