METHODS OF

INVESTIGATI
NGOfelia
Mrs. CELLSSolano
Saludar
Department of Natural Sciences
University of St. La Salle
INVESTIGATION OF CELLS AND THEIR PARTS
EXPERIMENTAL SUBJECTS OF CELL
BIOLOGISTS
model_organisms.exe
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 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:
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 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.
 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.
COMPOUND MICROSCOPE DISSECTING MICROSCOPE
 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.
 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
 Contrast in
Phase
Microscopy

Contrast in
Light
Microscopy
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
 The confocal
microscope
produces
optical
sections by
excluding out-
of-focus light
3. Polarizing
Compact
microscopy– bone
birefrigent substances
rotate direction of
polarized light
emerging from
polarizing filters
 Useful for visualizing
substances with
repetitive, oriented
molecular structures

Collagen fibers, polarizing

microscopy
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
Specimen Preparation for EM  Fixation in
osmium
tetroxide/
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.
 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.
SEM: T-lymphocyte, E.coli TEM: mitochondria &
attacked by macrophage chloroplast
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
 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 before
rapid freezing in liquid nitrogen chloroplast thylakoid membranes
or Freon12 at 1000C to 1550C.
 Not entirely free of artifacts;
valuable in the study of
membranes and their junctional
specializations.

vesicles
 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
techniques permit direct
Epithelial cell culture: keratin
analysis of cell behavior
(red). DNA (green)
• Used also for studies of cellular parasites, metabolism of
normal and cancerous cells; cytogenetic research, molecular
biology and recombinant DNA technology
 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.
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.
 Reproductive cloning
Donor
cell Nucleus from
donor cell
Implant embryo in Clone of donor
surrogate mother is born

Therapeutic cloning

Remove Grow in culture Induce stem cells to

Add somatic cell Remove embryonic form specialized
nucleus nucleus from adult to produce an stem cells from
from egg cells for therapeutic
donor to enucleated early embryo embryo and grow in use
cell egg cell culture

Transplantation & explantation techniques used

in grafting experiments as well as embryo transfer
utilize this method.
 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.
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.
Electron micrographs of 3 cell fractions isolated by
density gradient centrifugation. A: Mitochondrial
fraction, contaminated with microsomes. B:
Microsomal fraction. C: Lysosomal fraction.
 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.
QUESTIONS?