Microscopy

Histology as a subject is entirely dependent on the microscope as a tool of study. Histology wouldn’t have come into being had the microscope not been invented. No microscope, no histology. The microscope is an amazing tool that provides a widow for visualizing the complex microstructure of the human body. The microscope is also  an essential tool for the advancement of other branches of basic medical such as histopathology and microbiology. Special forms of microscopes (e.g. stereomicroscopes) are used for microdissection and microsurgery.

Types of Microscopes

There are two main classes of microscopes: the light microscope (LM) and the electron microscope (EM). There are different types of each of these two classes. Types of light microscopes include the bright field microscope, fluorescent microscope, confocal microscope, dark-filed microscope and phase-contrast microscope. Types of electron microscopes include the transmission electron microscope (TEM), the scanning electron microscope (SEM) and the cryogenic electron microscope (CEM). Images seen by the light microscope are always colored (due to light refraction), whereas those seen by the electron microscope are always black and white (B&W).

The bright field microscope

This type of microscope is also known as the conventional microscope or the compound light microscope (Fig A1). It utilizes visible light to illuminate and produce a magnified image of the tissue section. It uses an ordinary electric lamp or a halogen bulb as the source of light. The bright field microscope is the most common type of microscope. It is the type of microscope we see in ordinary histology students teaching labs and ordinary histology preparation and research laboratories. Histology students should acquaint themselves with components of the bright field microscope. They should be able to identify the light source, the stage, the objective lenses and the eyepiece, and should be able to utilize the microscope.

Light rays from the light source pass upwards across the stained tissue section on the stage, then into one of the objective lenses to be magnified (refraction) and into the eyepiece lenses producing a magnified colored image (Fig A2).

Fig. A1. The brightfield microscope and its components

Fig A2. Histological section of the pancreas stained by hematoxylin and eosin (H&E) and examined by a conventional brightfield microscope. 

The Fluorescent microscope

The fluorescent microscope is a modified light microscope. There are two well-known varieties of fluorescent microscopes: the conventional one and the confocal one. The confocal one differs from the conventional one in that it is capable examining the specimen at many different levels ultimately giving a sharp 3-dimentional image. In both the conventional and confocal fluorescent microscopes an ordinary light beam is produced by the light source. Both types of microscopes are equipped with a unique type of mirror known as the dichroic mirror (Fig A3). This type of mirror allows the passage of light with long waves lengths but does not allow the passage of ordinary light with short waves lengths. Thus, ordinary light is reflected by the mirror whereas fluorescent light passes through the mirror. The specimen should have the capability to fluoresce (must contain fluorophores). Ordinary light produced by the light source is reflected by the dichroic filament towards the specimen on the stage. It induces fluorescence in the specimen on the stage of the microscope. Fluorescent light emitted by the specimen is magnified by objective lenses then passes undeflected through the dichroic mirror producing a magnified image of the fluorescent specimen (Fig A4).

The fluorescent microscope is a very strong tool for studying cell biology and immuno-cytochemistry.

Fig. A3. Showing the mechanism of image formation in fluorescent microscopes. The beam produced by the light source is filtered and then reflected by the dichroic mirror towards the specimens that is excited to produce a fluorescent light. This passes up through the objective lens and through the dichroic mirror forming a final magnified colored image

Fig A4. Image of a section from the nervous system taken by a confocal laser microscope. The image of the multipolar neuron (green) is sharp and appears three dimensional.   

The phase-contrast microscope and the darkfield microscope are seldom utilized for histology, and as thus they won’t be dealt with here.

The electron microscope

Electron microscopy is the strongest tool for studying the structure of tissues, cells and microorganisms at the finest levels. It is also of great importance in studying macromolecular aggregations within cells and tissues. The structure of tissues and cells at the very high magnifications of electron microscopes is known as ultrastructure.

Examination of tissue under EM requires complex preparations. There are various types of electron microscopes which require different types of sample preparation and visualizing techniques. Highly sophisticated types of electron microscopes were invented in the last few decades and contributed greatly to the advancement of histology, cell biology, microbiology and histopathology. They contributed greatly to the better understanding of newly emerging diseases such as the devastating covid 19.

There are different types of electron microscopes; these include: the transmission electron microscope (TEM), the scanning electron microscope (SEM), the scanning transmission electron microscope (STEM), the reflection electron microscope (REM), the cryogenic electron microscope (CEM), the environmental scanning electron microscopes (ESEM), the low voltage electron microscope (LVEM) and the cryo-electron tomography (cryo-ET). TEM and SEM are the most widely used types of EMs. TEM is quite commonly used for studying the ultrastructure of cells and tissues at very high magnification (x100s of thousands) in ultrathin (very thin) sections (about 50 nm thick). SEM is mainly used for studying surfaces; it creates a 3D image of the specimen. Electron microscopes do not have a light source.

The transmission electron microscope is the prevalent. It is widely used for the study of the ultrastructure of cells and cell organelles. It is a huge instrument that contains a vacuum column, a tungsten filament and an electromagnetic field (Fig. A5). A high voltage electron current ignites the tungsten filament at the top TEM, to produce an electron beam. The electron beam travels down inside the vacuum column towards the tissue section (ultrathin section) and produces an image in black and white. The image is enormously magnified by the electromagnetic field (lens).

A transmission electron microscope fitted with a cold chamber (-80 to -180 0C) for frozen ultrathin sections is known as the cryogenic transmission electron microscope (CTEM). It is used for immunocytochemistry.

Fig. A5. Image and diagrammatic illustration of the transmission electron microscope.

The scanning electron microscope (SEM) is smaller and is also commonly used. It is used to study the surface morphology of cells and structures. The surface of the specimen to be examined with SEM is sputter coated with a heavy metal. The electron beam does not penetrate cells or tissues, instead the electrons are reflected from surface by the metal coat. Sensors capture the reflected secondary electrons, and a 3D image of the surface is created. The inside of cells is not examined with the SEM.

Fig. A6. Image and diagrammatic illustration of the scanning electron microscope.

Whereas SEM reveals the surface morphology of cells and tissues, TEM gives the very details of the structure cells and tissue components. Thus, TEM is the instrument of choice for studying cell organelles and inclusions. In the following two pictures (Figs A7 and A8) you will see the difference between the two.

Fig. A7. Transmission electron micrograph showing part of the cytoplasm of a cell containing clearly visible organelles. CG, Golgi apparatus; RER, cisterna of rough endoplasmic reticulum. The nuclear envelope shows distinct nuclear pores.

Fig. A7. Scanning electron micrograph showing the surface morphology of a podocytes - a specialized kidney cell (rad arrow). The cell and its processes appear 3D.

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