The document describes various types of light microscopes used to observe specimens at higher magnifications, including brightfield, stereoscope, darkfield, fluorescence, phase-contrast, differential interference contrast, and confocal microscopes. It also discusses electron microscopes, scanned-probe microscopes, and the preparation methods for slides in light microscopy, transmission electron microscopy, and scanned-probe microscopy. Key cell structures like prokaryotic and eukaryotic cells, organelles, and inclusions are defined.

1. The Microscope

2. Types of Compound Light Microscopy
• A brightfield microscope uses visible light as a source of
illumination.
– The specimen appears against a bright background.
– The main purpose is to observe various stained specimens.
• A stereoscope or dissecting microscope has two
objective lenses that allow three-dimensional view.
– Because specimens are usually viewed in a Petri dish rather
than on a slide, manipulation of the specimen is possible.
• A darkfield microscope uses a special condenser with
an opaque disc that blocks light from entering the
objective lens directly.
– The specimen appears light against a black background.
– The main purpose is to observe specimens that are invisible
in brightfield microscopy, do not stain easily, or are distorted
by staining.

3. Types of Compound Light Microscopy
• A fluorescence microscope reveals transparent
specimens when treated with special fluorescent dyes
known as fluorochromes and illuminated with
ultraviolet light.
– The principal use is immunofluorescence to detect the
locations of certain proteins in specimens.
• A phase-contrast microscope uses a special condenser
containing an annular diaphragm to convert normally
invisible differences in the optical path of light into
visible light-dark differences, which facilitates detailed
examination of the internal structures of living
specimens.
– No staining is required.
– The objective lens have a diffraction plate.
– Both direct light and diffracted light are brought together to
produce the image.

4. Types of Compound Light Microscopy
• A differential interference contrast microscope
uses two beams of light separated by prisms
to produce colored three-dimensional images
without staining.
• A confocal microscope uses laser light to
illuminate one plane of a specimen at a time.
– The microscope also uses fluorochromes.
– The principal use is to obtain two- and three-
dimensional images with the help of computer
software.

5. Preparation of a Slide for Compound
Light Microscope
1. The specimen is preserved in a fluid known as
fixative using a process known as fixation.
2. The specimen is dehydrated using alcohols of
progressively higher concentrations.
3. The specimen is embedded in paraffin wax and
cut into extremely thin slices by a microtome.
4. The specimen is cleared and mounted in a
transparent medium between a slide and cover
glass.
5. The specimen is immersed in colorful stains and
dyes to differentiate the components.

6. Technique: Immunofluorescence
• Antibodies are molecules produced by the human body
and many animals in response to a foreign substance
known as antigen for defense.
1. After mounting the specimen onto a slide, the specimen
is blocked to prevent antibodies from sticking to locations
where the protein of interest is not found.
2. The specimen is treated with a primary antibody to locate
the protein of interest.
3. The specimen is washed to remove any excess primary
antibody.
4. The specimen is treated with a secondary antibody
attached to a fluorochrome that will detect the primary
antibody.
5. The specimen is washed to remove any excess secondary
antibody.
6. The specimen is viewed under a fluorescence microscope.

7. Electron Microscopy
• An electron microscope uses electron beams
reflected on electromagnetic lenses to magnify
objects up to 100,000 times.
1. A scanning electron microscope (SEM) is used
to produce detailed pictures of the specimen’s
surface features.
– The electron beam scans the surface of the sample.
– The dislodged electrons from the specimen enter a
detector, producing the image.
2. A transmission electron microscope (TEM) gives
similar image as in compound microscope, but
can reach magnifications up to 100,000 times.

8. Preparation of a Slide for Transmission
Electron Microscopy
1. The specimen is fixed with a primary fixative.
2. The specimen is rinsed in appropriate buffer.
3. The specimen is fixed again with osmium
tetroxide.
4. The specimen is rinsed to remove osmium.
5. The specimen is dehydrated by alcohols of
progressively higher concentration.
6. The specimen is embedded in plastic resin.

9. Preparation of a Slide for Transmission
Electron Microscopy
7. The resin is polymerized to the specimen by
heat, catalysts, or UV light.
8. The specimen is cut into very thin slices by an
ultramicrotome.
9. The specimen is cleared and mounted in a
transparent medium between a slide and cover
glass.
10. The specimen is immersed in heavy metal
stains to differentiate the components.

10. Scanned-Probe Microscopy
• A scanned-probe microscope uses probes to examine the
surface of a specimen.
• No special preparation required.
• Resolving power is much greater than that of an electron
microscope.
1. A scanning tunneling microscope uses a thin metal probe
that scans a specimen and produces an image revealing the
bumps and depressions of the atoms on the surface of the
specimen.
– The principal use is to provide very detailed views of molecules
inside cells.
2. An atomic force microscope uses a metal-and-diamond
probe gently forced down along the surface of the
specimen.
– The image produced is three-dimensional.
– The principal use is to provide images of molecular processes
and biological molecules in nearly atomic detail.

11. Types of Cells
• Prokaryotic cells
– Mainly bacterial cells
– Lack a nucleus and instead contain a concentrated region
of DNA (the nucleoid region)
– Ribosomes and vacuoles are the only organelles present
– Contain cell wall
• Eukaryotic cells
– Include plant cells, animal cells, fungal cells, and protist
cells
– Contain a nucleus
– All organelles present
– Only plant cells and fungal cells contain cell wall

12. Parts of a Cell
• Cytosome or cytoplasm is a jelly-like fluid that is
enclosed by the plasma membrane.
• Organelles are organ-like structures found in the
cytoplasm capable of performing specific
functions.
• Inclusions are byproducts of cell activity.
1. Plasma membrane
– Composed of a fluid lipid bilayer made of
phospholipids that serves as a selective barrier
– Contains proteins that are either embedded in or
attached to the lipid bilayer