Optical microscope

Microscopes are devices that greatly magnified images of small (often not visible to the eye ) structures, or objects through the use of optical effects create.

In addition to conventional light microscopy, there are a variety of special light microscopic methods, such as phase contrast, interference contrast, fluorescence, polarization and confocal microscopy.

• 4.1 Principle of the composite microscope
• 4.2 illumination optics

• 5.1 resolution beyond the Abbe limit 5.1.1 Localization Microscopy SPDMphymod with Vertico SMI
• 5.1.2 Stimulated emission depletion

History

The principle of enlargement through water-filled glass shells was described by the Romans ( Seneca ) and magnifying lenses were already known in the 16th century.

The Dutch eyeglass wiper Hans Janssen and his son Zacharias Janssen are often regarded as the inventor of the first composite microscope in 1590. However, this is based on a declaration by Zacharias Janssen himself from the mid-17th century. The date is questionable, as Zacharias Janssen was even born until 1590. Galileo Galilei developed the occhiolino 1609, a compound microscope with a convex and a concave lens. However, Zacharias Janssen had demonstrated a device with the same principle a year earlier at the Frankfurt Fair. Galileo's microscope was celebrated by the " Academy of the Lynxes " in Rome, which was founded in 1603 by Federico Cesi. A drawing of Academician Francesco Stelluti of 1630 is the oldest drawing, which was made with the aid of a microscope. In her three views of bees ( from above, below and from the side) as well as enlarged details can be seen. The bee came before in the arms of the Barberini family, belonged to the Pope Urban VIII. Stelluti wrote in a banner above the figure: " For Urban VIII Pontifex Optimus Maximus [ ... ] by the Academy of Lynxes, and in eternal reverence we dedicate to you this symbol ".

Christiaan Huygens (1629-1695), also Dutch, developed a simple two - lens ocular system in the late 17th century. It was corrected achromatic, so had less color aberration and therefore was a major step forward in improving the appearance under the microscope. Huygens eyepieces are still in production today, but are compared to modern Weitfeldokularen optically clearly inferior.

Robert Hooke also used for the drawings of his in 1665 published " Micrographia " a compound microscope ( see figure). The strongest magnifications which he presented in his book, were 50-fold. Stronger magnifications were not possible because the aberrations in the front lens (objective ) and originated in the eyepiece, multiplied, so that no finer details could be seen.

Antoni van Leeuwenhoek (1632-1723) therefore took a different approach. The magnification of a lens is the stronger, the more it is arched. Small, roughly spherical lenses therefore have the highest magnification. Leeuwenhoek was brilliant in the exact smallest grinding lenses, a technique that had been inadequately mastered before. Its simple microscope with only one lens were indeed cumbersome to use, but since he copied only with a micro- lens, accounted for the multiplication of the aberrations. His microscopes had an up to 270 -fold magnification [ evidence ?]. So Leeuwenhoek discovered what he called " Animalkulen ", unicellular bacteria and protozoa.

In 1768, the Michel Ferdinand d' Albert d' Ailly, Duc de Chaulnes ( 1714-1769 ) described the first designed specifically for measurement measuring microscope.

Robert Brown used in 1830 still a simple microscope and thus discovered the nucleus and Brownian motion. It took 160 years before compound microscopes produced the same image quality as Leeuwenhoek's simple microscope.

Until well into the 19th century good compound microscopes were made by trial and error and based on past experience. Ernst Abbe developed in 1873 the data needed to build better microscopes that were still valid today physical principles. As a result, it was possible for the first time to produce a lens whose resolution limit was no longer limited by the material quality, but by the physical diffraction laws. This physical limit of resolution is referred to as the Abbe limit. The corresponding microscopes were produced with Carl Zeiss in the optical shops. They benefited from the products developed by Otto Schott optical glasses and developed by August Köhler illumination apparatus for Köhler illumination.

Light microscope types

Subdivision design or application - Overview

• The light microscope, the light is irradiated from the same side, of the observed. It finds use in non-transparent specimens and in fluorescence microscopy. In contrast, the more common arrangement in which the specimen is irradiated, referred to as a transmitted light microscope.
• A stereomicroscope has separate beam paths showing the specimen from different angles, so that a three-dimensional impression for both eyes.
• A dash microscope is a readout of a theodolite, an angle meter in surveying customer.
• A surgical microscope is used by doctors in the operating room.
• A trichinoscope is used in the meat inspection for the detection of Trichinella (roundworms ).
• A vibration microscope is used to study the vibration of strings in stringed instruments.
• A measuring microscope has an optional feature that allows a measurement of the preparation.
• A computer - microscope can be connected to a computer that is used to display the image, for example, via a USB cable.

Simple and Compound Microscopes

As a simple optical microscope lenses are referred to enable a high magnification. The transition to a basically just functioning but weaker magnifying glass is flowing. Microscopes are commonly used today, however, the compound microscopes, so called because they are composed of two lens systems: the front optical element, the objective ( see also figure), forms an intermediate image, which is enlarged from the eyepiece again.

Transmitted light or reflected light microscopy

Depending on the applied lighting equipment, a microscope for transmitted light or reflected light microscopy can be used. For transmission microscopy, the light is passed through the product before it is collected on the lens of the microscope. Therefore, transparent or thinly sliced ​​preparations are required. In the reflected-light microscopy, the light is either coming from the microscope passed through the lens to the specimen or irradiated from the side ( oblique illumination ). The reflected light is again on the specimen collected from the lens. When dermoscopy usually opaque preparations are used. Such preparations are often about in materials science, where get samples of material cut and polished or etched surfaces, which are then examined microscopically.

Construction of a transmitted-light microscope

The modules for a typical light microscope interact as follows: The eye facing lens ( eyepiece, A) enlarges an image of the object ( first zoom level (eg 10 -fold) The magnified by the eyepiece image is facing from the object.. . field lens, the lens produced (B ) the lens forms the second level of magnification Modern microscopes are usually equipped with several lenses, you can switch between them with a revolver mechanism the lenses typically have magnifications between 2 -. , and 12-fold. the total magnification of the microscope is calculated by multiplying the magnification of the 1st and 2nd level of magnification.

The object is in transmitted light microscopes usually deposited on the glass slide (C). So that the light from the mounted light source below the slide (B ) illuminates the object optimally transmitted light microscopes have a separate lens system ( D), the so-called condenser lens (see Koehler illumination ). The slide is attached to the object table ( E). Condenser and object table can be moved together to focus the object in the vertical direction. The light source is old and very simple new microscopes used a mirror ( F). Otherwise an electric light source is used.

Upright, inverted or inverted microscopes

Upright microscope is a general term for microscopes in which the lens of looking up to the preparation (as shown in the figure). In contrast, the lens is mounted under the table in a reverse or inverted microscope, the increased space between the illumination unit and the table allows microscopy of preparations with a greater thickness ( several to 10 inches ) and through the walls of laboratory vessels therethrough. Microscopes with this design is an indispensable tool for studies of living cells in culture vessels ( cell culture ), eg in the patch -clamp technique and with the use of micromanipulators, which are fed from the top of the specimen.

Efficiency

With optimal nature of the devices and the use of immersion oil can be personalized with classical light microscopy, as developed mainly in the 19th century, differ from each other at best objects that are 0.2 to 0.3 microns or further apart. The achievable resolution is not determined by the available quality of the equipment but by physical laws. It depends inter alia upon the wavelength of light used.

Methods that have been developed since the 1990s and based on nonlinear dye properties, also allow a resolution under this so-called Abbe limit.

Physical principles of light microscopy - Overview

• Bright-field microscope, the "normal" light microscope
• Dark-field microscope
• Phase contrast microscope
• Polarizing microscope
• Differential interference contrast
• Called interference reflection microscopy, and reflection contrast microscope
• Kathodolumineszenzmikroskop
• Ultramicroscope
• Lichscheiben microscopy ( SPIM)
• Fluorescence microscope
• Confocal microscope or a confocal laser scanning microscope (CLSM - Confocal Laser Scanning Microscope )
• Multiphoton microscope including two-photon microscope

The following recent developments in light microscopy allow a resolution beyond the classical Abbe limit

• Vertico SMI Structured illumination SMI with the SPDMphymod technology ( localization microscopy - based technology )
• Optical Rasternahfeldmikroskop ( SNOM)
• TIRF microscope
• Stimulated Emission Depletion Microscope ( STED )
• Photoactivated Localization Microscopy (PALM and STORM )
• 3D - SIM microscope
• 4Pi microscope

Transmitted light microscope

The basic objective of a light microscope (Greek: μικρόν (micron ) = small σκοπεῖν ( skopein ) = something all) is to increase the magnification and the resolution of details that can not be seen with the naked eye. This is due to physiological limitations of the human visual system, which has only a limited power range and therefore can not be reproduced sharply to the object in arbitrarily small intervals. Another limitation is the difference in aberrations, which limit the volume of the smallest imageable. In principle, the object is enlarged shown ( image magnification) and this then viewed with a magnifying glass to look at this from a larger " neighborhood " than would be possible with the naked eye can. In the last step, the angles are increased ( angular magnification ). Light microscopes are often given only with the abbreviation "LM".

Principle of compound microscope

The light coming from the object is optically imaged by means of a combination of at least two lens systems the lens (3) and the eyepiece (1). Here, a real intermediate image is generated from the object by the objective lens, which is viewed through the eyepiece magnifier to enlarge analogously. The magnification of the microscope is the product of the objective magnification and eyepiece. The lenses are interchangeable, as a rule, so that the magnification of each task is adjusted. The revolving nosepiece ( 2) provides quick lens changes by turning the respective desired lens in the beam path. Focusing is done by adjusting the height of the tube or of the stage. This is often also equipped with a displaceable object holder for positioning the observed object from the lens.

A distinction is made by light microscopy, in which the object is transparent or very thin and is illuminated from the side facing away from the lens, and the reflected light microscopy. In this, the surface of the object is examined by light from the side facing the lens. In the incident and transmitted light microscopy, a distinction is the dark field microscopy and phase contrast microscopy except the normal bright-field microscopy.

Illumination optics

The Köhler illumination is the usual method of a transmitted light arrangement. It consists of the following elements:

• Light source
• Collector
• Field diaphragm
• Aperture
• Condenser

To achieve the optimum performance of a microscope a correct tuning of the iris image and the object image with a chained beam path is necessary. Setting this vote is also referred to as charcoal burners. In the Köhler illumination the collector forms the light source into the aperture stop at the same time the condenser, the light field stop in the object.

Resolution

At the magnification of the microscope, see: magnification (optics)

Critical to the ability of a microscope to image structures of small objects distinct, (next to the contrast ) is not the magnification but the resolution. This relationship is to be understood not only by ray- reflection, but results from the wave nature of light. Ernst Abbe was the first to the decisive influence of the numerical aperture on the resolution. He gave as beneficial magnification

Of. This means that the smallest resolved by the objective structures according to the figure through the eyepiece can still be resolved in the eye, ie approximately at an angle of 2 ' ( minutes of arc ) appear. If the magnification is set higher (eg through an eyepiece at high magnification ), the image of the object is indeed shown even greater, but there are no further details recognizable object. Objectives and eyepieces must therefore be coordinated.

According to the laws of wave optics, the resolution of the light microscope by the size of the wavelength of the illumination is limited, see Numerical aperture.

Resolution beyond the Abbe limit

1971 published Christoph Cremer and Thomas Cremer theoretical calculations on the production of an ideal Holograms to overcome the diffraction limit, which holds an interference field in all directions in space, called a Hologram. Since the 90s of the 20th century, other methods have been developed that allow an optical resolution beyond the Abbe limit. They are all based on that Abbe theory, while limited the size of a focusable lens with light spot, but not the possibly non-linear response of molecules on ( STED and PALM). There is also the possibility to dispense with the focusing of light and very close to generate optical effects to the objects to be observed. (SNOM and TIRF ) we also speak of super-resolution.

Localization microscopy SPDMphymod with Vertico SMI

The Vertico SMI / SPDMphymod technology was developed by Christoph Cremer at the University of Heidelberg and is based on a combination of light optical techniques of localization microscopy ( SPDM, Spectral Precision Distance Microscopy) and structured illumination (SMI, Spatially Modulated Illumination). A special feature in contrast to focusing techniques such as 4Pi microscopy, the wide-field images, with which you can record whole cells nanoscopically. Among the photophysical conditions of localization microscopy SPDMphymd the super -resolution light microscopy for many is " quite common" dye molecules such as GFP, fluorescein or Alexa dyes applicable. This expands the applicability of the method on many areas SPDM biophysical, cell biological and medical research.

• Super -resolution light microscopy with the Vertico SMI / SPDMphymod technology

Colocalization microscopy with GFP and RFP fusion proteins ( nucleus of a cancer cell). 120.000 localized molecules in a wide-field recording (470 square microns )

Stimulated emission depletion

Stimulated emission depletion ( STED ) was developed by the Göttingen physicist Stefan Hell and his staff. He received the German Future Prize for his related work in 2006. This fluorescence -based technique is no longer limited by the resolution of the light wavelength, but only by the brightness of the incident light on the specimen. Leica Microsystems offers since October 2007 to a commercial device with this technology.

Since the size of protein complexes in the region of 10 to 200 nm, this technique may enable the optical microscope to penetrate into molecular sizes. So could be resolved with STED microscopy as single bubbles with nerve messengers (synaptic vesicles). Unlike electron microscope also intact, living cells can be theoretically evaluated by this method.

Also for the manufacture of tiny electronic circuits STED may be interesting. With suitable switchable molecules, the STED principle could be used for producing the finest nanostructures. Although the process would probably be too slow for mass storage, you could make arbitrarily small structures with visible light.