Bright field microscopy
Encyclopedia
Bright field microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light
and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample. Bright field microscopy is the simplest of a range of techniques used for illumination of samples in light microscopes and its simplicity makes it a popular technique. The typical appearance of a bright field microscopy image is a dark sample on a bright background, hence the name.
Bright field microscopy may use critical
or Köhler illumination
to illuminate the sample.
with most biological samples as few absorb light to a great extent. Staining is often required to increase contrast, which prevents use on live cells in many situations. Bright field illumination is useful for samples which have an intrinsic colour, for example chloroplasts in plant cells.
Bright field microscopy is a standard light microscopy technique and therefore magnification
is limited by the resolving power
possible with the wavelength
of visible light.
White
White is a color, the perception of which is evoked by light that stimulates all three types of color sensitive cone cells in the human eye in nearly equal amounts and with high brightness compared to the surroundings. A white visual stimulation will be void of hue and grayness.White light can be...
and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample. Bright field microscopy is the simplest of a range of techniques used for illumination of samples in light microscopes and its simplicity makes it a popular technique. The typical appearance of a bright field microscopy image is a dark sample on a bright background, hence the name.
Light path
The light path of a bright field microscope is extremely simple, no additional components are required beyond the normal light microscope setup. The light path therefore consists of:- Transillumination light source, commonly a halogen lampHalogen lampA halogen lamp, also known as a tungsten halogen lamp, is an incandescent lamp with a tungsten filament contained within an inert gas and a small amount of a halogen such as iodine or bromine. The chemical halogen cycle redeposits evaporated tungsten back on to the filament, extending the life of...
in the microscope stand. - Condenser lens which focusses light from the light source onto the sample.
- Objective lens which collects light from the sample and magnifies the image.
- OcularsEyepieceAn eyepiece, or ocular lens, is a type of lens that is attached to a variety of optical devices such as telescopes and microscopes. It is so named because it is usually the lens that is closest to the eye when someone looks through the device. The objective lens or mirror collects light and brings...
and/or a cameraCameraA camera is a device that records and stores images. These images may be still photographs or moving images such as videos or movies. The term camera comes from the camera obscura , an early mechanism for projecting images...
to view the sample image.
Bright field microscopy may use critical
Critical illumination
Critical illumination or Nelsonian illumination is a method of specimen illumination used for transmitted and reflected light optical microscopy. Critical illumination focuses an image of a light source on to the specimen for bright illumination...
or Köhler illumination
Köhler illumination
Köhler illumination is a method of specimen illumination used for transmitted and reflected light optical microscopy. Köhler illumination acts to generate an extremely even illumination of the sample and ensures that an image of the illumination source is not visible in the resulting image...
to illuminate the sample.
Performance
Bright field microscopy typically has low contrastContrast (vision)
Contrast is the difference in visual properties that makes an object distinguishable from other objects and the background. In visual perception of the real world, contrast is determined by the difference in the color and brightness of the object and other objects within the same field of view...
with most biological samples as few absorb light to a great extent. Staining is often required to increase contrast, which prevents use on live cells in many situations. Bright field illumination is useful for samples which have an intrinsic colour, for example chloroplasts in plant cells.
Bright field microscopy is a standard light microscopy technique and therefore magnification
Magnification
Magnification is the process of enlarging something only in appearance, not in physical size. This enlargement is quantified by a calculated number also called "magnification"...
is limited by the resolving power
Resolving power
Resolving power may refer to:* Angular resolution* Spectral resolution* Resolution...
possible with the wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...
of visible light.
Limitations
- Very low contrastContrast (vision)Contrast is the difference in visual properties that makes an object distinguishable from other objects and the background. In visual perception of the real world, contrast is determined by the difference in the color and brightness of the object and other objects within the same field of view...
of most biological samples. - Low apparent optical resolutionOptical resolutionOptical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged.An imaging system may have many individual components including a lens and recording and display components...
due to the blur of out of focus material. - The sample often has to be stained before viewing. Therefore, live cells cannot usually be viewed. There are some exceptions, however, including the observation of cytoplasmic streamingCytoplasmic streamingCytoplasmic streaming is the directed flow of cytosol and organelles around large fungal and plant cells. This movement aids in the delivery of nutrients, metabolites, genetic information, and other materials to all parts of the cell...
in Chara cells.
Enhancements
- Reducing or increasing the amount of the light source via the iris diaphragm.
- Use of an oil immersion objective lens and a special immersion oil placed on a glass cover over the specimen. Immersion oil has the same refractionRefractionRefraction is the change in direction of a wave due to a change in its speed. It is essentially a surface phenomenon . The phenomenon is mainly in governance to the law of conservation of energy. The proper explanation would be that due to change of medium, the phase velocity of the wave is changed...
as glass and improves the resolution of the observed specimen. - Use of sample staining methods for use in microbiologyMicrobiologyMicrobiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...
, such as simple stains (Methylene blueMethylene blueMethylene blue is a heterocyclic aromatic chemical compound with the molecular formula C16H18N3SCl. It has many uses in a range of different fields, such as biology and chemistry. At room temperature it appears as a solid, odorless, dark green powder, that yields a blue solution when dissolved in...
, SafraninSafraninSafranin is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. This is the classic counterstain in a Gram stain...
, Crystal violetCrystal violetCrystal violet or Gentian violet is a triarylmethane dye. The dye is used as a histological stain and in Gram’s method of classifying bacteria. Crystal violet has antibacterial, antifungal, and anthelmintic properties and was formerly important as a topical antiseptic...
) and differential stains (Negative stains, flagellar stains, endospore stains). - Use of a colored (usually blue) or polarizing filterFilter (optics)Optical filters are devices which selectively transmit light of different wavelengths, usually implemented as plane glass or plastic devices in the optical path which are either dyed in the mass or have interference coatings....
on the light source to highlight features not visible under white light. The use of filters is especially useful with mineralMineralA mineral is a naturally occurring solid chemical substance formed through biogeochemical processes, having characteristic chemical composition, highly ordered atomic structure, and specific physical properties. By comparison, a rock is an aggregate of minerals and/or mineraloids and does not...
samples.