Fluorescence Microscopy in Diagnostics and Immunology

What is Fluorescence Microscopy?

Fluorescence microscopy is a powerful technique for diagnosing infectious diseases and monitoring immune responses. It primarily focuses on the identification of infectious agents through methods such as fluorochroming and immunofluorescence, allowing for the visualization of bacteria, antigens, and antibodies.

What is the history of Fluorescence Microscopy?

• 1904: The technique was initiated by Köhler.

• 1938: Haitinger made significant contributions to its advancements.

• 1941: Coons, Creech, and Jones further developed the techniques.

• 1958-1967: Continued advancements were made by Riggs, Leitz, Zeiss, and Tomlinson.

• Current State: Continuous developments showcase its evolving nature.

What are the advantages of Fluorescence Microscopy?

• Ease of use: User-friendly techniques.

• Sensitivity: Capable of detecting low concentrations of substances.

• Specificity: Accurately identifies target substances.

• Rapid results: Provides quick diagnostic capabilities.

• Reliability and Universality: Applicable across various contexts.

• Adaptability: Techniques can be modified to suit different needs.

What are the applications of Fluorescence Microscopy?

• Infectious Disease Diagnosis: Effectively identifies pathogens.

• Autoimmune Disease Diagnosis: Investigates immune disorders.

• Environmental Monitoring: Tracks microorganisms in various environments.

• Emergency Support: Facilitates rapid response in critical situations.

• Biological Research: Useful in investigative research contexts.

• Industrial Process Control: Monitors industrial processes for contamination.

What is the principle of Fluorescence?

Fluorescence refers to the ability of a dye (fluorochrom) to emit light of a longer wavelength when excited by shorter wavelengths such as UV or blue-violet light, typically resulting in emission wavelengths in the green or orange light regions.

What are Fluorochromes?

Fluorochromes are chemicals that fluoresce upon excitation by specific light wavelengths, playing a crucial role in fluorescence microscopy.

What is Fluorochroming (FC)?

Fluorochroming is a method commonly used to detect bacterial pathogens, and it offers advantages such as:

• High contrast color transformation.

• Rapid examination of specimens.

• High susceptibility for detection.

• An example of this method is Boya's method for detecting mycobacteria.

How is Boya's Method used for Detection of Mycobacteria?

1. Prepare a smear of tissue culture.

2. Fix the slide over a flame for 10 minutes.

3. Stain with a mixture of auramin and rhodamin B.

4. Rinse and differentiate using acid alcohol.

5. Perform contrastive staining with acid fuchsin.
   The outcome shows tuberculous bacteria stained yellow-orange against a dark background.

What is Immunofluorescence (IM)?

Immunofluorescence involves using marked antibodies against specific antigens. It operates on the principle of visualizing the antigen + marked antibody complex under a fluorescence microscope, commonly using fluorochromes like Fluorescein Isothiocyanate (FITC). The process includes the formation of antibody-fluorochrome complexes.

What are the differences between Direct and Indirect Immunofluorescence?

• Direct Method: Involves binding the detected antigen directly with marked antibodies.

• Indirect Method: Involves initial binding to unmarked antibodies, followed by detection with marked antispecies antibodies, especially useful for detecting antigens and serum antibodies.

What are the advantages and disadvantages of Immunofluorescence?

• Advantages:

  • Rapid result delivery.

  • Does not require pure antigens.

  • High sensitivity and versatile applications.

  • Capable of detecting intracellular organisms.

  • Simple and low-cost methods.

• Disadvantages:

  • High initial equipment costs.

  • Requires trained technical operators.

What is the conclusion about Fluorescence Microscopy?

Fluorescence microscopy is a versatile tool in diagnostics, especially for infectious diseases. Its various techniques allow for rapid and sensitive identification of pathogens, with ongoing advancements enhancing its effectiveness and applicability in multiple fields.

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Fluorescence Microscopy in Diagnostics 🦠

History 🕰

The evolution of fluorescence microscopy:

• 1904: Köhler

• 1938: Haitinger

• 1941: Coons, Creech, and Jones

• 1958: Riggs et al.

• 1959: Leitz and Zeiss

• 1967: Tomlinson

The ongoing development of these techniques indicates that fluorescence microscopy technology is still advancing.

Advantages of Fluorescence Microscopy ✨

Fluorescence microscopy is advantageous, particularly for medical diagnostic tests, because it's:

• Easy

• Sensitive

• Specific

• Rapid

• Reliable

• Universal

• Adaptable

Applications 🔬

Fluorescence microscopy has a wide range of applications, including:

• Infectious disease diagnosis

  • Ag detection + identification, Ab titration, toxin detection

• Autoimmune disease diagnosis

  • Ab detection, identification, titration

• Environment monitoring

• Emergency support

• Biological research

  • location, identification of cells, tissues, nucleic acids

• Industrial process control

  • monitoring of clean rooms, detection of fluid contamination, sampling of food and beverages

• Disease diagnosis

  • etiological agent detection

• Environment microorganism monitoring

• Biological attack defense

  • rapid diagnosis, detection of bio aerosols, sampling of suspicious munitions.

• Industrial process control

Principle of Fluorescence 💡

The wavelength of emitted radiation is usually in the green, greenish-yellow, or orange light spectrum. It is measured in nanometers (nm).

• light wavelength: 120-380 nm is UV-violet

• spectral colors - wavelength in nm

Fluorescent microscopy (FM)

• FM - observation of preparates, dyed with fluorochromes or conjugates under fluorescence microscopu. Basic part - light source, high pressure mercury-arc lamp. Further parts are filter for infra-red rays, excitation filter - retains visible?

Fluorochromes 🌈

The specificity of fluorescent microscopy requires the use of nonfluorescing immersion oils.

Importance in Diagnostics 🌟

Immunofluorescence and fluorochroming are of utmost importance in diagnosing infectious diseases, especially bacterial infections.

Fluorochroming (FC) 🧪

• Low concentrations of fluorochromes are used for staining bacterial pathogens.

• It offers high contrast in color transformation, allows for fast specimen examination, and exhibits high susceptibility.

Boya's Method 🦠

Boya's method for detecting mycobacteria serves as a suitable example of bacterial visualization using fluorochroming.

1. Fixation: Fixate an impression or smear (of tissues culture) slide over the flame for 10 minutes during heating (3 times until rising of steam) with solution mixture of auramin (auramin 1.0 g, rhodamin B 0.1 g and distilled water ad 1000 ml).

2. Rinse: Rinse the slide preparation with water.

3. Differentiation: Differentiate the slide preparation with acid alcohol.

4. Rinse: Rinse the slide preparation with distilled water.

5. Contrastive Staining: Stain the background with acid fuchsin solution for 3 minutes.

6. Rinse and Dry: Rinse with distilled water and dry.

Tuberculous germs (single rods or clusters of bacteria) are stained yellow-orange against a dark, nonfluorescing background.

Immunofluorescence (IM) 🎯

The reaction principle:

Antigen+antibody (marked)

Under a fluorescence microscope, this is visualized by a fluorochrome bound to the marked antibodies.

Fluorochromes for Marking Antibodies

• Fluorescein isocyanate

• Fluorescein isothiocyanate (FITC)

• Tetramethyl rhodamin isocyanate

These dyes bind well, allowing antibodies to react with the antigen.

The chemical reaction between serum immunoglobulin and a fluorochrome (usually FITC) is called conjugation. The marked antibody is the conjugate.

Direct Method of Immunofluorescence

Direct method: Ag+Ab =AgAb

Indirect Method of Immunofluorescence (IMIF)

indirect method: Ag+Ab==AgAb

AgAb+Ab∗==AgAbAb∗'

• Shows how to detect antibodies using a known antigen. The antigen binds to the antibody you are trying to detect, and then a labeled antibody binds to the first antibody/antigen complex.

Essentially, IMIF uses two steps for staining, where an immune serum (unmarked antibodies) serves as an interlayer against the detected antigen, and then an antispecies conjugate stains against the immunoglobulin in the interlayer. This method can detect both antigens and serum antibodies

This interlayer is then affected with an antispecies conjugate against the immunoglobulin in the interlayer. Staining occurs in two phases. This process can detect both antigens and serum antibodies.

Applications of Immunofluorescence 👍

Due to its speed and reliability, immunofluorescence is widely used in theoretical and applied research, as well as in laboratory diagnostics of infectious diseases for:

• Detection of disease agents or their antigens

• Detection of antibodies

The direct method is less laborious and has fewer non-specific reactions, while the indirect method is more universal.

Advantages and Disadvantages of Immunofluorescence Microscopy ✅ ❌