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INVESTIGATION OF CELLS AND THEIR PARTS

INVESTIGATING CELLS

Department of Natural Sciences

University of St. La Salle
Bacolod City

Objective: At the end of this lesson, students will be  1990- The Human
able to explain the mechanisms of action and Genome Project begins.
applications of methods used to investigate cells. HGP leaders established
the 'Bermuda Principles'
Topic Outline and agreed that genome
1. Historical Landmarks in Cell Investigations sequence data should be
2. Experimental Subjects of Cell Biologists made freely available in
the public domain to
3. Microscopy: Light, phase contrast, fluorescence,
ensure that sequence
confocal, polarizing, electron microscopy
information led as rapidly
4. Cell isolation and sorting: fluorescence activated as possible to advances
sorter, laser capture microdissection, cell in healthcare and
fractionation, flow cytometry research.
5. Cell and Tissue Culture  2003- The Human
6. Microsurgery Genome Project is
7. Irradiation completed and published Where do we go from here?

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EXPERIMENTAL SUBJECTS OF CELL

BIOLOGISTS

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

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Units for measuring microscopic dimensions

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IMPORTANT TERMS IN MICROSCOPY TYPES OF

 Magnification – increases the apparent size of the MICROSCOPES Anton van
specimen; a property of both ocular & objective lenses Leeuwenhook
 Numerical aperture – a measure of the size or angle of 1.Light microscopes- (1632-1723)
the cone of light delivered by the illuminating compound,
condenser lens to the object plane and of the cone of dissecting,
light emerging from the object. brightfield, and
 Resolving power – a measure of linear distance of the
phase-contrast
smallest degree of separation at which 2 details can
still be distinguished from each other; dependent on  Best resolution is
quality of objective lens; R also varies according to the 0.2 µm.
refractive index at the interface of the media used.  Maximum
 Resolution becomes a problem in microscopes with magnifications are
high magnification between 1000X and
 Powerful microscopes have higher numerical
1250X.
aperture resulting to improved resolution.

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Why does the “letter

e” appear inverted
and magnified?

COMPOUND MICROSCOPE DISSECTING MICROSCOPE

 Compound  Lens system

microscopes
Phase contrast microcopy changes light speed
bring small as it passes through
structures with
objects "closer"
different refractive
to the observer indices
by increasing the  The phase of the
magnification of light is altered by its
the sample. passage through the
cell, and small
 Since the sample
phase differences
is the same can be made visible
distance from the by exploiting
viewer, a "virtual interference effects
image" is formed  Phase-contrast and
differential
as the light
interference optics
passes through produce 3-D images
the magnifying of transparent living
lenses. cells, tissues

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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 The
& localizing NA in cells confocal
 Fluorescence spectros- microscope
copy analyzes light produces
emitted by fluorescent optical
compounds in a micro- sections by
spectrophotometer excluding
 This permits highly out-of-focus
sensitive assays of light
cellular substances such as catecholamines

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3. Polarizing Specimen Preparation for EM  Fixation in

microscopy– osmium
birefrigent tetroxide/
substances dichromate,
acrolein and
rotate
glutaldehyde.
direction of  Since
polarized light registration
emerging from of color is
polarizing not possible
with the EM
filters
system,
 Useful for staining with
visualizing colored dyes
substances is not done in
with repetitive, EM studies.
oriented
molecular Collagen fibers,
structures 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

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TEM
Used to study cell ultrastructure
and its components; can see
proteins and objects at nanolevel
Electron beams pass through the
sample
0.2-1nm resolution
Low depth of field
5,000,000x magnification
Electron-scattering
2-D black and white images
Very thin samples required
Specimen mounted on copper
grids
Limited field of view
Direct imaging on fluorescent or
PC screen
SEM: T-lymphocyte, E.coli
attacked by macrophage
12/11/2019
SEM
Produces surface morphology Freeze-cleaving, Freeze-
images of cells and organisms etching or Cryofracture
methods
Electron beams scans through
the surface of the sample  Used with EM; replicas are
0.5-10nm resolution
made of surfaces of frozen
aqueous materials at very
High depth of field
low temperatures in vacuo
100,000x magnification
 The use of chemical
Electron adsorption
fixatives, dehydrating and
3-D black and white images embedding agents are
Easy preparation technique avoided by using a freezing
Aluminum stubs specimen microtome/cryostat which
mounting permit sections to be
Large field of view obtained without
Image projected by detectors on embedding.
PC screen
 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.
TEM: mitochondria &
chloroplast vesicles
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ISOLATING CELLS CELL FRACTIONATION

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

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 mixed organelle fraction can be further separated by equilibrium
a container. Cells can be cultured, cytoplasm and organelles
density gradient centrifugation. Pure fractions of organelles can be
extracted, or specific molecules purified for analysis. biochemically studied and analyzed for purity under EM.

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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.
Electron micrographs of 3 cell fractions • Cell and tissue culture
isolated by density gradient centrifugation. techniques permit direct Epithelial cell culture:
A: Mitochondrial fraction, contaminated analysis of cell behavior. keratin (red). DNA (green)
• Used also for studies of cellular
with microsomes. B: Microsomal fraction.
parasites, metabolism of normal and cancerous cells;
C: Lysosomal fraction. cytogenetic research, molecular biology and
recombinant DNA technology, stem cell research.
..\..\videos\methods\Subcellular Fractionation.mp4

FLOW CYTOMETRY Cytometry is the Because they contain rapidly

measurement of the growing cells, embryos or
characteristics of tumors are frequently used as
starting material. The cells
cells. Variables that
are dispersed into a
can be measured by suspension and added to a
cytometric methods culture dish containing
include cell size, cell nutrient media. The cells in a
count, cell primary culture usually grow
until they cover the culture
morphology (shape
dish surface. Normal human
and structure), cell fibroblasts can usually be
cycle phase, DNA cultured for 50 to 100
content, and the population doublings, after
existence or which they stop growing and
die. In contrast, cells derived
absence of specific
from tumors frequently
proteins on the cell proliferate indefinitely in
surface or in the culture and are referred to as
cytoplasm. immortal cell lines.

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Selective
MICROSURGERY IRRADIATION
irradiation of
 Cultures provide cells free of CT and spread out small areas of
on a glass surface, so that they are accessible to living cells using
microsurgical procedures. microbeams of
 Extremely minute instruments as microneedles, protons, UV
microhooks, and micropipettes are devised. beams, & high
 These are positioned within an operating chamber power organ
on the stage of the compound microscope by lasers produce
mechanical micromanipulators capable of discrete lesions
achieving controlled movements in various planes. in chromosomes
 Transplantation & explantation techniques used in or other cell
grafting experiments as well as embryo transfer components to
utilize this method. assess its effect
..\..\videos\methods\Somatic Cell Nuclear Transfer upon the cell as
(Schematic) _ HHMI BioInteractive.mp4 a whole.

Choose at least 3 methods

from the list. Summarize a
research article for each
method detailing its use in the
investigation. (1 short-sized
page per method, Arial/Calibri
11, 1.5 spacing, 2015 and
above, APA format for
references)

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