Light Microscope

The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast.

The object is placed on a stage and may be directly viewed through one or two eyepieces on the microscope. In high-power microscopes, both eyepieces typically show the same image, but with a stereo microscope, slightly different images are used to create a 3-D effect. A camera is typically used to capture the image (micrograph).

The sample can be lit in a variety of ways. Transparent objects can be lit from below and solid objects can be lit with light coming through ( bright field) or around ( dark field) the objective lens. Polarised light may be used to determine crystal orientation of metallic objects. Phase-contrast imaging can be used to increase image contrast by highlighting small details of differing refractive index.

A range of objective lenses with different magnification are usually provided mounted on a turret, allowing them to be rotated into place and providing an ability to zoom-in. The maximum magnification power of optical microscopes is typically limited to around 1000x because of the limited resolving power of visible light. While larger magnifications are possible no additional details of the object are resolved.

Alternatives to optical microscopy which do not use visible light include scanning electron microscopy and transmission electron microscopy and scanning probe microscopy and as a result, can achieve much greater magnifications.

Types

There are two basic types of optical microscopes: simple microscopes and compound microscopes. A simple microscope uses the optical power of single lens or group of lenses for magnification. A compound microscope uses a system of lenses (one set enlarging the image produced by another) to achieve much higher magnification of an object. The vast majority of modern research microscopes are compound microscopes while some cheaper commercial digital microscopes are simple single lens microscopes. Compound microscopes can be further divided into a variety of other types of microscopes which differ in their optical configurations, cost, and intended purposes.

Simple microscope

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. The use of a single convex lens or groups of lenses are found in simple magnification devices such as the magnifying glass, loupes, and eyepieces for telescopes and microscopes.

Compound microscope

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 (image 1). 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 (image 2). The use of a compound objective/eyepiece combination allows for much higher magnification. Common compound microscopes often feature exchangeable objective lenses, allowing the user to quickly adjust the magnification. A compound microscope also enables more advanced illumination setups, such as phase contrast.

Other microscope variants

There are many variants of the compound optical microscope design for specialized purposes. Some of these are physical design differences allowing specialization for certain purposes:
* Stereo microscope, a low-powered microscope which provides a stereoscopic view of the sample, commonly used for dissection.
* Comparison microscope, which has two separate light paths allowing direct comparison of two samples via one image in each eye.
* Inverted microscope, for studying samples from below; useful for cell cultures in liquid, or for metallography.
* Fiber optic connector inspection microscope, designed for connector end-face inspection
* Traveling microscope, for studying samples of high optical resolution.

Other microscope variants are designed for different illumination techniques:
* 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.
* Polarizing microscope, similar to the petrographic microscope.
* Phase-contrast microscope, which applies the phase contrast illumination method.
* Epifluorescence microscope, designed for analysis of samples which include fluorophores.
* Confocal microscope, a widely used variant of epifluorescent illumination which uses a scanning laser to illuminate a sample for fluorescence.
* Two-photon microscope, used to image fluorescence deeper in scattering media and reduce photobleaching, especially in living samples.
* Student microscope – 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, 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
* Tip-enhanced Raman microscope, is a variant of optical microscope based on tip-enhanced Raman spectroscopy, without traditional wavelength-based resolution limits. This microscope primarily realized on the scanning-probe microscope platforms using all optical tools.

Digital microscope

A digital microscope is a microscope equipped with a digital camera allowing observation of a sample via a computer. Microscopes can also be partly or wholly computer-controlled with various levels of automation. Digital microscopy allows greater analysis of a microscope image, for example, measurements of distances and areas and quantitation of a fluorescent or histological stain.

Low-powered digital microscopes, USB microscopes, are also commercially available. These are essentially webcams with a high-powered macro lens and generally do not use transillumination. The camera attached directly to the USB port of a computer so that the images are shown directly on the monitor. They offer modest magnifications (up to about 200×) without the need to use eyepieces, and at very low cost. High power illumination is usually provided by an LED source or sources adjacent to the camera lens.

Digital microscopy with very low light levels to avoid damage to vulnerable biological samples is available using sensitive photon-counting digital cameras. It has been demonstrated that a light source providing pairs of entangled photons may minimize the risk of damage to the most light-sensitive samples. In this application of ghost imaging to photon-sparse microscopy, the sample is illuminated with infrared photons, each of which is spatially correlated with an entangled partner in the visible band for efficient imaging by a photon-counting camera.

History

Invention

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.Raymond J. Seeger, Men of Physics: Galileo Galilei, His Life and His Works, Elsevier - 2016, page 24J. William Rosenthal, Spectacles and Other Vision Aids: A History and Guide to Collecting, Norman Publishing, 1996, page 391

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. Johannes' (some claim dubious) testimony pushes the invention date so far back that Zacharias would have been a child at the time, leading to speculation that, for Johannes' claim to be true, the compound microscope would have to have been invented by Johannes' grandfather, Hans Martens. Another claim is that Janssen's 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 also 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 and/or could look through the wrong end in reverse to magnify small objects. The only drawback was that his 2 foot long telescope had to be extended out to 6 feet to view objects that close. After seeing the compound microscope built by Drebbel exhibited in Rome in 1624, Galileo built his own improved version. In 1625, Giovanni Faber coined the name ''microscope'' for the compound microscope Galileo submitted to the Accademia dei Lincei in 1624 (Galileo had called it the "''occhiolino''" or "''little eye''"). Faber coined the name from the Greek words ''μικρόν'' (micron) meaning "small", and ''σκοπεῖν'' (skopein) meaning "to look at", a name meant to be analogous with "telescope", another word coined by the Linceans.

Christiaan Huygens, another Dutchman, developed a simple 2-lens ocular system in the late 17th century that was achromatically corrected, and therefore a huge step forward in microscope development. The Huygens ocular is still being produced to this day, but suffers from a small field size, and other minor disadvantages.

Popularization

Antonie van Leeuwenhoek (1632–1724) is credited with bringing the microscope to the attention of biologists, even though simple magnifying lenses were already being produced in the 16th century. Van Leeuwenhoek's home-made microscopes were simple microscopes, with a single very small, yet strong lens. They were awkward in use, but enabled van Leeuwenhoek to see detailed images. It took about 150 years of optical development before the compound microscope was able to provide the same quality image as van Leeuwenhoek's simple microscopes, due to difficulties in configuring multiple lenses. In the 1850s, John Leonard Riddell, Professor of Chemistry at Tulane University, invented the first practical binocular microscope while carrying out one of the earliest and most extensive American microscopic investigations of cholera.

Lighting techniques

While basic microscope technology and optics have been available for over 400 years it is much more recently that techniques in sample illumination were developed to generate the high quality images seen today.

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.

Fluorescence microscopy

Modern biological microscopy depends heavily on the development of fluorescent probes for specific structures within a cell. In contrast to normal transilluminated light microscopy, in fluorescence microscopy the sample is illuminated through the objective lens with a narrow set of wavelengths of light. This light interacts with fluorophores in the sample which then emit light of a longer wavelength. It is this emitted light which makes up the image.

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.

Components

All modern optical microscopes designed for viewing samples by transmitted light share the same basic components of the light path. In addition, the vast majority of microscopes have the same 'structural' components (numbered below according to the image on the right):
* Eyepiece (ocular lens) (1)
* Objective turret, revolver, or revolving nose piece (to hold multiple objective lenses) (2)
* Objective lenses (3)
* Focus knobs (to move the stage)
** Coarse adjustment (4)
** Fine adjustment (5)
* Stage (to hold the specimen) (6)
* Light source (a light or a mirror) (7)
* Diaphragm and condenser (8)
* Mechanical stage (9)

Eyepiece (ocular lens)

The eyepiece, or ocular lens, is a cylinder containing two or more lenses; its function is to bring the image into focus for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are interchangeable and many different eyepieces can be inserted with different degrees of magnification. Typical magnification values for eyepieces include 5×, 10× (the most common), 15× and 20×. In some high performance microscopes, the optical configuration of the objective lens and eyepiece are matched to give the best possible optical performance. This occurs most commonly with apochromatic objectives.

Objective turret (revolver or revolving nose piece)

Objective turret, revolver, or revolving nose piece is the part that holds the set of objective lenses. It allows the user to switch between objective lenses.

Objective lens

At the lower end of a typical compound optical microscope, there are one or more objective lenses that collect light from the sample. The objective is usually in a cylinder housing containing a glass single or multi-element compound lens. Typically there will be around three objective lenses screwed into a circular nose piece which may be rotated to select the required objective lens. 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 5× to 100× 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 of field in the resulting image. Some high performance objective lenses may require matched eyepieces to deliver the best optical performance.

Oil immersion objective

Some microscopes make use of oil-immersion objectives or water-immersion objectives for greater resolution at high magnification. These are used with index-matching material such as immersion oil or water and a matched cover slip between the objective lens and the sample. The refractive index of the index-matching material is higher than air allowing the objective lens to have a larger numerical aperture (greater than 1) so that the light is transmitted from the specimen to the outer face of the objective lens with minimal refraction. Numerical apertures as high as 1.6 can be achieved. The larger numerical aperture allows collection of more light making detailed observation of smaller details possible. An oil immersion lens usually has a magnification of 40 to 100×.

Focus knobs

Adjustment knobs move the stage up and down with separate adjustment for coarse and fine focusing. The same controls enable the microscope to adjust to specimens of different thickness. In older designs of microscopes, the focus adjustment wheels move the microscope tube up or down relative to the stand and had a fixed stage.

Frame

The whole of the optical assembly is traditionally attached to a rigid arm, which in turn is attached to a robust U-shaped foot to provide the necessary rigidity. The arm angle may be adjustable to allow the viewing angle to be adjusted.

The frame provides a mounting point for various microscope controls. Normally this will include controls for focusing, typically a large knurled wheel to adjust coarse focus, together with a smaller knurled wheel to control fine focus. Other features may be lamp controls and/or controls for adjusting the condenser.

Stage

The stage is a platform below the objective lens which supports the specimen being viewed. In the center of the stage is a hole through which light passes to illuminate the specimen. The stage usually has arms to hold slides (rectangular glass plates with typical dimensions of 25×75 mm, on which the specimen is mounted).

At magnifications higher than 100× moving a slide by hand is not practical. A mechanical stage, typical of medium and higher priced microscopes, allows tiny movements of the slide via control knobs that reposition the sample/slide as desired. If a microscope did not originally have a mechanical stage it may be possible to add one.

All stages move up and down for focus. With a mechanical stage slides move on two horizontal axes for positioning the specimen to examine specimen details.

Focusing starts at lower magnification in order to center the specimen by the user on the stage. Moving to a higher magnification requires the stage to be moved higher vertically for re-focus at the higher magnification and may also require slight horizontal specimen position adjustment. Horizontal specimen position adjustments are the reason for having a mechanical stage.

Due to the difficulty in preparing specimens and mounting them on slides, for children it is best to begin with prepared slides that are centered and focus easily regardless of the focus level used.

Light source

Many sources of light can be used. At its simplest, daylight is directed via a mirror. Most microscopes, however, have their own adjustable and controllable light source – often a halogen lamp, although illumination using LEDs and lasers are becoming a more common provision. Köhler illumination is often provided on more expensive instruments.

Condenser

The condenser is a lens designed to focus light from the illumination source onto the sample. The condenser may also include other features, such as a diaphragm and/or filters, to manage the quality and intensity of the illumination. For illumination techniques like dark field, phase contrast and differential interference contrast microscopy additional optical components must be precisely aligned in the light path.

Magnification

The actual power or magnification of a compound optical microscope is the product of the powers of the eyepiece and the objective lens. For example a 10x eyepiece magnification and a 100x objective lens magnification gives a total magnification of 1,000×. Modified environments such as the use of oil or ultraviolet light can increase the resolution and allow for resolved details at magnifications larger than 1,000x.

Operation

Illumination techniques

Many techniques are available which modify the light path to generate an improved contrast image from a sample. Major techniques for generating increased contrast from the sample include cross-polarized light, dark field, phase contrast and differential interference contrast illumination. A recent technique (Sarfus) combines cross-polarized light and specific contrast-enhanced slides for the visualization of nanometric samples.
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