Light and electron microscopy
The prefix “micro-” is often used in reference to things that are extremely small; while the suffix “-scopy” (derived from scope) refers to looking at objects. Therefore microscopy is that technical component of science that deals with viewing structures and objects that are too small (or near impossible) to see with the naked eyes. The fundamental concepts of microscopy came about with the advent of glass during the time of the Roman Empire around 200 BC. The concept evolved over the years from using single lens magnification to more complex models that incorporated multiple magnifying lenses.
There are several different types of microscopy in use today, ranging from the simpler forms that will be discussed in this article (light and electron microscopy) to more complexed forms such as infrared, ultraviolet, wide field fluorescence microscopy and laser, digital holographic and virtual microscopy. The major goals of a microscope include magnifying the target object, produce a detailed image and making the details visible to the observer.
This discussion will cover the general anatomy of light and electron microscopes, different subtypes of each, as well as the advantages and disadvantages of each. Additionally, it will also look at how each of these devices has helped in the advancement of the field of medicine.
Light microscopy is probably the most popular form of microscopy encountered by students. It uses light from the visible spectrum and couples it with compound (serial) magnifying lenses to observe an object.
There are several components to the modern light microscope that come together to enhance its function:
- The base of the microscope provides stability to the device and allows the user’s hands to be free to manipulate other aspects of the microscope or document relevant observations.
- The base also houses the electrical circuitry for the illuminator (light source) and switch.
- Some devices are equipped with a brightness adjustment nob that allows the observer to increase or decrease the intensity of the illuminator.
- The base is attached to a frame (arm) that is connected to the head of the device. Also attached to the arm is the mechanical stage. The specimen (which is usually mounted on a microscope slide) is placed on the stage and held in place by metal arms.
- The stage can be moved laterally and anteroposteriorly with the aid of stage adjustment knobs.
- The stage also has a central slit that allows light passing from the illuminator and condenser to penetrate the specimen. The condenser is located on the inferior surface of the stage; as the name suggests, it condenses light from the illuminator onto the specimen. The aperture diaphragm (an iris-like structure) of some condensers can be adjusted to enhance the contrast of the specimen.
- Attached to the ventral surface of the head of the microscope is a revolving nosepiece, which also has several objective lenses attached to it. The power of the objective lenses is usually 4x, 10x, 20x, 40x and 100x; in other words, a 20x power objective lens will increase the size of the specimen twenty times its original size.
- The observer is able to visualize the specimen by looking through the ocular lens (eye piece) attached to the head of the microscope. Some microscopes are monocular (one eye piece) while others are binocular (two eye pieces). Binocular microscopes are equipped with dioptre adjustments that allow the user to adjust the eye pieces to fit their face. These lenses usually carry a magnification power of 10x and may be equipped with a scale on the lens so that the observer can document desired measurements.
- Finally, the observer can utilize the coarse and fine adjustment knobs on the lateral aspect of the arm to refine the resolution of the specimen.
In summary, light leaves the illuminator and passes through a condenser which focuses the light on the specimen through the slit in the mechanical stage. The observer is able to choose the desired magnification from the revolving nosepiece and observe the specimen via the ocular lens. The image can be further focused with the coarse and fine adjustment knobs.
The total magnification of the image is the product of the power of the objective and ocular lenses. In other words, if a 40x objective lens is used, the total magnification would be the power of the ocular lens (10x) multiplied by the power of the objective lens (40x), which would be 400x in this case. This is the basic underlying principle in Bright Field (Köhler illumination) microscopy.
Although the light microscope is able to magnify an image, visualization of the sample is dependent on the degree of absorption and transmission of visible light through the specimen. Most of these samples are poorly visualized because they have low contrast qualities. However, the advent of staining techniques has allowed researchers to artificially colour the specimen so that contrast can be enhanced. Varieties of staining techniques exist and are selected based on the properties of the tissues or organisms to be visualized. Some examples include:
- Papanicolaou’s stain used in “Pap” (cervical) smears
- Toluidine blue for thin sections of all tissue types and
- Haematoxylin and Eosin (H&E) stains used for a variety of histology specimen
Unlike Bright Field microscopy which depends on the contrast and pigmentation of the specimen, Phase Contrast microscopy depends on the refractive index ( the ability of materials to bend light) of the biological entities. The concept is that some aspects of the cell (for example) have a higher refractive index than other areas. As a result, light that is highly refracted deviates towards the edges of the objective lens, while light that is poorly refracted travels toward both the center of the objective lens. The light waves arriving at the periphery and the center are said to be out of phase and consequently cancel each other out. The resulting image is darker based on the refractive index of the object. Phase Contrast is the preferred imaging modality for specimen that requires high magnification (400x or 1000x) such as cilia, flagella or amoebae.
Fluorescence microscopy is a modified version of optical microscopy that employs the property of fluorochromic entities (possessing innate fluorescent properties) in highlighting structures. Most biological specimens lack the ability to fluoresce by themselves and as a result, they are labeled with fluorochromes. When fluorochromes such as green fluorescent protein (GFP) or fluorescein are stimulated with high energy light, they in turn release energy in the form of light of a longer wavelength (lower frequency).
This microscopic principle is particularly useful in immunology studies and studies involving the growth of protein crystals. Although the researcher can directly observe the magnified structures via the eye piece, most light microscopes can be attached to digital cameras in order to store the imaging for later analysis or publication.
Electron microscopy utilizes beams of charged particles (electrons) as opposed to visible light used in optical microscopy. This is made possible by the wave-like behaviour of accelerated electrons when they are placed in a vacuum. Furthermore, the pathway of the “electron waves” can be modified with the use of magnetic and electric fields (analogous to the effect of glass lenses on visible light).
The electron microscope has some features that are similar to that of an optic microscope. The illuminating source in the case of the electron microscope is an electron gun that emits electrons into a vacuum. The electron gun along with a high voltage source is intended to increase the velocity of the electrons within the vacuum. By doing this, the wavelength of the electron beam will be reduced and consequently the resolution of the resulting image will be increased. The vacuum space is the center of a tube with coils of wire around the external part of the tube (a solenoid) extending from the emission source to the detection camera. Electromagnetic condenser lenses are used as opposed to glass lenses for two reasons:
- The focal length of these magnetic lenses can be altered by changing the current passing through the solenoid.
- Glass lenses have no effect on electron beams and would therefore be useless in this system.
The lenses condense the electron beams prior to them making contact with the specimen; which is held in place by a specimen holder, which is analogous to the mechanical stage of the light microscope. Objective lens then collects the transmitted electron beans and magnifies the image before projecting it to the fluorescent screen. The former is similar to the objective lenses on the revolving nosepiece of the light microscope, while the latter replaces the eyepiece of the same device.
Transmission Electron Microscopy
In summary, an electron gun emits electrons at high voltage into a vacuum space. The electrons pass through the first condenser lens, then the corresponding aperture followed by a second condenser lens. The subsequent electrons then make contact with the specimen and the transmitted waves pass through the objective lens, then the objective aperture. The resulting electron beam then makes contact with a fluorescent screen (or light sensitive sensor in more modern devices) which produces light when hit by electrons. The resulting light emission is captured by specialized imaging software. This is the fundamental principle behind Transmission Electron Microscopy. This technique produces black and white, two-dimensional images.
Scanning Electron Microscopy
Instead of detecting electrons being transmitted from an electron source, Scanning Electron Microscopy uses the primary electron beam to excite the specimen. The electrons that are subsequently emitted from the surface of the specimen are then detected and interpreted. This technique is able to generate three-dimensional structures of the desired samples. The column of the Scanning Electron Microscope is significantly shorter than that of the Transmission Electron Microscope owing to the fact that lenses are only needed at the proximal end (above the specimen).
Light vs Electron Microscopy
- Relatively cheap
- Little to no training required
- Can be used on desktop surface
- Easy specimen preparation
- Can view live specimen
- Need for staining or contrast enhancement
- Cannot visualise structures smaller than 100 nm
- Limited magnification and resolution
- Can only view 2D structures
- Higher magnification
- Great resolution
- Able to visualise smaller structures
- Shows greater detail
- Can view 3D structures (SEM)
- Large, stand-alone equipment
- Specialised training required
- More difficult to prepare specimen
- Cannot view live specimen
- Require large amount of electricity to operate
- External magnets may interfere with equipment
The advent of microscopy has facilitated the advancement of pathological histology. Pathologists are able to prepare and analyse samples of biological tissue and give definitive diagnoses regarding the underlying disease processes.
Similarly, microbiologists can stain and examine body fluids for pathogenic microorganisms that may be responsible for a patient’s presenting complaint. Furthermore, pathologists are able to assess the margins of specimen taken during surgical procedures in order to advise the surgeons as to whether or not they were successful in their resection. Essentially, microscopy has enhance modern day surgical procedures and therapeutics.