The first microscopists: Hooke and Leeuwenhoek

Magnification and resolution are two different properties, and confusing them is the commonest beginner error in microscopy. Magnification is simply how many times larger the image is than the object; it is a ratio, and it can be increased almost without limit by adding lenses. Resolution, also called resolving power, is the smallest distance between two separate points at which they can still be distinguished as two points rather than one blurred spot. For a light microscope that limit is set by two things: the wavelength of the visible light used, roughly 400 to 700 nanometres, and the numerical aperture of the objective, which describes how wide a cone of light the lens can gather. The classic figure is about 0.2 micrometre, or 200 nanometres, for the best light microscope; below that, two organisms merge into one image no matter how much you enlarge it.

Pushing magnification past roughly 1000 to 1500 times produces what microscopists call empty magnification: the picture grows larger but no new detail appears. This is why Leeuwenhoek’s little single lenses of 50 to 300 times could beat the compound microscopes of his day, which magnified more but suffered from chromatic and spherical aberration — colour fringes and blurring introduced when light passes through several lenses. A single tiny lens held close to the eye, with the specimen almost touching it, gave a bright, sharply focused image instead.

The same physics explains the modern laboratory routine: bacteria, about 0.5 to 2 micrometres wide, are examined with an oil-immersion objective at 1000 times, where immersion oil raises the numerical aperture and therefore the resolution; viruses, measured in nanometres, cannot be resolved by any light microscope and require electron microscopy, whose much shorter electron wavelength lowers the limit to around 0.2 nanometre. For a nurse this is practical: a Gram stain reported as “no organisms seen” means that no organisms large enough for the light microscope to resolve were present in that field — it does not exclude a virus, and it does not mean the specimen was sterile.

The objects Hooke drew in 1665 were not microorganisms, and the distinction is not a quibble. Cork is the outer bark of the cork oak, a tissue built from plant cells that have died and had their contents replaced by a waxy, waterproof substance, so the walls left behind are made mainly of cellulose and suberin. What Hooke’s instrument, magnifying about 30 times, showed was therefore a honeycomb of empty compartments: the rigid walls of dead plant tissue, with the living material gone. The word he chose, cell, comes from the Latin cellula, a small room, and he was describing architecture, not life.

Leeuwenhoek’s animalcules were the opposite — whole, living organisms that swam, changed shape, divided and responded, which is why they could reasonably be called little animals. A living cell, in the sense used in this course, must have a membrane that controls what enters and leaves, cytoplasm where metabolism happens, a genome, and machinery for making proteins; Hooke saw none of that. The confusion is easier to understand because the same word now carries a later discovery: in 1838 and 1839 the botanist Matthias Schleiden and the zoologist Theodor Schwann argued that all plants and animals are built of cells, and Rudolf Virchow added in 1855 that every cell comes from a pre-existing cell.

That is cell theory, a statement about living units, and it was published more than 170 years after Hooke drew a slice of bottle cork. The difference still matters in a clinical laboratory: a Gram stain or a culture is looking for living organisms with metabolism and growth, whereas a sample of dead tissue, wood or cork under the same microscope shows only empty walls.

A lens works because light bends when it crosses the boundary between air and glass. A biconvex lens — thicker in the middle than at the edge — bends the rays from each point of an object so that they reconverge on the far side and form an image, and the distance at which that image forms is the focal length. The shorter the focal length, the closer the lens must sit to the object and the greater the enlargement; Leeuwenhoek’s best lenses were tiny beads of glass with a focal length of little more than a millimetre, which explains how a single lens could reach 300 times. A short focal length has a price, however.

The depth of field becomes extremely shallow, so only a very thin plane of the specimen is in focus at once, and the specimen must be thin and translucent because light has to pass through it. Leeuwenhoek ground and polished his own lenses by hand, mounted specimens on pins, and lit them with a candle or with daylight directed by a small mirror, adjusting position with screws until the image was sharp. Compound microscopes of the period, with two lenses separated by a tube, magnified more conveniently but suffered from spherical and chromatic aberration — blurring and coloured fringes produced by the curvature and by the fact that different colours of light bend by different amounts.

A single bead of glass avoided most of that distortion, which is why the amateur from Delft, not the great instrument-makers of London, first saw bacteria. The lesson for the modern laboratory is that a technique is more than an apparatus: the light, the specimen preparation, the depth of field and the skill of the observer all contribute to whether a structure can ultimately be seen, which is why standard operating procedures describe all of them and not merely the instrument.