Optical microscope
The earliest microscopes were single lens magnifying glasses with limited magnification, which date at least as far back as the widespread use of lenses in eyeglasses in the 13th century. Compound microscopes first appeared in Europe around 1620 including one demonstrated by Cornelis Drebbel in London around 1621 and one exhibited in Rome in 1624. The actual inventor of the compound microscope is unknown although many claims have been made over the years. These include a claim 35 years after they appeared by Dutch spectacle-maker Johannes Zachariassen that his father, Zacharias Janssen, invented the compound microscope and or the telescope as early as 1590. Zacharias probable birth date of 1585 makes it unlikely he invented it in 1590 and the claim of invention is based on the testimony of Zacharias Janssens son, Johannes Zachariassen, who may have fabricated the whole story. Another claim is that Janssens competitor, Hans Lippershey who applied for the first telescope patent in 1608 also invented the compound microscope. Other historians point to the Dutch innovator Cornelis Drebbel with his 1621 compound microscope. Galileo Galilei is sometimes cited as a compound microscope inventor. After 1610, he found that he could close focus his telescope to view small objects such as flies close up. In 1625, Giovanni Faber coined the name microscope for the compound microscope Galileo submitted to the in 1624. Faber coined the name from the Greek words micron meaning small, and skopein meaning to look at, a name meant to be analogous with telescope, another word coined by the Linceans.
A simple microscope uses a lens or set of lenses to enlarge an object through angular magnification alone, giving the viewer an erect enlarged virtual image. A compound microscope uses a lens close to the object being viewed to collect light called the objective lens, which focuses a real image of the object inside the microscope. That image is then magnified by a second lens or group of lenses called the eyepiece that gives the viewer an enlarged inverted virtual image of the object. The use of a compound objective-eyepiece combination allows for much higher angular magnification. Common compound microscopes often feature exchangeable objective lenses, allowing the user to quickly adjust the magnification. At the lower end of a typical compound optical microscope, there are one or more objective lenses that collect light from the sample. These arrangements are designed to be parfocal, which means that when one changes from one lens to another on a microscope, the sample stays in focus. Microscope objectives are characterized by two parameters, namely magnification and numerical aperture. The former typically ranges from 5x to 100x while the latter ranges from 0.14 to 0.7, corresponding to focal lengths of about 40 to 2 mm respectively. Objective lenses with higher magnifications normally have a higher numerical aperture and a shorter depth field in the resulting image.
In August 1893, August Köhler developed Köhler illumination. This method of sample illumination gives rise to extremely even lighting and overcomes many limitations of older techniques of sample illumination. Before development of Köhler illumination the image of the light source, for example a lightbulb filament, was always visible in the image of the sample. The Nobel Prize in physics was awarded to Dutch physicist Frits Zernike in 1953 for his development of phase contrast illumination which allows imaging of transparent samples. By using interference rather than absorption of light, extremely transparent samples such as live mammalian cells can be imaged without having to use staining techniques. Just two years later, in 1955, Georges Nomarski published the theory for differential interference contrast microscopy, another interference-based imaging technique. Modern biological microscopy depends heavily on the development of fluorescent probes for specific structures within a cell. Since the mid-20th century chemical fluorescent stains such as DAPI which binds to DNA have been used to label specific structures within the cell. More recent developments include immunofluorescence which uses fluorescently labelled antibodies to recognise specific proteins within a sample, and fluorescent proteins like GFP which a live cell can express making it fluorescent.
Stereo microscope is a low-powered microscope which provides a stereoscopic view of the sample, commonly used for dissection. Comparison microscope has two separate light paths allowing direct comparison of two samples via one image in each eye. Inverted microscope is for studying samples from below useful for cell cultures in liquid or for metallography. Fiber optic connector inspection microscope is designed for connector end-face inspection. Traveling microscope is for studying samples of high optical resolution. Petrographic microscope whose design usually includes a polarizing filter, rotating stage, and gypsum plate to facilitate the study of minerals or other crystalline materials whose optical properties can vary with orientation. Phase-contrast microscope applies the phase contrast illumination method. Epifluorescence microscope is designed for analysis of samples that include fluorophores. Confocal microscope is a widely used variant of epifluorescent illumination that uses a scanning laser to illuminate a sample for fluorescence. Two-photon microscope is used to image fluorescence deeper in scattering media and reduce photobleaching especially in living samples. Student microscope is an often low-power microscope with simplified controls and sometimes low-quality optics designed for school use or as a starter instrument for children. Ultramicroscope is an adapted light microscope that uses light scattering to allow viewing of tiny particles whose diameter is below or near the wavelength of visible light around 500 nanometers mostly obsolete since the advent of electron microscopes.
The maximum resolving power of optical microscopes is typically limited to around 200 nanometers because of the diffraction limit of visible light. While larger magnifications are possible no additional details of the object are resolved. At very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction rings. These are called Airy disks. The resolving power of a microscope is taken as the ability to distinguish between two closely spaced Airy disks or in other words the ability of the microscope to reveal adjacent structural detail as distinct and separate. It is these impacts of diffraction that limit the ability to resolve fine details. The extent and magnitude of the diffraction patterns are affected by both the wavelength of light lambda, the refractive materials used to manufacture the objective lens and the numerical aperture NA of the objective lens. There is therefore a finite limit beyond which it is impossible to resolve separate points in the objective field known as the diffraction limit. Assuming that optical aberrations in the whole optical set-up are negligible, the resolution d can be stated as usually a wavelength of 550 nm is assumed which corresponds to green light. With air as the external medium, the highest practical NA is 0.95, and with oil, up to 1.5. In practice the lowest value of d obtainable with conventional lenses is about 200 nm.
On the 8th of October 2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, William Moerner and Stefan Hell for the development of super-resolved fluorescence microscopy. Stimulated emission depletion STED is a fluorescence microscopy technique which uses a combination of light pulses to induce fluorescence in a small sub-population of fluorescent molecules in a sample. Each molecule produces a diffraction-limited spot of light in the image, and the centre of each of these spots corresponds to the location of the molecule. As the number of fluorescing molecules is low the spots of light are unlikely to overlap and therefore can be placed accurately. This process is then repeated many times to generate the image. Stefan Hell of the Max Planck Institute for Biophysical Chemistry was awarded the 10th German Future Prize in 2006 and Nobel Prize for Chemistry in 2014 for his development of the STED microscope and associated methodologies. SPDM spectral precision distance microscopy is a light optical process of fluorescence microscopy which allows position, distance and angle measurements on optically isolated particles well below the theoretical limit of resolution for light microscopy. Optically isolated means that at a given point in time, only a single particle or molecule within a region of a size determined by conventional optical resolution typically approx. 200, 250 nm diameter is being registered. Many standard fluorescent dyes like GFP, Alexa dyes, Atto dyes, Cy2/Cy3 and fluorescein molecules can be used for localization microscopy provided certain photo-physical conditions are present.
Common questions
When was the compound microscope first invented and who claimed to have created it?
The earliest compound microscopes appeared in Europe around 1620 with demonstrations by Cornelis Drebbel in London in 1621 and in Rome in 1624. Dutch spectacle-maker Johannes Zachariassen claimed his father Zacharias Janssen invented the device as early as 1590, though this claim is considered unlikely due to birth date discrepancies.
What year did August Köhler develop Köhler illumination for optical microscopes?
August Köhler developed Köhler illumination in August 1893 to provide extremely even lighting that overcomes limitations of older sample illumination techniques. This method ensures the image of the light source is not visible within the sample image.
Who won the Nobel Prize in Physics for phase contrast illumination in 1953?
Dutch physicist Frits Zernike received the Nobel Prize in physics in 1953 for developing phase contrast illumination. This technique allows imaging of transparent samples like live mammalian cells without using staining techniques.
What is the maximum resolving power limit of standard optical microscopes?
The maximum resolving power of optical microscopes is typically limited to around 200 nanometers because of the diffraction limit of visible light. Conventional lenses cannot resolve separate points below approximately 200 nm even with high magnification.
When was the Nobel Prize in Chemistry awarded for super-resolved fluorescence microscopy development?
The Nobel Prize in Chemistry was awarded on the 8th of October 2014 to Eric Betzig, William Moerner and Stefan Hell for their work on super-resolved fluorescence microscopy. Stefan Hell also received the 10th German Future Prize in 2006 for his STED microscope methodologies.