Fundamentals of Microscopy

from chitin, which is a unique property of fungal organisms. Single-celled fungi are typically yeast organisms, of which there are at least a thousand species known. They live in many different environments; only a few are considered pathogenic to humans and animals.

Molds are multicellular fungal organisms that form colonies that can be seen by the human eye. You can see molds in damp environments, such as bathrooms and rotting plants. Molds participate in the decompensation process and are known allergens to humans. Some molds make mycotoxins that cause diseases. Molds can be helpful too, particularly in the making of certain antibiotics, including penicillin.

Helminths are multicellular pathogens and are visible with the naked eye. The are not truly microorganisms but they do have microscopic larvae and eggs. There are multiple types of helminths that cause disease, such as tapeworms and pinworms, which reside in the gastrointestinal system.

Viruses are acellular so they do not technically qualify as living things. Viruses are basically genetic material covered in a protein coat and sometimes wrapped in an envelope. They do not function independently but must incorporate their genetic material into a host cell in order to replicate their genome. There are viruses that infect bacteria, called bacteriophages, and viruses that attack other types of organisms. Surprisingly, not all viruses are strictly pathogenic.

FUNDAMENTALS OF MICROSCOPY

As you will learn in this section, there are several different types of microscopy. The concept of invisible species being the cause of disease was first developed in the 1500s. At the time, there were no scientific techniques for actually seeing them, however. The first microscope was made by Antonie van Leeuwenhoek, who is believed to be the “father of microbiology”. He first observed single-celled organisms in the late 1600s. Galileo also pioneered the field of microscopy. Galileo used a compound microscope to evaluate insects, while van Leeuwenhoek developed light microscopy that could see bacteria.

Robert Hooke also lived in the late 1600s and contributed to microscopy by first evaluating cells. He studied cork, which led him to believe that cells were empty. The actual inventors of the microscope, however, were Hans and Zaccharias Janssen, who were lens makers. Their work predates the others; however, they did not publish their findings.

The term, light microscope, actually applies to several different types of microscope. The typical type of light microscope you will use in the laboratory is a brightfield microscope but there are other types as well that are used primarily in research settings.

Brightfield microscopy involves a microscope with at least two lenses that will reveal a darker image on a lighter background. Some will be monocular, while others are binocular, having two eyepieces. There is an ocular lens in the eyepiece that offers a 10 times magnification, and a number of objective lenses that have different levels of magnification. The total magnification seen is the product of both types of magnifications provided. Figure 3 shows a monocular light microscope:

Figure 3.

When you see an item under the microscope, it is referred to as a specimen. The specimen is fix onto the slide and clipped on the platform or stage of the microscope. There are knobs that adjust the ocular focusing coarsely and finely. There is usually a light source beneath the specimen to improve visualization. Light is focused on the specimen with a condenser lens. There Is also a diaphragm that will monitor the amount of light in reaching the specimen and a rheostat, that dims or brightens the light source.

Light is differentially transmitted, reflected refracted, or absorbed by the different parts of the specimen, allowing for different features being seen. Chromophores or pigments will provide the different colors seen. Many chromophores actually come from the staining of the item. Magnifications up to 1000 times can be seen with a light microscope. Oil immersion is used at this magnification in order to decrease the scattering of light rays at higher-order magnitudes, improving resolution.

Darkfield microscopy is related to brightfield microscopy with modification of the condenser. There is a tiny opaque disc that has been placed between the condenser lens and the illuminator. This is called the opaque light stop. The result is a brighter image on a dark background. The advantage is that it can be used on living specimens that are not stained. Figure 4 shows an image on darkfield microscopy:

Figure 4.

Phase contrast microscopy makes use of interference and refraction within a specimen to create better resolution without having to stain the specimen. The trick is to alter the wavelengths of light that pass through the specimen. The condenser contains an annular stop that makes a hollow cone of light that focuses on the specimen. There is a phase plate and a phase ring that cause the light waves to be out of phase from those that pass through the plate. There are peaks and troughs in the waves that can either augment or cancel each other out. The end result is a dark background and a lighter specimen that can be living and unstained. It can particularly outline cellular features.

Differential interference contrast microscopy is related to phase contrast microscopy because interference patterns are created. There are two beams of light involved in polarization of the light waves. The images are high contrast and living organisms that appear three-dimensional. There is no staining required. Figure 5 shows an image seen with DIC microscopy:

Figure 5.

A fluorescence microscope makes use of fluorochromes, which can absorb light energy, revealing parts of the specimen as a fluorescent substance. Chlorophyll is naturally fluorescent and there are dyes also that will fluoresce under the microscope. The chromophores will emit light that has a longer wavelength than the UV light or shortwavelength light that is emitted from the light source. Bright colors can be seen against a dark background. This type of microscopy can be used in order to identify pathogens. Multiple fluorochromes can be used to distinguish between structures.

Fluorescence microscopy can be used in immunofluorescence, which involves the use of fluorescent antibodies that tag parts of the specimen so they fluoresce under the microscope. There is direct immunofluorescence assays and indirect immunofluorescent assays. The difference is the actual target of the fluorescence. With indirect or IFA assays, the fluorescence tags antibodies that themselves do not fluoresce. This allows for a wider range of organisms to be identified because many different antibodies can be used to detect pathogens. Figure 6 shows the two types of immunofluorescence:

Figure 6.

A confocal microscope uses a laser to create a two-dimensional image of the specimen at different levels. The different levels of images can be reconstructed with a computer to create a three-dimensional image. It can be used to examine and unfixed images of things like biofilms of bacteria. Figure 7 shows a fluorescent image that has been creates with a confocal microscope:

Figure 7.

The confocal lens is not very good at looking at thicker images. This problem has led to the development of what’s called a two-photon microscopy, which uses fluorochromes, scanning, and infrared light in order to see the specimen. Because it involves low energy levels, it takes two photons to highlight the specimen. It can be used to see living tissues, such as the brain, whole organs, and embryos. Because it is costly to purchase and use, it is used mainly in research settings.

Electron microscopy will allow for much better magnification than any form of light microscopy. It uses very short-wavelength electron beams to visualize the specimen.

The image that can be gotten can be magnified by 100,000 times magnification. This allows it to see things as small as a strand of DNA. It cannot visualize a living thing because of staining and preparation.

There are two types of electron microscopy: these are the transmission electron microscope or TEM and the scanning electron microscope or SEM. The TEM shows a sharp image not unlike that of a light microscope. The electrons pass through the specimen. The technique requires a very thin specimen and staining is done using a heavy metal stain. The SEM will look at the surfaces of specimens in three-dimensional detail. The specimen is coated with gold in order to stain it. There are some SEMs that can magnify up to 2 million times magnification. Figure 8 shows a scanning electron microscopy image:

Figure 8.

Scanning probe microscopy doesn’t use electrons or light but passes sharp probes over the surface of the specimen. The images are the best magnification that can be seen, up to 100 million times magnification. It can look at atoms on certain surfaces.