Microscopic Methods in Microbiology

Principles of Phase Contrast Microscopy

The creation of an image in phase contrast microscopy relies on the manipulation of light waves. Objects are rendered visible when different phases of light are brought into alignment with one another. This resulting interference produces a visible image of the object in the observer's eye.

Fundamentals of Fluorescent Microscopy

Fluorescent microscopy is based on the physical principle of fluorescence within substances. These fluorescent materials possess the ability to absorb light rays of short wavelengths, such as ultraviolet (UV) light, and subsequently emit light of longer wavelengths. This emitted light can then be visualized through the use of a specialized filter. While certain organisms exhibit natural fluorescence in specific light spectrums, others that do not naturally fluoresce must be treated with fluorescent dyes known as fluorochromes.

When microorganisms are stained with these fluorochromes and examined under a fluorescent microscope—using either ultraviolet rays or light with a wavelength close to UV—they appear as distinct, sharp, luminescent objects set against a dark background.

Fluorochromes and Pathogen Identification

Fluorochromes exhibit a specific affinity for different types of microorganisms, making them valuable for identification. For instance, the fluorochrome Auramine O, which glows yellow when exposed to UV rays, binds strongly to the bacterium $Mycobacterium \, tuberculosis$, the causative agent of tuberculosis. When this dye is applied to a sample containing these bacteria, they are visualized as bright yellow forms against a dark backdrop.

Similarly, $Bacillus \, anthracis$, the pathogen responsible for anthrax, appears as a light green rod-shaped structure when stained with the fluorochrome fluorescein isothiocyanate.

Immunofluorescence and Diagnostic Applications

The most significant application of fluorescent microscopy is found in a diagnostic procedure known as the fluorescent antibody technique or immunofluorescence. This technique utilizes antibodies, which are natural defense molecules produced by human and animal organisms in response to a foreign substance, known as an antigen.

To produce fluorescent antibodies for a specific antigen, the specific antigen (such as a bacterium) is injected into an animal. The animal's immune system then begins to produce specific antibodies against that antigen. After a designated period, these antibodies are isolated from the animal's serum. These fluorescently labeled antibodies are then placed on a slide containing unknown bacteria. If the unknown bacteria are identical to those initially injected into the animal, the fluorescent antibodies will bind to the bacterial surface, causing them to fluoresce. This procedure can detect bacteria or other pathogens even in the absence of cellular tissues or other clinical materials, and it is specifically applied in the diagnosis of syphilis and rabies.

Evolution and Theory of Electron Microscopy

The development of the electron microscope was prompted by the limited resolution of light microscopes, which is capped at approximately $0.2 \, \mu m$ because the wavelength of the visible spectrum is fixed and cannot be further refined. Electrons are exceedingly small, negatively charged particles that exhibit wave-like behavior similar to light waves. When electrons are accelerated using a high voltage, such as $100,000 \, V$ ($100 \, kV$) or higher, they produce a wavelength of roughly $0.005 \, nm$. This is significantly shorter than the wavelength of ultraviolet rays.

In practical application, the resolution achieved with an electron microscope for biological preparations is approximately $0.4 \, nm$. The first electron microscope was constructed by Knoll and Ruska in 1931, and the fundamental operating principles of the instrument have remained consistent since then.

Mathematical Principles of Electron Resolution

According to wave mechanics, any particle moving at a sufficiently high velocity takes on the characteristics of a wave. The wavelength ($\lambda$) of such a particle depends on its velocity and can be calculated using the following expression:

$\lambda = \frac{h}{m_0 \times v}$

In this formula:

• $h$ is Planck's constant ($6.62 \times 10^{-34} \, J \, s$).
• $m_0$ is the rest mass of an electron ($9.1 \times 10^{-31} \, kg$).
• $v$ is the velocity of the particle (electron).

The velocity of the electron is determined by the expression:

$v = \sqrt{\frac{2 \times e \times U}{m_0}}$

In this expression:

• $U$ is the voltage at the cathode ($300 \, kV$ to $400 \, kV$).
• $e$ is the charge of an electron ($1.6 \times 10^{-19} \, C$).

Practically, this indicates that an increase in the voltage applied to the cathode results in a higher electron velocity. Based on the wavelength expression, a higher velocity leads to a shorter wavelength. According to the resolution formula ($d = \frac{\lambda}{NA}$), a shorter wavelength result in a higher resolving power. For example, applying a voltage of $100 \, kV$ to the cathode yields a wavelength of $0.004 \, nm$. Substituted into the resolution formula with a numerical aperture ($NA$) of 1, the theoretical resolution would be $0.004 \, nm$. However, the actual resolution of the best electron microscopes is typically between $0.4 \, nm$ and $1 \, nm$ due to the limitations of electromagnetic lenses and electron optics.

Structural Design of Electron Microscopes

The primary components of an electron microscope include a light source (electron beam), optical components, an observation device, a vacuum system, and an electronic system to maintain constant voltage and wavelength. All optical parts, the specimen, and the photographic material are housed within a hermetically sealed tube under a high vacuum. This vacuum is necessary to prevent electrons from losing velocity or kinetic energy through collisions with air particles.

Instead of a traditional light source, the electron microscope uses a beam of electrons accelerated by high voltage. The source of these electrons is the "electron gun" located at the top of the instrument. It consists of a tungsten filament (cathode)—which can be heated to $2000 \, ^{\circ}C$—and a hollow anode. The heated cathode emits swarms of electrons that the anode collects into a narrow beam, directing it toward the sample. To focus these electrons, electromagnetic lenses are used instead of glass lenses. The resulting image is viewed on a fluorescent screen or a photographic plate.

Transmission Electron Microscopy (TEM)

Electron microscopy is divided into two main types: the Transmission Electron Microscope (TEM) and the Scanning Electron Microscope (SEM). In a Transmission Electron Microscope, a focused electron beam from the electron gun passes through a specially prepared, ultra-thin cross-section of the specimen. The beam is focused onto a small area of the sample using electromagnetic condenser lenses, which perform a function analogous to the condenser in a light microscope, such as directing the beam in a straight line.