Fundamentals of Microscopy

Abstract

The light (or optical) microscope is the icon of science. The aphorism “seeing is believing” is often quoted in scientific papers involving microscopy. Unlike many scientific instruments, the light microscope will deliver an image however badly it is set up. Fluorescence microscopy is a widely used research tool across all disciplines of biological and biomedical science. Most universities and research institutions have microscopes, including confocal microscopes. This introductory paper in a series detailing advanced light microscopy techniques explains the foundations of both electron and light microscopy for biologists and life scientists working with the mouse. An explanation is given of how an image is formed. A description is given of how to set up a light microscope, whether it be a brightfield light microscope on the laboratory bench, a widefield fluorescence microscope, or a confocal microscope. These explanations are accompanied by operational protocols. A full explanation on how to set up and adjust a microscope according to the principles of Köhler illumination is given. The importance of Nyquist sampling is discussed. Guidelines are given on how to choose the best microscope to image the particular sample or slide preparation that you are working with. These are the basic principles of microscopy that a researcher must have an understanding of when operating core bioimaging facility instruments, in order to collect high‐quality images. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Setting up Köhler illumination for a brightfield microscopeBasic Protocol 2: Aligning the fluorescence bulb and setting up Köhler illumination for a widefield fluorescence microscopeBasic Protocol 3: Generic protocol for operating a confocal microscope

Lightsheet Microscopy

“…Because the illumination and detection pathways are decoupled and entirely separate, the lightsheet can be formed in several ways without being constrained by the detection objective and signal collection optics. Although the theoretical lateral resolving power of the lightsheet microscope is given by the well‐known formula for widefield fluorescence microscopy (0.61.λ/NA; Sanderson, 2020) and is in practice limited by the resolution of the camera chip (0.5‐2.0 μm), the axial resolving power is determined by the numerical aperture (NA) of the detection objective and the thickness of the lightsheet (2‐6 μm), which also sets the optical section thickness of the Z‐stack.…”

Super‐Resolution Fluorescence Microscopy Methods for Assessing Mouse Biology

“…Resolution just occurs (the Rayleigh criterion) when the central maximum of the diffraction pattern from the first feature coincides with the first minimum of the diffraction pattern of the second, closely apposing, feature in an object (Demmerle, Wegel, Schermelleh, & Dobbie, 2015; Fig. 2 from a Current Protocols article by Sanderson, 2020). At this point, the dip in intensity between the two diffraction patterns is about 26%.…”

“…For a simple explanation of the phenomenon of diffraction see Bradbury (1984), and for an in‐depth treatment see Hammond (2015). How diffraction and interference underlie the formation of microscopical images is explained in a Current Protocols article by Sanderson (2020).…”

“…Whichever aide‐memoire you adopt, it is crucial that you consider these parameters when planning your imaging experiment and (if you have the choice) selecting which microscope to use. Figure 8 in a Current Protocols article by Sanderson (2020) gives a flow chart to help you decide which microscope to use for each imaging task. Figure 3 here also shows the inter‐relationships between different techniques and how these techniques can be combined.…”

“…Phototoxicity, with references, has been covered in a Current Protocols article by Sanderson (2020). In addition Wäldchen, Lehmann, Klein, van de Linde, & Sauer (2015) have shown that transfection reduces photoresistance and increases the likelihood of cell death with high irradiance.…”