Light Microscopy and Tissue PreparationChapter 1

Condenser: Collects and focuses light from the light source onto the specimen being viewed. The condenser is close to the stage and has an aperture (iris diaphragm) that controls the amount of the light coming up through the condenser.

Objective lenses: These lenses are attached to the nosepiece of the microscope. The objective lens is responsible for magnifying the specimen/section to be visualized. The type and quality of an objective lens influences the performance of a microscope. A standard microscope has three, four, or five objective lenses that range in power from 4× to 100×. The objective lens collects maximum amount of light from the object to form a high quality magnified real image.

Eye piece: The viewer looks through the eyepiece to observe the magnified image. Eye pieces may be monocular, binocular or combined photo binocular (trinocular). It is the final stage of the optical path of the microscope and produces a magnified virtual image which is seen by the eye. The eye piece has a power of 10×. In a binocular microscope diopter adjustments are present to adjust the focusing of the eye piece.2

Arm: The arm connects the body tube to the base of the microscope.

Coarse adjustment: Mechanical knobs that bring the specimen into general focus.

Fine adjustment: Fine tunes the focus and increases the detail of the specimen. Individual user has to adjust according to his/her own vision to observe the tissue section.

Nosepiece: A rotating turret that houses the objective lenses. The viewer rotates the nosepiece to select different objective lenses.

Stage: The flat platform where the slide is placed and lies perpendicular to the optical pathway. It has Stage clips that hold the slide in place. Stage Control Knobs move the stage left and right or up and down. The stage also has a vernier caliper attached to it so that the viewer can come back to any reference point by the help of the caliper. An aperture in the middle of the stage allows light from the illuminator to reach the specimen.

Base: The base supports the microscope. The illuminator with its power switch is located on the base.

Chromatic aberration: Production of a colored spectrum of white light. Different lenses corrected for chromatic aberrations are listed in Table 1.1.

Spherical aberration: A defect of single lenses due to their curved surface. Light passing through the periphery of the lens is refracted to a greater extent than through the central part. This is corrected by using a compound lens.3

Mount the specimen with the coverslip facing up on the stage

Optimize the lighting

Adjust the condenser

Focus, locate, and center the specimen

Adjust eyepiece separation and focus

Select an objective lens for viewing

Move up the magnification in steps

Dark-field microscopy: The specimen is illuminated from the side and only scattered light enters the objective lens which results in bright objects against dark background. Images produced by dark-field microscopy are low resolution and details cannot be seen. Dark-field microscopy is especially useful for visualization of small particles, such as bacteria.

Phase contrast microscopy and differential-interference-contrast allow objects that differ slightly in refractive index or thickness to be distinguished within unstained or living cells.

Fluorescence microscopy: A fluorochrome is excited with ultraviolet light and the resulting visible fluorescence is viewed. This produces a bright image in a dark background. There are some natural fluorescence substances which fluoresce when ultraviolet light falls on them, called primary fluorescent substances. Certain fluorescent dyes when added to the tissue lead to a secondary fluorescence which are visualized by a fluorescent microscope.

Confocal microscopy: The confocal scanning optical microscope is designed to illuminate an object in a serial fashion, point by point, where a small beam of light (from a LASER) is scanned across the object rapidly in an X-Y raster pattern. The images are digitized and stored.4

Electron microscopy: The property of accelerated electrons in vacuum to behave like light and travel in a straight line has been exploited in the invention of the electron microscope. Instead of glass lenses here one uses electromagnetic lenses. The wave length of the electrons in vacuum is 10,0000 times less than light. The resolving power of an electron microscope is 0.2 nm. In transmission electron microscopy the beam of electron passes through the tissue which is a thin section less than 100 nm. To prepare such thin section one uses an ultramicrotome and instead of steel blades, the sections are cut with laboratory prepared fresh glass knives. The sections are picked up in grids and are stained with uranyl acetate and lead citrate before viewing. Transmission electron microscope is used for ultrastructural studies. In scanning electron microscope the beam of electrons are reflected back from the surface, thus giving us the surface view.

Tissue collection: Commonly tissue is obtained from, autopsy, surgical procedures, experimental animals (rabbit, rats, mice, etc.) either perfused or decapitated (Guideline of ethics are always observed in any experimental study).

Fixation: The primary objective of fixation is that stained section of any tissue must maintain clear and consistent morphological features to almost that what was existing during life.

Effects of fixation: It coagulates the tissue proteins and constituents, thus minimizing their, loss during tissue processing, hardens the tissue and makes it insensitive to hypotonic or hypertonic solution.

Tissue processing: Principle of tissue processing involves replacement of all extracellular water from the tissue and replacing it with a medium that provides sufficient rigidity to enable sectioning without any damage or distortion to the tissue.

Dehydration: Removal of water by a dehydrating agent. Commonly used dehydrating agent is alcohol in descending grades (e.g. 100%, 90%, 70%, 50%, 30%).

Clearing: Making the tissue clear by removing the dehydrating agent, e.g. xyline, chloroform.

Infiltration: Permeating the tissue with a support media.

Embedding: Paraffin wax is routinely used as an embedding media. It has a melting point of 45–55 degree. Other embedding media used are celloidin and resins.

Sectioning: A rotary microtome is used to cut sections of 5–7 μ thick for routine histology. The sections are cut and picked up on clean glass slides under a water bath. They are dried before staining.

Staining: Hematoxylin and eosin is routinely used for all teaching slides. Morphological identification becomes easier. Hematoxylin is a basic dye and stains the nucleus blue while eosin is an acidic dye and stains the cytoplasm pink. Once the sections are stained they are mounted and are ready for viewing under a light microscope.