Optical Microscopy Techniques

Dark Field Microscopy

• Dark field microscopy uses a special condenser lens that directs a cone of light away from the objective lens, creating a dark background.
• The objective lens must have a numerical aperture (NA) less than 1.0, and oil immersion is used to maximize scattered light-gathering power.
• Objective lenses with a higher NA can be used if they have an adjustable diaphragm to reduce the NA.
• Applications of dark field microscopy include:
  • Detection of certain microorganisms, such as Treponema palladium, Leptospira, and Borrelia.
  • Examination of stool samples for Vibro chlorea and Campylobacter species.

Fluorescence Microscopy

• Fluorescence microscopy is used to image samples that fluoresce, absorbing one wavelength of light and emitting another.
• The phenomenon of fluorescence occurs when a substance absorbs the energy of a shorter light wavelength and emits light of a longer wavelength.
• Fluorescence microscopy is used in the detection of antigen and antibody reactions, known as immunofluorescence.
• The process involves exciting fluorescent molecules with light of one wavelength and collecting the light of a different wavelength that they emit to form a picture.

Microscope Maintenance

• Regular maintenance is essential to ensure the longevity of a microscope.
• The microscope should be stored in a box, and the objectives lens should not touch the object.
• The user should be seated at the right height and directly away from sunlight.
• It is recommended to have the microscope checked once a year by a professional technician.

Uses of Microscopes

• Microscopes are used to study atomic structure, tissue structure, and the role of proteins in cells.
• They are useful in medical, biological, geological, and crime detection applications.
• Microscopes are used to examine forensic evidence.

Hematuria

• Hematuria is defined as the presence of blood or red blood cells in the urine.
• It can be a sign or indicator of an underlying cause, which may be benign or require further investigation.
• The presence of blood or red blood cells can enter and mix with urine at multiple anatomical sites, including the urinary system, female reproductive system, and integumentary system.
• Imaging of the upper urinary tract and cystoscopy of the lower urinary tract are indicated for hematuria.

Urinary Casts

• Urinary casts are formed when cells or other substances become trapped in the protein as the cast is formed.
• The numbers and types of cells and/or material can yield detailed information and may suggest a specific diagnosis.

Crystals

• Urine contains many dissolved substances that can form crystals, solid forms of a particular substance, if the concentration of the substance is high enough.
• Crystals are identified by their shape, color, and urine pH.
• Normal crystals may be present in the urine of healthy individuals, while abnormal crystals may indicate an abnormal metabolic process.

Western Blotting

• Western blotting is a technique used to detect proteins.
• It involves separating proteins in a mixture using SDS-PAGE, transferring the protein bands to a membrane, and probing with antibodies specific to the protein of interest.
• The antigen-antibody complexes can be visualized using enzyme-linked antibodies or chemiluminescence.

Volumetric Analysis

• Volumetric analysis, also known as titrimetry, is a quantitative analytical technique used to determine the concentration of a substance in a solution.
• The method relies on measuring the volume of a reagent required to react completely with the analyte.
• Volumetric analysis is a versatile and widely used technique in analytical chemistry, providing precise and accurate measurements of the concentration of substances in solutions.