Optical microscope

• 1. Introduction

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  The optical microscope is the oldest type of microscope, and its present compound form first appeared in the 17th century. It works on a deceptively simple idea. Take visible light, pass it through a system of lenses, and a fly's wing or a single cell swells into something a human eye can study. Yet there is a hard wall built into this instrument. Visible light can only resolve detail down to around 200 nanometers, because of what physicists call the diffraction limit. Below that, you can magnify all you like and see nothing new. So how did a tool with such a strict ceiling become the workhorse of biology, medicine, and mineralogy? Who actually invented it, and why is the answer still disputed? And how did later scientists find ways to slip past a limit that was supposed to be absolute? The instrument on the laboratory bench hides four centuries of argument, accident, and quiet genius.

• 2. The Two Families

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  A single lens is all a simple microscope needs. It enlarges an object through angular magnification alone, handing the viewer an erect, enlarged virtual image. The magnifying glass belongs to this family, as do loupes and the eyepieces of telescopes. The compound microscope plays a more elaborate game. A lens close to the object, the objective, collects light and focuses a real image inside the instrument. A second lens or group of lenses, the eyepiece, then magnifies that image, giving the viewer an enlarged, inverted virtual image. Stacking one set of lenses to enlarge what another has already enlarged is what unlocks much higher magnification. The vast majority of modern research microscopes are compound, while some cheaper commercial digital models remain simple single-lens devices. From the compound design springs a whole catalogue of specialized variants, each tuned to a different problem.

• 3. A Disputed Invention

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  Around 1620 in Europe, the compound microscope appears in the record, including one demonstrated by Cornelis Drebbel in London about 1621 and another exhibited in Rome in 1624. The true inventor remains unknown, though many have claimed the honour. The Dutch spectacle-maker Johannes Zachariassen testified, 35 years after the fact, that his father Zacharias Janssen had built the compound microscope as early as 1590. The trouble is that this date would have made Zacharias a child at the time. Some historians treat the testimony as dubious. For it to hold, the real inventor would have to be Johannes' grandfather, Hans Martens. Janssen's competitor Hans Lippershey, who applied for the first telescope patent in 1608, has also been credited. Galileo Galilei joined the story too. After 1610 he found he could close-focus his telescope onto small objects like flies, or look through the wrong end to magnify them. His 2 foot telescope had to be stretched to 6 feet to manage the trick. After seeing Drebbel's instrument in Rome in 1624, Galileo built an improved version. He called it the occhiolino, or little eye. In 1625, Giovanni Faber coined the name microscope from the Greek words for small and to look at.

• 4. The Single Lens Triumph

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  Antonie van Leeuwenhoek, who lived from 1632 to 1724, is credited with bringing the microscope to the attention of biologists. His instruments were not the elaborate compound kind. They were simple microscopes built around a single, very small, very strong lens. They were awkward to handle, yet they let him see detailed images that astonished his contemporaries. The lead held for a remarkably long time. It took about 150 years of optical development before the compound microscope could match the image quality of van Leeuwenhoek's homemade lenses, because configuring multiple lenses well proved so difficult. Help came from Christiaan Huygens, another Dutchman, who in the late 17th century devised a simple two-lens ocular that was achromatically corrected. The Huygens ocular suffers from a small field size and other minor faults, yet it is still produced today. The binocular era arrived later still, when John Leonard Riddell, Professor of Chemistry at Tulane University, invented the first practical binocular microscope in the 1850s while conducting one of the earliest American investigations of cholera.

• 5. Taming The Light

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  Even lighting was the breakthrough of August 1893, when August Köhler developed the illumination method that bears his name. Before Köhler illumination, the image of the light source itself, a lightbulb filament for instance, was always visible in the picture of the sample. His method swept that flaw away and overcame many limits of older techniques. The next leap let scientists see the invisible. The Nobel Prize in physics went to the Dutch physicist Frits Zernike in 1953 for phase contrast illumination. By exploiting interference rather than absorption, it allows extremely transparent samples, such as live mammalian cells, to be imaged without staining. Just two years later, in 1955, Georges Nomarski published the theory for differential interference contrast microscopy, another interference-based technique. A different path opened with fluorescence. Here the sample is lit through the objective with a narrow band of wavelengths, and fluorophores inside emit light of a longer wavelength that forms the image. Chemical stains like DAPI, which binds to DNA, have labelled cell structures since the mid-20th century. Later came immunofluorescence, using fluorescently labelled antibodies, and fluorescent proteins like GFP, which a living cell can express to make itself glow.

• 6. Anatomy Of The Instrument

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  The eyepiece sits at the top, a cylinder of two or more lenses whose job is to bring the image into focus for the eye. Eyepieces are interchangeable, with typical magnifications of 5, 10, 15, and 20 times, the 10 being most common. At the lower end, objective lenses collect light from the sample, usually around three of them screwed into a rotating nose piece. These are built to be parfocal, so the sample stays in focus when you switch lenses. Objectives are defined by magnification, typically from 5 to 100 times, and numerical aperture, ranging from 0.14 to 0.7, which corresponds to focal lengths of about 40 to 2 millimeters. Oil-immersion and water-immersion objectives push further. An index-matching material like immersion oil sits between lens and sample, raising the numerical aperture above 1 and as high as 1.6, which lets the lens gather more light and reveal smaller details. Below the objective is the stage, a platform with a central hole for light and arms to hold glass slides of about 25 by 75 millimeters. Above 100 times magnification, moving a slide by hand becomes impractical, so a mechanical stage shifts it on two horizontal axes by control knob. The total power of the instrument is simply the eyepiece power multiplied by the objective power. A 10 times eyepiece with a 100 times objective yields a total magnification of 1,000 times.

• 7. The Wall And The Ways Past It

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  Point objects, viewed at very high magnification with transmitted light, do not appear as points. They show as fuzzy discs ringed by diffraction patterns called Airy disks. Resolving power is the ability to tell two closely spaced Airy disks apart. The diffraction that creates them sets a finite barrier, governed by the wavelength of light, the refractive materials in the objective, and its numerical aperture. Assuming a wavelength of 550 nanometers for green light, the lowest practical resolution with conventional lenses is about 200 nanometers, though a new lens using multiple scattering of light has reached below 100 nanometers. Scientists have found ways around the wall. Holographic techniques, described by Courjon and Bulabois in 1979, can break the limit, though their experimental resolution was restricted. Fluorescent samples open more doors, through near-field scanning optical microscopy using evanescent waves, and stimulated emission depletion. The sarfus method places a thin sample on a contrast-enhancing surface to visualize films as thin as 0.3 nanometers. The honours followed the breakthroughs. On the 8th of October 2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, William Moerner, and Stefan Hell for super-resolved fluorescence microscopy. Hell, of the Max Planck Institute for Biophysical Chemistry, had already won the 10th German Future Prize in 2006 for the STED microscope.

• 8. Where Light Cannot Reach

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  Some questions demand a wavelength shorter than visible light can offer. To resolve finer detail, scientists turned to instruments that use other waves entirely. The scanning electron microscope, the transmission electron microscope, and scanning probe techniques reach magnifications far beyond anything optical microscopy can manage. Electrons and X-rays carry shorter wavelengths, which pushes the diffraction limit lower and the resolution higher. The trade-offs are severe. Electron and X-ray microscopy demand a vacuum or partial vacuum, ruling out most live and biological samples, with the exception of the environmental scanning electron microscope. Scanning tunneling and atomic force microscopes scan a tiny probe across a surface, their resolution set by the probe size, where micromachining can produce tips with radii of 5 to 10 nanometers. These methods also strip away colour, so some information is simply lost. Yet they remain essential for molecular and atomic effects, such as age hardening in aluminium alloys or the microstructure of polymers. The optical microscope, meanwhile, keeps its own territory, working extensively in microelectronics, nanophysics, biotechnology, pharmaceutical research, mineralogy, and microbiology, and serving medical diagnosis in the field known as histopathology.