AQA 3.2.1.3; Edexcel B 2.6 (vi); OCR A 2.1.1 (a, b, e, f); OCR B 2.1.1 (a(i), d, i)

Definitions

Let’s get some boring definitions out of the way before getting onto the good stuff. First of all, what is a microscope? Although this may sound like a rather condescending question, many would find them difficult to specifically define. A microscope is an optical instrument with a lens capable of magnifying an object. The term ‘microscopic’ is used to describe those objects that cannot be seen without the aid of a microscope.

So what about magnifying? Does this just mean making objects appear larger and being able to see them in more detail? If so, why the need for so many different types of microscope? Well there are multiple truths there. Magnify does simply mean to make something appear larger than it really is, and can be defined by the equation: magnification = size of image / size of actual object. Let’s break this equation down slightly – magnification has no units, and is described by the number of times the object has been enlarged – for example X100. The size of the image can be measured using a ruler and the picture the good people (HA!) of your exam boards will give you.

An eyepiece graticule and stage micrometer can also be used. The graticule comes built in to many microscope models, and basically consists of a series of increments that can be compared to increments of fixed length (the stage micrometer, which is usually placed under the microscope) which allows you to calibrate the size of the increments on the graticule (work out the length each of them represents) at different magnifications. There’s a great, concise tutorial here on calibrating the eyepiece graticule, but suffice to say if any readers plan on taking biology at HE you’ll find yourself spending far too much time squinting down microscopes at these!

So then, if our image is nice and magnified, we can observe it in as much detail as we like by simply increasing the magnification, right? Sadly, no. This is where resolution comes into play. Resolution is the smallest point between two objects in view that remain distinct from one another. Basically, a microscope with high resolving power will be able to allow you to distinguish between two very small, very close objects without them being reduced to a blurry mess (think about pixelated images having low resolution). Although we’re now firmly into detail you really won’t need at A-level, for those interested resolution can be calculated as such: resolution = (0.61 x λ) / Numerical Aperture. “More of those funny symbols,” I hear you cry. Fear not, λ (lambda) is another Greek symbol, used to represent wavelength. Numerical aperture refers to the range of angles light can be detected from by the microscope. As an added bonus I’ll also mention penetration depth (insert jokes here) which essentially refers to the ability of the microscope to see deep into the sample being magnified. There is often a trade-off between resolution and penetration depth.

 

History and diversity of the microscope

The very earliest, and very primitive, microscopes were formed by Zaccharias and Hans Jenssen (although this claim has been disputed), who put magnifying glass into a tube and noticed the magnifying effect this had circa 1590. This is the earliest example of the compound microscope, but we’ll get to that later. However, Antony Van Leeuwenhoek is often credited as the inventor of the microscope, who used curved glass to magnify exceptionally small organisms. Around 1670 Robert Hooke (of Hooke’s law fame) added a water flask and barrel to capture and focus light onto the specimen, which was aided by the addition of another lens. Here’s a great link if you’re interested in reading more about the contributions of Robert Hooke to microscopy. I’d also recommend visiting this link to learn more about the history of microscopy in general.

Today, microscopes are generally divided into two main categories – light and election which we’ll examine now in more detail.

Light microscopy

This is the type of microscope you’ll be most familiar with, and will have almost certainly encountered before. The resolution is not as powerful as in electron microscopes, however light microscopes are invaluable in that they allow for the study of live cells. Here are some of the main types of light microscopy:

  • Bright-field microscopy – this is the kind you will be most familiar with, and are frequently used in classrooms. They have an ocular lens (near the eyepiece), an objective lens (close to the object being studied) and a condenser lens (to focus light) as well as a light source.
  • Phase-contrast microscopy – this creates a light phase shift (makes light ‘fall out of sync’) to make light regions appear lighter and dark regions appear darker.
  • Differential interference-contrast microscopy – as above, but using two beams of polarised light.
  • Stained bright-field microscopy – As with bright-field, but using a stain to give pigment to the sample being studied. More on stains later.
  • Fluorescence microscopy – Now we’re onto the good stuff! Cells or specific regions and organelles within them are stained (often with different coloured stains to show different organelles at the same time) with a fluorescent dye. Fluorescent dyes are remarkable in that, when they are stimulated by a beam of light, the wavelength they emit is longer than that which they absorb, giving off beautiful fluorescent colours.
  • Confocal microscopy – As above, but with an enhanced ability to focus the light by using a pinhole system, giving a clearer image.

Electron microscopy

Here lies much greater resolution. However, the trade-off is that these microscopes cannot observe living tissue – or, at least, if the tissue was alive when it went in, the electron beam would soon take care of that. Here are the three main types:

  • Transmission electron microscopes – A beam of electrons passes through the object – parts of which will absorb electrons and appear darker.
  • Scanning electron microscopes – Electrons pass onto the surface of the object, causing other electrons to be reemitted. This technique is only capable of viewing the surface of objects, however it produces very beautiful images. I recommend visiting the website of scientist and artist David Scharf who has transformed SEM into an art form.
  • Freeze-fracture microscopes – involves freezing the specimen in liquid nitrogen and membranes are split, allowing for the observation of membrane proteins. However, this technique is known to produce artifacts (anomalies) in the final image due to the effects of freezing.

I hope this was helpful. Next time I’ll be discussing some of the applications of microscopy, including flow cytometry and staining.