This document provides an overview of microscopic anatomy and various microscopy techniques. It discusses that [1] cells are the basic building blocks of living organisms and come in varied shapes and sizes, [2] microscopy involves using probes like light or electron beams that interact with tissue components to produce images, and [3] important considerations in microscopic analysis include the probe size and its ability to interact with and observe the object being investigated. It then describes various microscopy methods like light, fluorescence, polarization, and electron microscopy as well as tissue preparation techniques and important microscopy terms.

1. Department of Natural Sciences
University of St. La Salle

2. MICROSCOPIC ANATOMY

3. All living organisms are constructed from cells.
Cells come in varied shapes and sizes

5. MICROSCOPY
 Interaction of probe used (photons: light,
phase contrast, polarizing & fluorescence
microscopy; electron beams: EM), and tissue
components produce image
 Considerations in microscopic analysis:
that the probe being utilized must not be
larger than the detail to be seen
that the probe and object being
investigated must interact
it must be possible to observe and
interpret this interaction
 Units for measuring microscopic dimensions:

6. WHAT
CAN
WE
SEE?

7. IMPORTANT TERMS IN MICROSCOPY
 Magnification – increases the apparent size
of the specimen; a property of both ocular
& objective lenses
 Working distance is the distance between
the specimen and the magnifying lens.
 Depth of field is a measure of the amount
of a specimen that can be in focus. Highly
sensitive video cameras enhance power of
microscopes, and create digitized images
that can be fed into computers for
quantitative image analysis

8. • Numerical aperture – a measure of the size or
angle of the cone of light delivered by the
illuminating condenser lens to the object
plane and of the cone of light emerging from
the object.
• Resolving power – a measure of linear
distance of the smallest degree of separation
at which 2 details can still be distinguished
from each other; dependent on quality of
objective lens; R also varies according to the
refractive index at the interface of the media
used
• Generally resolution increases with
magnification, although there comes a point
of diminishing returns where magnification is

10. PREPARATION OF TISSUES FOR MICROSCOPIC
EXAMINATION (MICROTECHNIQUE)
1.Fixation - prompt treatment of tissues in
fixatives for about 12 hrs. (depending on
tissue size) prevent autolysis by enzymes or
bacteria, and preserve their morphologic
and molecular composition.
• To render the structural components
insoluble, chemicals that precipitate the
proteins are used.
• The best fixatives are those that produce
fine precipitates, e.g. buffered isotonic
solution of 4% formaldehyde and
glutaraldehyde react with amine groups
(NH2) of proteins, or cross-link with protein

11.  Gross distortions without basis in the
structure of the living cell are termed
fixation artifacts.
 Examples of artifacts:
• swelling and shrinkage of tissue
components due to poor fixation,
dehydration and/or embedding
techniques;
• wrinkles, tears, air bubbles due to poor
sectioning technique;
• dust and stain precipitate in section
resulting from use of old stain solutions,
use of improperly filtered or unfiltered
stain solutions, mistakes made during
preparation of the stain, or poor

12. Folded artifact Cracked tissue artifact
Knife mark + folded artifact

13. 2.Dehydration and Clearing
 Bathing of tissue in graded
concentrations of organic solvents (70-
100% ethanol) to replace tissue water
within 6-24 hrs.
 Ethanol is then replaced with solvent
miscible in embedding medium (xylene,
benzene, toluene)
 WHY? Most fixatives are water soluble,
most embedding media are non-polar and
are not miscible with water.
 Dehydration moves the tissue from a polar
(water-based) medium to a non-polar
medium (e.g. toluene) that is miscible with
the embedding medium.

14. 3.Embedding in melted paraffin at 58-600C, or
plastic resin at room temperature
 Tissue will be sectioned, and needs to be
durable enough to withstand the
sectioning process.
 Embedding in wax or plastic immobilizes
structural components of tissue. Holds
them in place as sectioning is done.
 Embedding medium must penetrate all
cellular/intercellular spaces to impart
rigid consistency to tissue before
sectioning
 Tissue shrinkage and artifacts may result
from heat needed for paraffin embedding;
virtually absent in resin embedding

15. 4.Sectioning by
microtome to a
thickness of 1-10 m
 Sections are then
floated in warm water
and transferred to
glass slides
 Allows histologist to:
see internal
structure of tissue.
stains or specific
markers such as antibodies to more easily
infiltrate the tissues.
light to pass through tissue making structure
visible.

16.  Images from thin sections
are 2-D; living tissues are 3-D
 In order to understand the
architecture of an organ,
sections made in different
planes should be studied
 How different 3-dimensional
structures may appear when
thin-sectioned. A: Different
sections through a hollow
ball and a hollow tube. B: A
section through a single
coiled tube may appear as
sections of many separate
tubes. C: Sections through a
solid ball (above) and
sections through a solid
cylinder (below).

18. 5.Staining– to differentiate the
colorless tissue elements as
certain cellular elements
take up more stain than
others, producing a contrast
that allows observation of
structure not visible in
unstained tissue. It may also
reveal differences in
chemical nature of regions
of the tissue.
6.Mounting – stained sections
are placed on a slide in a
gummy medium that
hardens. The preparation is
then covered with a thin

19. TYPES OF
MICROSCOPES
1.Light microscopes-
compound,
dissecting,
brightfield, and
phase-contrast
 Best resolution is 0.2
µm.
Anton van
 Maximum Leeuwenhook
magnifications are (1632-1723)
between 1000X and
1250X.

20. COMPOUND MICROSCOPE DISSECTING MICROSCOPE

21.  Compound
microscopes
bring small
objects "closer"
to the observer
by increasing the
magnification of
the sample.
 Since the sample
is the same
distance from the
viewer, a "virtual
image" is formed
as the light
passes through
the magnifying
lenses.

23. Bright-field phase-contrast
Nomarski differential-
phase-contrast dark-field
 Phase contrast microcopy- uses a lens system that
changes light speed as it passes through structures
with different refractive indices
• The phase of the light is altered by its passage
through the cell, and small phase differences can
be made visible by exploiting interference effects
• Phase-contrast and differential interference optics
produce 3-D images of transparent living cells,

24. Contrast in
Phase
Microscopy
Contrast in
Light
Microscopy

25. 2.Fluorescence microscopy– uses strong UV light
source that irradiate substances dyed with
fluorescent stains, e.g.
acridine-orange
• These appear as
brilliant, shiny particles
on a dark background;
useful for identifying
& localizing NA in cells
• Fluorescence spectros-
copy analyzes light
emitted by fluorescent
compounds in a micro-
spectrophotometer
• This permits highly
sensitive assays of
cellular substances such as catecholamines

27. The
confocal
microscope
produces
optical
sections by
excluding
out-of-focus
light

28. 3. Polarizing Compact
bone
microscopy–
birefrigent
substances rotate
direction of
polarized light
emerging from
polarizing filters
• Useful for
visualizing
substances with
repetitive, oriented
Collagen fibers,
molecular polarizing microscopy
structures

29. 4.Electron microscopy– uses high energy
electron beams (between 5,000 - 109 electron
volts) focused through electromagnetic lenses.
 Interaction of electrons deflected by lenses
beamed on tissue components permits high
resolution (0.2 - 1 nm) and 400x greater
magnification than light microscopes
 The increased resolution results from the
shorter wavelength of the electron beam
 Disadvantages of EM: requirements of a
vacuum-enclosed system, high voltage,
mechanical stability; special treatment &
sample preparation make it highly complex and
costly; requires the services of well-trained
personnel

32. T
E
M
S
E
M

33. Scanning vs. Transmission EM:
• In the TEM, the image is formed directly on the
image plane.
• In the SEM, the image is formed indirectly by
accumulation of information from the specimen
point by point.
• There is no need to cut ultra thin sections because
the beam of the SEM does not pass through the
specimen.
• The resolution of the SEM is about 100 Angstrom
vs. 4-5 Angstrom achieved by the transmission
type.
• The SEM has great depth of field making it possible
to obtain 3-D images.
• TEM magnifications are commonly over 100,000X
• SEM displays images on high resolution TV

34. SEM: T-lymphocyte, E.coli TEM: mitochondria &
attacked by macrophage chloroplast

35. Specimen Preparation for EM:
• Fixation in osmium tetroxide, osmium dichromate,
acrolein and glutaldehyde.
• Since registration of color is not possible with the
EM system, staining with colored dyes is not done in
EM studies.
• Specimen is mounted on a copper grid covered with
carbon and/or plastic film

37. Freeze-cleaving, Freeze-
etching or Cryofracture
methods
• Used with EM; replicas
are made of surfaces of
frozen aqueous materials
at very low temperatures
in vacuo
• The use of chemical
fixatives, dehydrating and
embedding agents are
avoided by using a
freezing
microtome/cryostat which
permit sections to be
obtained without

38. • Freezing does not
inactivate most enzymes,
hinders diffusion of small
molecules, eliminates
dissolution of tissue lipids
by solvents
• The tissue is impregnated chloroplast thylakoid membranes
with a 25% glycerol
solution before rapid
freezing in liquid nitrogen
or Freon12 at 1000C to
1550C.
• Not entirely free of
artifacts; valuable in the
study of membranes and vesicles
their junctional

39. QUESTIONS?