The document discusses various microscopy techniques used to study cells and their parts at the microscopic level. It describes light microscopes like compound, dissecting, and fluorescence microscopes. It also discusses electron microscopes like scanning and transmission electron microscopes. It explains techniques like cell fractionation, cell and tissue culture, laser capture microdissection, and microscopy that allow isolation and study of individual cells and organelles.

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

2. INVESTIGATION OF CELLS AND THEIR PARTS

3. EXPERIMENTAL SUBJECTS OF CELL
BIOLOGISTS

6. 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:

7. W
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8. IMPORTANT TERMS IN MICROSCOPY
 Magnification – increases the apparent size of the
specimen; a property of both ocular & objective lenses
 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
 Resolution becomes a problem in microscopes with
high magnification
 Powerful microscopes have higher numerical
aperture resulting to improved resolution.

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

11. COMPOUND MICROSCOPE DISSECTING MICROSCOPE

12.  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.

13.  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, tissues

14. 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

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

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

18. 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

19. Specimen Preparation for EM  Fixation in
osmium
tetroxide/
dichromate, a
crolein and
glutaldehyde.
 Since
registration
of color is
not possible
with the EM
system, stain
ing with
colored dyes
is not done in
EM studies.

22. Scanning vs. Transmission EM:
 In the TEM, the image is formed directly on the image
plan
 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 monitors.

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

24. 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
embedding

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

26. ISOLATING CELLS
Antibody is coupled to a
fluorescent dye to label
specific cells. Labeled
cells are then sorted out
in a fluorescence
activated cell sorter.
Individual cells traveling
single file in a fine
stream pass through a
laser beam and the
fluorescence of each is
rapidly measured.
Fluorescent cells are
deflected by a strong
electric field into an
appropriate container.

27. In laser capture microdissection, selected cells isolated from
tissue slices are coated with a thin plastic film, and a region of
interest is irradiated with a laser beam. This melts the film, and
captured cells are removed. A related method uses a laser
beam to directly cut out a group of cells and catapult them into
a container. Cells can be cultured, cytoplasm and organelles
extracted, or specific molecules purified for analysis.

28. CELL
FRACTIONATION
Homogenization/ differential centrifugation separate organelles
based on their sedimentation coefficients. The latter depends
on its size, form and density, and viscosity of the medium.

29. A mixed organelle fraction can be further separated by equilibrium
density gradient centrifugation. Pure fractions of organelles can be
biochemically studied and analyzed for purity under EM.

30. Electron micrographs of 3 cell fractions
isolated by density gradient centrifugation.
A: Mitochondrial fraction, contaminated
with microsomes. B: Microsomal fraction.
C: Lysosomal fraction.

31. CELL AND TISSUE CULTURE
• Cells/tissues are dispersed
mechanically or chemically
(treatment with trpysin or
collagenase), & grown in
chemically defined synthetic
media to which growth
factors, hormones and serum
components are added.
• Cell and tissue culture
Epithelial cell culture: keratin
techniques permit direct (red). DNA (green)
analysis of cell behavior.
• Used also for studies of cellular
parasites, metabolism of normal and cancerous
cells; cytogenetic research, molecular biology and
recombinant DNA technology, stem cell research.

32. Because they contain rapidly
growing cells, embryos or
tumors are frequently used as
starting material. The cells are
dispersed into a suspension
and added to a culture dish
containing nutrient media. The
cells in a primary culture
usually grow until they cover
the culture dish surface.
Normal human fibroblasts can
usually be cultured for 50 to
100 population doublings, after
which they stop growing and
die. In contrast, cells derived
from tumors frequently
proliferate indefinitely in
culture and are referred to as
immortal cell lines.

33. MICROSURGERY
 Cultures provide cells free of
CT and spread out on a glass
surface, so that they are
accessible to microsurgical
procedures.
 Extremely minute instruments
as microneedles, microhooks,
and micropipettes are devised.
 These are positioned within an
operating chamber on the stage
of the compound microscope
by mechanical
micromanipulators capable of
achieving controlled
movements in various planes.

34. Reproductive cloning
Donor
cell Nucleus from
donor cell
Implant embryo in Clone of
surrogate mother donor is born
Therapeutic cloning
Remove Grow in culture Induce stem
Add somatic cell Remove embryonic cells to form
nucleus nucleus from to produce an stem cells from specialized cells
from egg adult donor to early embryo embryo and grow for therapeutic
cell enucleated egg in culture use
cell
Transplantation & explantation techniques
used in grafting experiments as well as
embryo transfer utilize this method.

35. IRRADIATION
 Selective irradiation of small areas of living cells using
microbeams of protons, UV beams, & high power
organ lasers produce discrete lesions in chromosomes
or other cell components without previous
sensitization with a vital dye.
 By irradiation, it is possible to achieve the selective
destruction of specific cell organelles and to assess
its effect upon the cell as a whole.