Principles of Microscopy and Staining

Microscopy

Components of a Light Microscope

In a light microscope, light passes through a condenser (converging the beams to pass through the specimen on the slide), the iris diaphragm controls the amount of light that passes into the lens, and total magnification is calculated by multiplying the magnification of each lens.

Microscopes are important tools for visualizing aspects of the world that we cannot be directly observed, such as how cells are organized or how microorganisms (microbes) move. Light microscopes, or optical microscopes, are the simplest kind of microscope and can tell us information about all those things. Light microscopes use visible light, such as the kind that comes from a light bulb, and a series of lenses to magnify an object. The compound light microscope is a type of light microscope that uses two or three lenses and a beam of light to magnify the object being observed.

Lenses are the most important component of a compound light microscope. Compound light microscopes typically have two or three lenses. One lens, the condenser, is responsible for focusing the light inside the microscope, while other lenses function to magnify the specimen being observed. Many light microscopes are able to magnify objects to different degrees, as they contain multiple objective lenses that can be changed per the need of the user. The objective lens is the first component of the microscope that magnifies the image of the specimen. For example, a microscope might provide the option to either magnify an object fourfold in order to see a large field of view in slightly more detail than would be visible by the naked eye, such as looking at a small leaf, or by tenfold in order to zoom in on a small area with more detail, such as viewing individual veins on the leaf. A parfocal microscope is a type of compound light microscope that contains objective lenses that keep the specimen being observed in focus even when the magnification is changed. Parfocal technology saves substantial time refocusing after objective lens are switched.

The light source is at the bottom of a compound light microscope. This light will travel through many different components of the microscope before reaching the eyes of the observer. The light is emitted from a source and first passes through a condenser, a lens that focuses the light so that it hits one point on the specimen being observed, such as microbes on a glass slide. Once the focused beam of light hits the specimen, the light is next refracted so that some of the light will pass through an iris diaphragm, an adjustable hole that allows some—but not all—of the light to travel to the objective lens. The objective lens magnifies the image of the specimen by diffracting the incoming light, dispersing it so that the light appears to have come from an object larger than the actual size of the specimen. Light passing through the objective lens finally hits an eyepiece lens, also known as a projector or ocular lens, that is located just below the observer’s eye and further magnifies the specimen. The total magnification, or how much larger the image of a specimen appears through the microscope compared to its actual size, is calculated by multiplying together the magnification of all the lenses in the microscope.

Compound Light Microscope

In a compound light microscope, light passes through the specimen and two or three lenses in order to magnify the specimen.
The resolving power of a microscope can be increased by immersion, placing a small drop of water, oil, or glycerin—referred to as the immersion medium—on top of the glass covering the specimen. Immersion oil is the most common immersion medium, and it has a similar refractive index to glass. Since glass and immersion oil have the same refractive index, a continuous connection is created between the specimen and the objective lens. Light passing through is not bent by encountering another substance, such as air with a different refractive index. A magnified specimen appears more clear to an observer’s eye because the light will not refract before hitting the objective lens.

Because everyone's vision is different, the focus of the microscope will have to be adjusted to see the magnified image. This is typically done by using a series of knobs found on the side of the microscope. These knobs move the stage holding the specimen up and down so that it is closer to or farther away from the objective lens. This movement alters the path that the light takes between the condenser lens and objective lens, therefore refocusing the light that ultimately moves into the eyepiece. Coarse adjustment is used when the image appears very blurry, and it moves the specimen quickly into a range that must be further fine-tuned before a clear image is observed. Fine adjustment is used when the image is nearly in focus and can make small adjustments to the position of the specimen in order to achieve sharp resolution of the image.

Preparation of Light Microscope Slides

Wet mounts are used to examine live organisms; however, smears fix (kill) the organisms.
Microbes cannot simply be placed on a microscope and viewed. Preparation is necessary in order to be able to view them under a microscope. At a minimum, microbes need to be placed on a glass microscope slide in such a way that they remain there while being viewed. The wet mount is a common slide preparation in which microbes are suspended in a small droplet of liquid. A coverslip, a thin piece of glass that protects the specimen from directly touching the lens of the microscope, is placed over the specimen in a wet mount. Microbes prepared in this way remain alive during the observation process. Microbes that live in the water, such as algae, can be observed under a microscope using a wet mount; this helps scientists learn how these microbes move. Because microbes are so tiny, around 1,000 algae can live in a single drop of water.

Wet Mount Slide

To prepare a wet mount slide, a specimen is suspended in a droplet of liquid placed between a glass slide and a coverslip.
Another preparation for viewing microorganisms on a slide is called a smear. This process denatures the microorganism—leaving it permanently fixed to the slide. A smear is a slide preparation for which microorganisms are spread in a thin layer onto a glass slide using a cotton swab and then left to dry. After they are dry, the microorganisms are adhered to the slide. This technique is commonly used in clinical settings for preliminarily determining the causative agent of illness in patients. Basic shape and growth pattern of disease causing bacteria, such as those causing tuberculosis or strep throat, can be determined from a smear.

Other Types of Microscopes

Besides light microscopes, other types of microscopes are dark field, phase-contrast, fluorescence, confocal, scanning electron, and transmission electron.

Compound light microscopes are the simplest and most common microscopes found in scientific laboratories. The light they use is the same kind of light that comes from a light bulb or a flashlight. There are many other kinds of microscopes that use the same kind of light for magnifying and visualizing microbes.

Dark field microscopy is similar to light microscopy, with one small modification. Dark field microscopy is a technique using a small disc, called a patch stop, that blocks direct light coming from the light source of the microscope. Instead, only diffracted light hits the sample, illuminating it and producing a bright image that appears on a dark background, like stars in the night sky. Dark field microscopy can be used to look at smears or wet mounts.

Conversely, the confocal microscopy technique blocks out all light except for a small, direct beam of light—or a laser—that illuminates one small area of the specimen being observed. Since scattered light is eliminated and only direct light hits the specimen, confocal microscopes acquire images with higher resolution than light microscopes. However, because only a small area of the specimen is being viewed at a time, many pictures of the same slide must be taken to see the entire specimen using confocal microscopy. Imaging of the entire specimen is done automatically by scanning across discrete sections which are then stitched together by software. Three dimensional images of specimens are achieved by scanning a specimen in multiple planes and assembling.

Phase-contrast microscopy is a technique for observing live microbes, in which the specimens do not need to be fixed before observation. This makes it especially useful for observing the motion of organisms in great detail. Phase-contrast microscopy takes advantage of differences between light that hits the microbe being observed and background light, which passes through an area of the microscope slide where no microbe is located. Light waves that pass through a microbe begin to move more slowly than light that does not because of differences in the index of refraction. Those differences in the movement of light waves are detected as the final image.

Instead of using bright light like a compound light microscope, fluorescence microscopes use specific wavelengths of light to illuminate a specimen. Fluorophores, molecules that can absorb and emit light, are activated by the specific wavelengths of light emitted by a fluorescence microscope. The light emitted by the fluorophore is then detected by eye or by a camera to produce the final image. To observe microbes using fluorescence microscopy, the microbe can be stained with a fluorescent dye that selectively binds to only bacteria, fungi, or other microbes present in a sample and therefore aids in differentiating them.

Electron microscopy is a technique that uses a beam of electrons, instead of light, to illuminate a specimen. Electrons are negatively charged subatomic particles, and similar to light, they behave as waves and particles. The wavelength of an electron is a little more than 1 nanometer, and therefore shorter than the wavelengths of visible light. This means that electron microscopes have a much higher resolving power than light microscopes. Electron microscopes can magnify objects up to 10,000,000 times their actual size. Light microscopes can only magnify things up to about 2,000 times. The ability of electron microscopes to magnify objects up to 5,000 times more than a light microscope can make electron microscopes useful for seeing inside cells and understanding the parts that make up a microbe. Some types of electron microscopes include transmission electron microscopes (TEM) and scanning electron microscopes (SEM). All electron microscopes bombard a specimen with electrons. In a scanning electron microscope, the electrons bounce off of the surface of the specimen and the reflected electrons are detected to produce the image. In transmission electron microscopes, the electrons pass through the specimen, interact with internal components, and are detected to produce an image.
The scanning electron micrograph provides high resolution surface imagery of Legionella pneumophila while the transmission electron micrograph visualizes its internal structures.

Common Classes of Microscopes

Microscope Type Resolution Method Uses
Bright field Up to 1,000x Uses light to produce a dark image on a bright background Algae, protozoa, bacteria cells, and some cellular structures
Dark field Up to 1,000x Uses light to produce a bright image on a dark background Live specimens of algae, protozoa, and bacterial cells
Phase-contrast Up to 1,000x Uses refraction and interference to create high contrast without the need for stains Cellular structures and organelles
Fluorescence Up to 2,000x Uses fluorescence dyes to stain organisms or parts of organisms Specific cellular components can be seen with different probes
Confocal Up to 2,000x Laser scans multiple planes to create many 2-D images that are combined into a 3D image. 3D images of cells and structures
Scanning electron Up to 1,000,000x Electron beam sweeps surface of specimen. High resolution imaging of cell surfaces
Transmission electron Up to 1,000,000x Electron beam passes through specimen. High resolution imaging of internal cell structures

Several classes of microscopes are available for visualizing microscopic organisms and materials. Each type utilizes different technology allowing visualization of unique characteristics.