Optical microscope

Microscope
Uses Small sample observation
Notable experiments
 Discovery of cells
Inventor Hans Lippershey
Zacharias Janssen
Related items Microscope Electron microscope
A modern microscope with a mercury bulb for fluorescence microscopy. The microscope has a digital camera, and is attached to a computer.

The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were designed around 1600. Basic optical microscopes can be very simple, although there are many complex designs which aim to improve resolution and sample contrast. Historically optical microscopes were easy to develop and are popular because they use visible light so the sample can be directly observed by eye.

The image from an optical microscope can be captured by normal light-sensitive cameras to generate a micrograph. Originally images were captured by photographic film but modern developments in CMOS and later CCD cameras allow the capture of digital images. Purely Digital microscopes are now available which just use a CCD camera to examine a sample, and the image is shown directly on a computer screen without the need for eye-pieces.

Alternatives to optical microscopy which do not use visible light include scanning electron microscopy and transmission electron microscopy.

Contents

Optical configurations

There are two basic configurations of the conventional optical microscope, the simple (one lens) and compound (many lenses). The vast majority of modern research microscopes are compound microscopes while some cheaper commercial digital microscopes are simple single lens microscopes. A magnifying glass is, in essence, a basic single lens microscope. In general microscope optics are static; to focus at different focal depths the lens to sample distance is adjusted and to get a wider or narrower field of view a different magnification objective lens must be used. Most modern research microscopes also have a separate set of optics for illuminating the sample.

Single lens (simple) microscope

A simple microscope is a microscope that uses only one lens for magnification, and is the original design of light microscope. Van Leeuwenhoek's microscopes consisted of a small, single converging lens mounted on a brass plate, with a screw mechanism to hold the sample or specimen to be examined. Demonstrations by British microscopist have images from such basic instruments. Though now considered primitive, the use of a single, convex lens for viewing is still found in simple magnification devices, such as the magnifying glass, and the loupe.

Compound microscope

A compund microscope is a microscope which uses multiple lenses to collect light from the sample and then a separate set of lenses to focus the light into the eye or camera. Compound microscopes are heavier, larger and more expensive than simple microscopes due to the increased number of lenses used in construction. The main advantages of multiple lenses are improved numerical aperture (see resolution limit below), reduced chromatic abberation and exchangeable objective lenses to adjust the magnification. A compound microscope also makes more advanced illumination setups, such as phase contrast, possible.

History

The oldest published image known to have been made with a microscope: bees by Francesco Stelluti, 1630[1]

Invention

It is difficult to say who invented the compound microscope. Dutch spectacle-makers Hans Janssen and his son Zacharias Janssen are often said to have invented the first compound microscope in 1590, but this was a declaration made by Zacharias Janssen himself during the mid 1600s. The date is unlikely, as it has been shown that Zacharias Janssen actually was born around 1590. Another favorite for the title of 'inventor of the microscope' was Galileo Galilei. He developed an occhiolino or compound microscope with a convex and a concave lens in 1609. Galileo's microscope was celebrated in the Accademia dei Lincei in 1624 and was the first such device to be given the name "microscope" a year later by fellow Lincean Giovanni Faber. 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.[2]

Christiaan Huygens, another Dutchman, developed a simple 2-lens ocular system in the late 1600s 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 problems.

Popularisation

Anton van Leeuwenhoek (1632-1723) is credited with bringing the microscope to the attention of biologists, even though simple magnifying lenses were already being produced in the 1500s. Van Leeuwenhoek's home-made microscopes were very small simple instruments, with a single, 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. Still, despite widespread claims, van Leeuwenhoek is not the inventor of the microscope.

Lighting techniques

While basic microscope technology and optics have been available for over 400 years it much more recently 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 Fritz Zernike in 1953 for his development of phase contrast illumination which allows imaging of transparent samples. By using interference rather than absorbtion 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, George Nomarski published the theory for differential interference contrast microscopy, another interference-based technique for imaging transparent samples.

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 emmitted light which makes up the image.

Since the mid 1900s 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

Basic optical transmission microscope elements(1990's)
Two Leica oil immersion microscope objective lenses; left 100x, right 40x.

All modern optical microscopes designed for viewing samples by transmitted light share the same basic components of the light path, listed here in the order the light travels through them:

In addition the vast majority of microscopes have the same 'structural' components:

These entries are numbered according to the image on the right.

Ocular (eyepiece)

The occular, or eyepiece, 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 2×, 5× and 10×. 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

The objective is a cylinder containing one or more lenses that are typically made of glass; its function is to collect light from the sample. At the lower end of the microscope tube one or more objective lenses are screwed into a circular nose piece which may be rotated to select the required objective lens. Typical magnification values of objective lenses are 4×, 5×, 10×, 20×, 40×, 50×, 60× and 100×. Some high performance objective lenses may require matched eyepieces to deliver the best optical performance.

Stage

The stage is a platform below the objective 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 mm by 75 mm, on which the specimen is mounted).

Light source

Many sources of light can be used. At its simplest, daylight is directed via a mirror. Most microscopes, however, have their own controllable light source - normally a halogen lamp.

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.

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.

Objective lenses

On a typical compound optical microscope there are a selection of lenses available for different applications. Many different objective lenses with different properties and magnification are available.

Typically there will be around three objective lenses: a low power lens for scanning the sample, a medium power lens for normal observation and a high power lens for detailed observation. The typical magnification of objective lenses depends on the intended application, normal groups of lens magnificaitons may be [4×, 10×, 20×] for low magnification work and [10×, 40×, 100×] for high magnification work.

Objective lenses with higher magnifications normally have a higher numerical aperture and a shorter depth of field in the resulting image.

Oil immersion objective

Some microscopes make use of oil immersion lens. These objectives must be used with oil (immersion oil) between the objective lens and the sample. The refractive index of the immersion oil is higher than air and this allows the objective lens to have a larger numerical aperture. The larger numerical aperture allows collection of more light making detailed observation of faint details possible.

Immersion lenses are designed so that the refractive index of the oil and of the cover slip are closely matched so that the light is transmitted from the specimen to the outer face of the objective lens with minimal refraction. An oil immersion lens usually has a magnification of 40 to 100×.

Magnification

The actual power or magnification of a compound optical microscope is the product of the powers of the ocular (eyepiece) and the objective lens. The maximum normal magnifications of the occular and objective are 10× and 100× respectively giving a final magnification of 1000×.

Magnification and micrographs

When using a camera to capture a micrograph the effective magnification of the image must take into account the size of the image. This is independent of whether it is on a print from a film negative or displayed digitally on a computer screen.

In the case of photographic film cameras the calculation is simple; the final magnification is the product of: the objective lens magnification, the camera optics magnification and the enlargement factor of the film print relative to the negative. A typical value of the enlargement factor is around 5× (for the case of 35mm film and a 6×4 inch print).

In the case of digital cameras the size of the pixels in the CMOS or CCD detector and the size of the pixels on the screen have to be known. The enlargement factor from the detector to the pixels on screen can then be calculated. As with a film camera the final magnification is the product of: the objective lens magnification, the camera optics magnification and the enlargement factor.

Operation

Optical path in a typical microscope

The optical components of a modern microscope are very complex and for a microscope to work well, the whole optical path has to be very accurately set up and controlled. Despite this, the basic operating principles of a microscope are quite simple.

The objective lens is, at its simplest, a very high powered magnifying glass i.e. a lens with a very short focal length. This is brought very close to the specimen being examined so that the light from the specimen comes to a focus about 160 mm inside the microscope tube. This creates an enlarged image of the subject. This image is inverted and can be seen by removing the eyepiece and placing a piece of tracing paper over the end of the tube. By carefully focusing a brightly lit specimen, a highly enlarged image can be seen. It is this real image that is viewed by the eyepiece lens that provides further enlargement.

In most microscopes, the eyepiece is a compound lens, with one component lens near the front and one near the back of the eyepiece tube. This forms an air-separated couplet. In many designs, the virtual image comes to a focus between the two lenses of the eyepiece, the first lens bringing the real image to a focus and the second lens enabling the eye to focus on the virtual image.

In all microscopes the image is intended to be viewed with the eyes focused at infinity (mind that the position of the eye in the above figure is determined by the eye's focus). Headaches and tired eyes after using a microscope are usually signs that the eye is being forced to focus at a close distance rather than at infinity.

The essential principle of the microscope is that an objective lens with very short focal length (often a few mm) is used to form a highly magnified real image of the object. Here, the quantity of interest is linear magnification, and this number is generally inscribed on the objective lens casing. In practice, today, this magnification is carried out by means of two lenses: the objective lens which creates an image at infinity, and a second weak tube lens which then forms a real image in its focal plane.[3]

Illumination techniques

Main article: microscopy

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.

Four examples of transilumination techniques used to generate contrast in a sample of tissue paper. 1.559 μm/pixel.

Bright field illumination, sample contrast comes from absorbance of light in the sample.

Cross-polarized light illumination, sample contrast comes from rotation of polarized light through the sample.

Dark field illumination, sample contrast comes from light scattered by the sample.

Phase contrast illumination, sample contrast comes from interference of different path lengths of light through the sample.

Other techniques

Modern microscopes allow more than just observation of transmitted light image of a sample; there are many techniques which can be used to extract other kinds of data. Most of these require additional equipment in addition to a basic compound microscope.

  • Epifluorescence microscopy
  • Confocal microscopy

Applications

Optical microscopy is used extensively in microelectronics, nanophysics, biotechnology, pharmaceutic research and microbiology.[4]

Optical microscopy is used for medical diagnosis, the field being termed histopathology when dealing with tissues, or in smear tests on free cells or tissue fragments.

Optical microscope variants

A miniature USB microscope.
Main article: Microscopy

There are many variants of the basic compound optical microscope design for specialised purposes. Some of these are physical design differences allowing specialisation for cetrain purposes:

Other microscope variants are designed for different illumination techniques:

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 quantitaton 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. The lack of illumination optics limits their use in a similar manner to stereo microscopes.

Limitations

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 of and magnitude of the diffraction patterns are affected by both by 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, is given by:

d = \frac { \lambda } { 2 NA }

Usually, a λ of 550 nm is assumed, corresponding to green light. With air as medium, the highest practical NA is 0.95, and with oil, up to 1.5. In practice the lowest value of d obtainable is about 200 nm.

Surpassing the resolution limit

Multiple techniques are available for reaching resolutions higher than the transmitted light limit described above. Techniques for surpassing the resolution limit for bright field microscopy include include ultraviolet microscopes, which use shorter wavelengths of light so the diffraction limit is lower. Holographic techniques, as described by Courjon and Bulabois in 1979, are also capable of breaking this resolution limit, although resolution was restricted in their experimental analysis.[5]

Using fluorescent samples more techniques are available. Examples include Vertico SMI, near field scanning optical microscopy which uses evanescent waves, and stimulated emission depletion. In 2005, a microscope capable of detecting a single molecule was described as a teaching tool.[6]

While most techniques focus on increases in lateral resolution there are also some techniques which aim to allow analysis of extremely thin samples. For example sarfus methods place the thin sample on a contrast-enhancing surface and thereby allows to directly visualize films as thin as 0.3 nanometers.

STED

Stimulated emission depletion is a simple example of how higher resolution of surpassing the diffraction limit is possible, but has major limitations. 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 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 for his development of the STED microscope.[7]

Alternatives

In order to overcome the limitations set by the diffraction limit of visible light other microscopes have been designed which use other waves.

The use of electrons and x-rays in place of light allows much higher resolution - the wavelength of the radiation is shorter so the diffraction limit is lower. To make the short-wavelength probe non-destructive, the atomic beam imaging system (atomic nanoscope) has been proposed and widely discussed in the literature, but it is not yet competitive with conventional imaging systems.

STM and AFM are scanning probe techniques using a small probe which is scanned over the sample surface. Resolution in these cases is limited by the size of the probe; micromachining techniques can produce probes with tip radii of 5-10 nm.

Additionally, methods such as electron or X-ray microscopy use a vacuum or partial vacuum, which limits their use for live and biological samples (with the exception of ESEM). The specimen chambers needed for all such instruments also limits sample size, and sample manipulation is more difficult. Color cannot be seen in images made by these methods, so some information is lost. They are however, essential when investigating molecular or atomic effects, such as age hardening in aluminium alloys, or the microstructure of polymers.

See also

  • Digital microscope
  • Köhler illumination
  • Microscope slide
  • Objective

References

  1. ↑ "The Lying stones of Marrakech", by Stephen Jay Gould, 2000
  2. ↑ Fi.it, "Il microscopio di Galileo"
  3. ↑ Stephen G. Lipson, Ariel Lipson, Henry Lipson, Optical Physics 4th Edition, Cambridge University Press, ISBN 9780521493451
  4. ↑ O1 Optical Microscopy By Katarina Logg. Chalmers Dept. Applied Physics. 2006-01-20
  5. ↑ D. Courjon and J. Bulabois (1979). "Real Time Holographic Microscopy Using a Peculiar Holographic Illuminating System and a Rotary Shearing Interferometer". +Journal of Optic 10 (3). 
  6. ↑ "Demonstration of a Low-Cost, Single-Molecule Capable, Multimode Optical Microscope". http://chemeducator.org/bibs/0010004/1040269mk.htm. Retrieved February 25, 2009. 
  7. ↑ "German Future Prize for crossing Abbe's Limit". http://www.heise.de/english/newsticker/news/81528. Retrieved Feb 24, 2009. 

Further reading

External links