Microscopy

Modern Light Microscope

What is Light Microscopy?

 

The Birth of Light Microscopy

image of Hooke's compound microscope in 1665
From "The Microscope and Its Revelations" Carpenter, 1891: image of Hooke's compound microscope 1665
From "The Microscope and Its Revelations" Carpenter, 1891: images of Leeuwenhoek's simple microscope
From "Anthony Van Leeuwenhoek and his 'little animals'", edited by Clifford Dobel: plate showing the drawings of bacteria from the human mouth
"Using the microscope in an upright position" from "The Microscope and Its Revelations" Carpenter, 1891
plate showing the drawing of a flea
From "Micrographia", by Robert Hooke, 1665: plate showing the drawing of a flea

The English scientist, Robert Hooke (1635-1703), was the first to publish work based upon the use of an optical microscope, in 1665. His publication was entitled "Micrographia" or "Some physiological descriptions of minute bodies made by magnifying glasses with observations and inquiries thereupon".

This book contained a description and illustration of the microscope that he used for his observations and several illustrations of parts of plants and animals.  He was the first to use the word “cell” in the biological context. 

Hooke used a compound microscope, having two lenses, whereas Antoni van Leeuwenhoek, a Dutch cloth merchant and amateur scientist, used only a single glass lens in his simple microscope, some 10 years or so later.

Leeuwenhoek made observations on bacteria, plants, blood cells and minerals and is generally considered to have made a greater contribution to our understanding of the microscopical world than Hooke.

The understanding of how to solve the problems of spherical and chromatic aberrations, two types of distortion related to the shape and type of the glass used in lens production, led to a major leap forward in the quality and resolving power of optical microscopes and hence, by about 1830, we have compound microscopes capable of relatively high magnification and good resolution. 

Around 1870, Ernst Abbe formulated his famous sine theory for the resolving power of the light microscope, which demonstrates the importance of the numerical aperture of the lenses used and the wavelength of light.  The higher the numerical aperture and the shorter the wavelength, the better the resolving power.  Thus, the theoretically possible resolution in light microscopy is approximately 0.2 mm.  The resolving power of an objective lens is the ability to show two object details separately from each other in the microscope image.

Resolution: the resolving power of the microscope depends on the width of the cone of illumination and therefore on both the condenser and the objective lens. It is calculated using the formula

Resolution = 0.61l / n sinq

Where n = the refractive index of the medium (usually air or oil) separating the specimen from the objective and condenser lenses.
l = the wavelength of light used (for white light the figure of 0.53 microns is commonly assumed)
q = half the angular width of the cone of rays collected by the objective lens from a typical point in the specimen (since the maximum width is 180 degrees, sinq has a maximum value of 1).

Numerical aperture: n sinq in the equation above is called the numerical aperture (NA) of the lens and is a function of its light-collecting ability.  For dry lenses this cannot be more than 1, but for oil-immersion lenses it can be as a high as 1.4.  The higher the numerical aperture, the greater the resolution and the brighter the image (brightness is important in fluorescence microscopy). However, this advantage is obtained at the expense of very short working distances and a very small depth of field.

By 1900 the theoretical principles of the microscope were well understood and the microscope had become a well-established research tool for professional scientists.  Further clever refinements have improved the performance of the instrument and these, together with the development of photographic and now digital imaging techniques, have led us to the modern research microscope used as an essential tool in laboratories all over the world.

Techniques:

Transmitted Light Microscopy

Transmitted light microscopy is the general term used for any type of microscopy where the light is transmitted from a source on the opposite side of the specimen to the objective lens. Usually, the light is passed through a condenser to focus it on the specimen to get maximum illumination. After the light passes through the specimen it goes through the objective lens to magnify the image of the sample and then to the oculars, where the enlarged image is viewed.

In order to get a usable image in the microscope, the specimen must be properly illuminated. The light path of the microscope must be correctly set up for each optical method and the components used for image generation. The condenser was invented to concentrate the light on the specimen in order to obtain a bright enough image to be useful. The optimum set-up for specimen illumination and image generation is known as Köhler illumination after the man who invented it. It is used for most of the optical configurations listed below. The microscope techniques requiring a transmitted light path include bright field, dark field, phase contrast, polarisation and differential interference contrast optics.

Bright Field (Köhler illumination) Microscopy

This is "normal" microscopy when no optical contrast technique is employed.  It uses transmitted light to view a specimen that contains inherent contrast/colour or is stained. In order to get the best image possible from any transmitted light form of microscopy, and it is crucial that the light path be set up properly. The method for doing this is called Köhler illumination after August Köhler; the man who invented it. It is also know as double diaphragm illumination because it employs both a field and an aperture iris diaphragm to set up the illumination. The condenser is used to focus parallel rays of light on the specimen, as if coming from infinity, thereby giving you the advantages of an evenly illuminated field, a bright image without glare and minimum heating of the specimen. As most cells and tissues have insufficient contrast in themselves, staining techniques are generally used.  Common stains include Papanicolaou’s stain, which is used for cervical smears, and toluidine blue, a general stain used for semi-thin sections of all tissue types.

Dark Field Microscopy

The specimen is illuminated obliquely, with no direct light entering the objective.  Features in the specimen plane which scatter light can clearly be seen against a dark background.  Darkfield illumination is provided by either a simple patch stop, a darkfield element in a phase contrast condenser or purpose-built darkfield condenser.  The latter is required for high-resolution objectives to prevent the oblique rays entering the wide aperture of the objective.  Applications include detection of micro-organisms in unstained smear preparations and classical diatom studies.

Phase Contrast

Devised by Zernicka, this technique exploits the fact that light slows slightly when passing through biological specimens.  The specimen is illuminated by a hollow cone of light coming through a phase annulus in the condenser.  Phase contrast objectives must be used, which have a corresponding phase plate.  Light rays passing through the specimen are slightly retarded, and further retardation takes place in the phase plate.  When these rays combine with rays which have not taken this path, degrees of constructive and destructive interference occur which produce the characteristic light and dark features in the image.

Polarised Light Microscopy

Polarised light microscopy uses plane-polarised light to analyse structures that are birefringent; structures that have two different refractive indices at right angles to one another (e.g. cellulose microfibrils). Normal, un-polarised, light can be thought of as many sine waves, each oscillating at any one of an infinite number of orientations (planes) around the central axis. Plane-polarised light, produced by a polar, only oscillates in one plane because the polar only transmits light in that plane. The polarised light microscope must be equipped with both a polarizer, positioned in the light path somewhere before the specimen, and an analyser (a second polarizer), placed in the optical pathway after the objective rear aperture. Image contrast arises from the interaction of plane-polarized light with a birefringent (or doubly-refracting) specimen to produce two individual wave components that are each polarized in mutually perpendicular planes. The velocities of these components are different and vary with the propagation direction through the specimen. After exiting the specimen, the light components become out of phase, but are recombined with constructive and destructive interference when they pass through the analyzer. Polarised light microscopy can be used to measure the amount of retardation that occurs in each direction and so give information about the molecular structure of the birefringent object (e.g. orientation).

Differential Interference Contrast

In this complex form of polarised light microscopy two slightly separate, plane polarised beams of light are used to create a 3D-like image with shades of grey.  Wollaston prisms situated in the condenser and above the objective produce the effect, and additional elements add colour to the image.  Care must be taken to interpreting DIC images as the apparent hills and valleys in the specimen can be misleading.  The height of a "hill" (e.g. the nucleus) is a product of both the actual thickness of the feature (i.e. ray path length) and its refractive index.  Variations of the DIC system are named after their originators, Nomarski and de Senarmont.  Options can be selected to maximise either resolution or contrast.

Fluorescence Microscopy

Fluorescence can be used as a label or tag when preparing specific biological probes. Some biological substances, such as chlorophyll and some oils and waxes, have primary fluorescence (auto-fluorescence). However, most biological molecules do not fluoresce on their own, so they must be linked with fluorescent molecules fluorochromes) in order to create specific fluorescent probes. The fluorochromes emit light of a given wavelength when excited by incident light of a different (shorter) wavelength. Specimens labelled with a fluorochrome such as fluorescein or green fluorescent protein (GFP) are illuminated with the relevant wavelength of light (blue in these examples) and emit the energy as a longer wavelength (green). 

The key feature of fluorescence microscopy is that it employs reflected rather than transmitted light, which means transmitted light techniques such as phase contrast and DIC can be combined with fluorescence microscopy.  To view the fluorescence in the microscope, several light filtering components are needed.  At the heart of the fluorescence microscope is the dichroic mirror cube which comprises three components: a dichroic beam splitter (partial mirror), an excitation filter and a barrier filter.  Specific filters are used to isolate the excitation and emission wavelengths for each fluorochrome.  The dichroic mirror reflects shorter wavelengths of light and allows longer wavelengths to pass and is required because the objective acts as both the condenser lens (excitation light) and objective lens (emission light); therefore the beam splitter isolates the emitted light from the excitation wavelength. This epi-illumination type of light path is required to create a dark background so that the fluorescence can be easily seen. The wavelength at which a beam splitter allows the longer wavelengths to pass must be set between the excitation and emission wavelengths of any given fluorochrome so that excitation light is reflected and emission light is allowed to pass through it. A bright light source producing the correct wavelengths for excitation is also required for fluorescence microscopy, normally a mercury arc lamp. When using confocal microscopy to view fluorescence, where up to 95% of the emission light is filtered out, specific wavelength lasers are used, as these are extremely bright and monochromatic.

Confocal Microscopy

Although conventional light and fluorescence microscopy allow the examination of both living and fixed specimens, certain problems exist with these techniques. One of the main problems is out-of-focus blur degrading the image by obscuring important structures of interest, particularly in thick specimens. In conventional microscopy, not only is the plane of focus illuminated, but much of the specimen above and below this point is also illuminated resulting in out-of-focus blur from these areas. This out-of-focus light leads to a reduction in image contrast and a decrease in resolution. In the confocal microscope, all out-of-focus structures are suppressed at image formation. This is obtained by an arrangement of diaphragms, which, at optically conjugated points of the path of rays, act as a point source and as a point detector respectively. The detection pinhole does not permit rays of light from out-of-focus points to pass through it. The wavelength of light, the numerical aperture of the objective and the diameter of the diaphragm (wider detection pinhole reduces the confocal effect) affect the depth of the focal plane. To obtain a full image, the point of light is moved across the specimen by scanning mirrors. The emitted/reflected light passing through the detector pinhole is transformed into electrical signals by a photomultiplier and displayed on a computer monitor.

What is Green Fluorescent Protein (GFP)?

Green Fluorescent Protein (GFP) is a protein naturally produced by the jellyfish Aequorea victoria; which produces glowing points of light around the margin of it’s umbrella. The light arises from yellow tissue masses that each consist of about 6000-7000 photogenic cells. These cells generate light by a process of bioluminescence, whose components include a calcium-activated photoprotein (aequorin) that emits blue-green light and an accessory green fluorescent protein (GFP), which accepts energy from aequorin and re-emits it as green light. GFP is a 238 amino acid long protein which is very stable in neutral buffers up to 65oC, and displays a broad range of pH stability from 5.5 to 12. The protein is intensely fluorescent, with a quantum efficiency of approximately 80% and molar extinction coefficient of 2.2 x 104 cm-1 M-1. GFP fluoresces maximally when excited at 400nm with a lesser peak at 475nm, and fluorescence emission peaks at 509nm. The intrinsic fluorescence of the protein is due to a unique covalently attached chromophore, which is formed post-translationally within the protein upon cyclisation and oxidation of residues 65-67, Ser-Tyr-Gly. The gene for GFP has been isolated and has become a useful tool for making expressed proteins fluorescent by creating chimeric genes composed of those of GFP and its different colour variants linked to genes for proteins of interest. This makes it possible to have an in vivo fluorescent protein, which may be followed in a living system.

There have been several recent developments for the use of GFP and its colour variants. Wild type GFP has two excitation peaks, a major one at 395nm (long wave UV; causes rapid quenching of the fluorescence) and a smaller one at 475nm (blue) and an emission peak at 509nm (green). For general fluorescence microscopy purposes, investigators have been using normal FITC filter sets for viewing GFP. These are inadequate for wild type GFP both in excitation 475-495nm, and emission 520-560nm. To alleviate this problem, several modified versions of GFP were constructed which have increased fluorescence (serine to threonine substitution at position 65 increased fluorescence 5-6 times), but perhaps more important, the major excitation peak has been red-shifted to 490nm with the emission staying at 509nm. This is better for use of FITC filter sets as this modified GFP has the same excitation range as FITC. Furthermore, in confocal microscopy the main laser line used for GFP excitation is from the argon laser at 488nm, there is no good commonly used laser line near 395nm. In Arabidopsis plants and cells, which we regularly use for our research at The John Innes Centre, poor or no fluorescence was seen when transformed with GFP-cDNA because the expression of GFP was curtailed by aberrant mRNA splicing. Therefore, modified forms of GFP were created to restore and improve expression of the fluorescent protein. The modified gene now contains an altered codon to remove a cryptic plant intron. Since then, other modifications have given further improvements in the brightness of the emission and different colour variants of GFP have been produced e.g. in order from shortest to longest emission spectra: blue (FP or BFP), cyan (CFP), green (GFP), yellow (YFP) and red (RFP). This makes it possible to prepare double-labelled specimens expressing two or more fluorescently labelled proteins. Added peptide sequences also allow targeting of GFP intracellular organelles like the lumen of the endoplasmic reticulum.

Other useful information about GFP in plants can be found on Jim Haseloff’s web-site, of Cambridge University.