When we use a magnifying glass to look at an insect or a piece of a flower we like to say its been magnified 10 times, or if we look at a fly’s foot with a microscope we might say it’s 200x larger; it’s been magnified 200 times. It’s the way we make sense of what we are looking at and how we might describe it to an interested someone looking over our shoulder.
When it comes to looking at a micrograph, be it printed in a book or viewed on a computer monitor, we really need to know how much larger the subject is in the micrograph as compared with that in real life. Stating magnification is often meaningless as it changes whenever the image is viewed at a size other than at the original capture dimensions and is why a Field of View dimension is more meaningful as suggested in the previous blog.
The human eye is a biological form of a simple camera with a single biconvex lens and a variable aperture at the front but with a light sensitive screen, the retina, at the back rather than film. Light striking the retina creates electrical impulses that flow to the brain for interpretation into a recognisable image.
If two small objects, such as a couple of grains of sand, are placed on a plain background almost touching each other and viewed from a distance of around 25cm, providing the distance between them is not less than about 0.25mm, with average eyesight they should be just visible as two separate entities. If the objects were a little closer together or being viewed from a little further away they would be seen as a single object and be described as being unresolved. It is upon this principle that a photograph printed in a book or newspaper is based. When examined with a hand lens, such a photograph is exposed simply as an array of dots and is seen as such because it has been resolved into individual points by the increase in magnification. However, to the unaided eye the individual dots are too close together to be resolved and consequently merge into one another to produce a pattern of light and dark, the photograph.
If all a hand lens could do was to magnify what the eye could see then there would be very little point in using one as it would not show anything more than was already visible to the eye but within physical limits of the instrumentation in use, magnification does more than this. It increases resolution; in our example, it not only enlarges the two grains of sand but also the space between them allowing the grains to be more easily observed as separate entities. However, if the primary magnification, i.e. the original magnification used on the microscope, is too low to resolve the space between two points they will still be seen as one. No matter how large an image is printed it will not show any more detail as the initial magnification was too low to capture the relevant information. Magnification beyond a point where additional detail is revealed is called empty magnification.
As an example, the pair of micrographs below show the identical region of the gullet and associated cilia of a paramecium, a uni-cellular organism found in ponds. Both have the same field of view (FoV = 0.043mm) but there is considerable difference in quality between the two. Image ‘A’ has a primary magnification of x20,000 whilst image ‘B’ was extracted from an image with a primary magnification of x2,000 (original micrograph at end of this blog).
At its original primary magnification, the micrograph below shows the entire organism and resolves all detail necessary for its intended purpose, specifically to illustrate the wave motion of cilia.
An area of the low magnification image was selected and enlarged to give the same field of view as image ‘A’ with a magnification of x20k. Since the field of view for both images is the same, the magnification for each image as they appear on the page must also be the same. However, the primary magnification of image ‘B’ is insufficient to resolve the detail revealed in image ‘A’ which has a primary magnification an order of magnitude greater.
However, everything in life comes at a cost. Whilst image 'A' provides a lot of data regarding fine detail, the low magnification micrograph of the entire organism gives us a contextual view that might otherwise be difficult to interpret from high magnification alone.