BCM 367
Cell Structure and Function
Theme A
CULTURING AND VISUALIZING CELLS
CHAPTER 4
MOLECULAR CELL BIOLOGY
Lodish  Berk  Kaiser  Krieger  Bretscher  Ploegh  Amon  Martin
Copyright © McMillan Education
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CULTURING AND
VISUALIZING CELLS
What do we know about cells,
And why do we know it?
Lodish et al. Molecular Cell Biology
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS
Culturing and Visualizing Cells
4.1 Growing and Studying Cells in Culture
4.2 Light Microscopy: Exploring Cell Structure and
Visualizing Proteins Within Cells
4.3 Electron Microscopy: High-Resolution Imaging
4.4 Isolation of Cell Organelles
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS
History
1665 Robert Hooke – dead cells
1674 Antonie van Leeuwenhook – living cells
1838 Matthias Schleiden – All plants are made of cells
1839 Theodore Schwann – All animals are made of cells
1858 Rudolf Virchow – All cells came
from pre-existing cells
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS - GROWING CELLS IN CULTURE
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Culturing and Visualizing Cells
4.1 Growing and Studying Cells in Culture
• Animal cells grow in culture when supplied with necessary
nutrients and appropriate 2D or 3D substrates.
• Primary cells have a finite life span; transformed tumor cells and
cell line cells can grow indefinitely.
• A fluorescence-activated cell sorter (FACS) sorts labeled cells.
• Hybridoma cells produce monoclonal antibodies that bind one
antigen epitope and are used for basic research and therapeutics.
• Basic biological processes can be studied by using genetic
manipulation or drugs to interfere with specific cell components.
• Large chemical libraries can be screened for compounds that target
specific processes.
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS - GROWING CELLS IN CULTURE
Growing Cells in Culture
Cell culture conditions
Unicellular – fairly easy to grow (bacteria, fungi, protist)
Multicellular organisms – more complex (ex. animal cells)
Why do we grow cells and
what do they need to grow?
Animal Cells: Nutrient-Rich Media and Solid Surfaces
o Mimic conditions in organisms tissues – pH, temperature, ionic strength, nutrients, etc.
First needs: specially coated plastic dishes to adhere to and Liquid media (antibiotics added
to prevent contamination)
Liquid media:
o 9 amino acids vertebrate animal cells are not capable to synthesise (phenylalanine,
valine, threonine, tryptophan, isoleucine, methionine, leucine, lysine, histadine)
o 3 amino acids only synthesised by specialised animal cells (cysteine, tyrosine, arginine)
o Nitrogen source in the form of glutamine
o Vitamins, salts, fatty acids, glucose
o Serum – protein factors essential for proliferation of mammalian cells, insulin, transferrin
(bio-accessible form of iron), growth factors
o Growth factors specific for certain cell types
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS - GROWING CELLS IN CULTURE
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Growing Cells in Culture
Primary vs. transformed cell cultures
Primary cell cultures formed from tissues like skin kidney liver etc.
o Cell-cell and cell-matrix interaction must be disrupted to form these primary cultures –
protease (eg. Trypsin,collagenase) and a divalent cation chelator (removes free Ca2+ eg.
EDTA) treatments assist in this
o Released cells moved to dishes with nutrient broth and serum for adherance to take
place
o Most cultured cells divide a set amount of time and then performs cell senescence and
ceases to grow – the big (–)’ve
Oncogenic mutations
Figure 9.1 Stages in the establishment of a cell culture.
o Embryonic stem cell – can be cultured indefinitely
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS - GROWING CELLS IN CULTURE
Growing Cells in Culture
Primary vs. transformed cell cultures
Research purposes require cells that double more than 50 times
o Indefinite growth by oncogenically TRANSFORMED cells due to mutations – give rise to
immortal cell lines
Often spontaneous in rodents not so in humans
For humans malignant tumor can be the source of such cell lines
- HeLa cell line 1952 – cervical cancer
Cultured HeLa cells with a
fluorescent protein targeted to the
Scanning electron micrograph of an
Golgi (orange), microtubules (green)
apoptotic HeLa cell
and DNA (cyan)
National Institutes of Health (NIH)
National Institutes of Health (NIH)
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS - GROWING CELLS IN CULTURE
We have cells
We know how to grow them
What now?
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Isolation – Flow Cytometry and Cell Sorting
How do we separate cells?
o Density – red blood cells vs. white blood cells – equilibrium density centrifugation
o Often its not that easy – FLOW CYTOMETRY AND CELL
SORTING
o Cell surface molecules used to mark cells (fluorescent dye
conjugated to antibody)
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS - GROWING CELLS IN CULTURE
Isolation – Flow Cytometry and Cell Sorting
How do we separate cells?
o Density – red blood cells vs. white blood cells – equilibrium density centrifugation
o Often its not that easy – FLOW CYTOMETRY AND CELL SORTING
o Cell surface molecules used to mark cells (fluorescent dye conjugated to antibody)
FLOW CYTOMETER: cells flow in liquid stream past a laser beam scattered light is measured and cells marked with specific
fluorescence is identified
Fluorescence-activated cell sorting (FACS)
o Ink jet technology combined with the old
Coulter cell counter
o Cells identified through flow cytometry
o Stream breaks into small drops each containing
one cell
o Charge laid onto drop and positive and
negatively charged drops are separated by
voltage plates
Experimental Figure 9.3 T cells bound to fluorescence-tagged antibodies to two cell-surface proteins
are separated from other white blood cells by FACS.
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS - GROWING CELLS IN CULTURE
Isolation – Flow Cytometry
Figure 9.2 Fluorescence-activated cell sorter (FACS) separates cells that are labeled differentially
with a fluorescent reagent.
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS - GROWING CELLS IN CULTURE
Isolation – Flow Cytometry
20 000 cells per second
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS - GROWING CELLS IN CULTURE
Two and Three Dimensional Growth Mimics In Vivo Environment
Most tissue types function with cells in relation to each other and in
close contact
Good example: Epithelial sheets found in epithelial tissue – covering for external
and internal surfaces of organs
Epithelial cells transport classes of molecules across the epithelial sheet
On plastic or glass – this function is limited
Special containers with porous surfaces (acting as basal lamina) have been
designed – gives rise to a two dimensional uniform sheet
2D vs
3D
Figure 9.4 Madin-Darby canine
kidney (MDCK) cells grown in
specialized containers provide a
useful experimental system for
studying epithelial cells.
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS - GROWING CELLS IN CULTURE
I have isolated a new drug
effective against a human cell
pathogen.
I want to test its effectiveness.
How important is cell culturing
to me?
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Complexity meets the specific research question
Natural media - Natural culture media is composed of biological
fluids that are naturally occurring.
Artificial media - Also referred to as synthetic media
Selective media - Only allows for certain cells to grow.
Differential media - Depending on metabolism.
Serum containing media
Serum-free media - avoid misinterpretation of immunological
results.
Chemically defined media - Contamination- free pure organic and
inorganic ingredients.
Protein-free media - Promote superior growth of the cells as well as
protein expression in addition to facilitating for the purification of
any expressed product.
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Light Microscopy - Overview
Magnification of a small object – light microscopy: 0.2 m resolution
Bright Field Light Microscopy:
o Total magnification = objective lens
lens (ocular or eye piece)
(closest to object) as well as the
projection
o Magnification vs. resolution:
The ability to distinguish between two very
closely positioned objects
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
Light Microscopy - Overview
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
Light Microscopy - Overview
Magnification of a small object – light microscopy: 0.2 m resolution
Bright Field Light Microscopy:
o Total magnification = objective lens (closest to object) as well as the projection lens (ocular
or eye piece)
Range of angles over which
the microscope can accept
light
Refractive index
Angular aperture
o Magnification vs. resolution: The ability to distinguish between two very closely positioned
objects
Resolution (D) = the minimum distance between two distinguishable objects
the smaller the resolution - the better the image (Ernst Abbe late 19th century)
D=
0.61
N sin 
(the numerical aperture; range from 1.4 for good to 1.7 for19
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
excellent)
Light Microscopy - Resolution
 = the angular aperture of the cone of the light entering the objective lens
N = refractive index of the medium between the objective lens and specimen (air = 1 and oil
or glass = 1.56
 = wavelength of incident light
How do I improve resolution?
o Using shorter wavelengths of light
o Gathering more light by increasing N or 
o Using oil which has a higher refractive index than air or water
Lenses can focus very close to the cover slip
Figure 9.8a Optical microscopes are commonly configured for bright-field (transmitted), phasecontrast, and epifluorescence microscopy.
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
Light Microscopy - Resolution
Mathematical exercise:
What is the resolution (D) of light microscope X if it has an angular
aperture of 60, refractive index of oil, and a wavelength of incident
light of 700nm
Resolution is in micrometers
Wavelength is in micrometers
D=
0.61
N sin 
D=
0.61 X 0.7
1.56 X sin 60
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
Phase Contrast and Differential-Interference-Contrast Microscopy
Visualising unstained living cells
Bright field optics is the most simple method of viewing
How light propagates
through a medium
Cellular materials have differences in their refractive indexes and thickness
Utilized by Phase-contract microscopy and Differential-interference-contrast (DIC) microscopy
Figure 9.9 Live cells can be visualized by microscopy techniques that generate contrast by interference.
The degree of darkness or brightness of a region of the sample depends on
the refractive index of that region –light moves slower in object with high
refractive index
DIC is based on interference between polarized light specially good for visualizing small
details and thick objects – contrast generated by differences in the refractive index of
the object and its surrounding medium
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
Phase Contrast and Differential-Interference-Contrast Microscopy
Light passing through specimen is refracted in
comparison with light just moving through - this
places it in a different phase
In phase brighter than out of phase
Allows specimen
to be illuminated
by defocused,
parallel light
wave fronts
Figure 9.8b-d Optical microscopes are commonly configured for bright-field (transmitted), phasecontrast, and epifluorescence microscopy.
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
Diatoms are singlecelled algae, live in
houses made of glass.
They are the only
organism on the
planet with cell walls
composed of
transparent, opaline
silica.
Phase contrast
vs DIC
1min 6:13min
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Fixing, Sectioning and Staining Cells
Live cells lack compound to absorb light – how can we visualize more detail?
Specimens are commonly fixed – treated with chemicals that link proteins and nucleic acids
o Formaldehyde cross-links amino groups on adjacent molecules stabilize protein-protein
and protein-nucleic acid interaction = molecules insoluble and stable
o Can be left in solution or cut into sections
Figure 9.10 Tissues for light microscopy
are commonly fixed, embedded in a
solid medium, and cut into thin sections.
Staining
o Various amount of stains for specific molecules in cells
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
Fluorescence
o Fluorescence occur when an electron is excited by light to a higher orbital
and falls back to the ground state, giving off light in the process
o Fluorochromes are dyes that accept light energy and re-emit light at longer
wavelength and at different color than the absorbed light
o Emitted light has longer wavelength AND less energy than absorbed light
EXCITATION (LASER)
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VS.
EMISSION (DETECTOR)
Fluorescence Microscopy Localizing and Quantifying Live Cell Molecules
When are chemicals fluorescent?
o It absorbs light at one wavelength
(excitation)
o It emits light at a specific longer
wavelength (emission)
o Laser = excitation
o Detection = emission
Fluorescent Microscopes:
o Excitation light is passed through
the objective into the sample,
then selective viewing of
emission through objective again
o Reflected excitation light is
deflected by dichroic mirror –
allowing longer wavelength to
pass through
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
495nm/519nm
Excitation/Emission
Laser/Detector
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7 examples of fluorescent microscopy
1. Ion Sensitive Fluorescent Dyes
2. Immunofluorescence and specific proteins in fixed cells
3. Fluorescent proteins tag specific proteins in living cells
4. Light Microscopy – 3D confocal and deconvolution
5. TIRF Microscopy
6. FRAP
7. FRET
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Example no. 1
Ion Sensitive Fluorescent Dyes
There are Fluorochromes whose fluorescence depends on the concentration of Ca2+ and H+
What else do we know about Ca2+ and H+? – Many cellular processes are affected by their
concentrations
fura-2 – five carboxylate groups – can form ester
linkages with ethanol = fura-2 ester
o Fura-2 ester is lipophilic – diffuses across plasma
membranes
o In cytosol: - esterases hydrolyse fura-2 ester = non-liphophilic fura-2 unable to cross
- one fura-2 binds one Ca2+ ion
fura-2 +Ca2+
340nm Excitation
back over membrane
510nm Emission
fura-2 +Ca2+ AND fura-2 380nm Excitation
510nm Emission
BOUND AND UNBOUND
fluorescence has TWO EXCITATION WAVELENGTHS
– this can determine the ratio of Ca2+ and small changes
that are occurring in the cell – increase of bound Ca2+
over time
Experimental Figure 9.11 Fura-2, a Ca2+-sensitive fluorochrome,
can be used to monitor the relative concentrations of cytosolic
Ca2+ in different regions of live cells.
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
Ion Sensitive Fluorescent Dyes
SNARF-1 – sensitive to H+ is used to monitor intracellular pH levels
Fluorochrome linked to a weak base
– partially protonated at pH7
- can freely permeate membranes
when entering acidic organelles – probes become protonated; cannot cross back
over membranes and accumulates
Used to stain lysosomes and mitochondria
Experimental Figure 9.12 Location of lysosomes and
mitochondria in a cultured living bovine pulmonary artery
endothelial cell.
WHAT TOOL ARE WE USING?
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
7 examples of fluorescent microscopy
1. Ion Sensitive Fluorescent Dyes
2. Immunofluorescence and specific proteins in
fixed cells
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Immunofluorescence and specific proteins in fixed cells
Example no. 2
In response to infection, vertebrate immune systems produces antibodies
Can identify and bind to specific infectious agents
In vivo production of antibodies, and isolation via affinity chromatography
-polyclonal – when animals blood is used
-monoclonal – when one specific cell line is produced to collect antibody
1. FIXING THE CELLS: for protein localization, the cell needs to be
‘frozen’ or fixed to keep all components in place
2. PERMEABILIZING THE CELLS: allow entry of antibody
(incubate cells in non-ionic detergent or extracting lipids through use of organic
solvent)
Figure 9.13 A specific protein can be localized in fixed tissue
sections by indirect immunofluorescence microscopy.
Indirect immunofluorescence microscopy
1. Cells treated with unlabelled antibody
2. Cells treated with second
fluorescence tagged antibody that
binds to first antibody
Unbound antibodies washed away
between steps
Covalent attachment of
fluorescent tag to antibody or
protein - conjugation 33
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
7 examples of fluorescent microscopy
1. Ion Sensitive Fluorescent Dyes
2. Immunofluorescence and specific proteins in fixed
cells
3. Fluorescent proteins tag specific
proteins in living cells
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Fluorescent proteins tag specific proteins in living cells
Fluorescent expression through green fluorescent protein (GFP)
Aequorea victoria
GFP: contains serine, tyrosine, and glycine with
spontaneously cyclizing side chains – forming a
green fluorescing chromophore
Example no. 3
Ssblakely
Recombinant DNA techniques produces – GFP coupled
proteins - Allows the localization of the fused, expressed protein in the cell
Figure 9.15 Many different
colors of fluorescent proteins
are now available.
Experimental Figure 9.14 Double-label
fluorescence microscopy can visualize
the relative distributions of two
proteins.
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
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7 examples of fluorescent microscopy
1. Ion Sensitive Fluorescent Dyes
2. Immunofluorescence and specific proteins in fixed cells
3. Fluorescent proteins tag specific proteins in living cells
4. Light Microscopy – 3D confocal and
deconvolution
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Light Microscopy – 3D confocal and deconvolution
Example no. 4
Experimental Figure 9.16 Deconvolution fluorescence
Conventional fluorescent microscopy
microscopy yields high-resolution optical sections that
Limited by:
can be reconstructed into one three-dimensional image.
1. Emitted fluorescent light comes from
(a)
(b)
more than one plane – blurred image
2. Thick specimen observation requires
consecutive images at various depths
2 Methods for High resolution 3D
information
- image data collected electronically for
both to allow computational editing
Deconvolution Microscopy
Computational methods to remove out of focus
planes
Achieved by creating a test slide with a series of images of fluorescent
beads
o Create a point spread function, to calculate the distribution of
fluorescent point sources that contributed to the ‘blur’
o The microscope calibration enables image series computational
deconvolution
Creates un-blurred images
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(a)
(b)
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3D confocal
Confocal microscopy – optical methods to obtain images from specific planes. Excluding light from other
planes
Collect a series of images through the vertical depth of the sample
TWO TYPES: point scanning and spinning disk
HOW DOES IT WORK?
Illuminate and collect emitted fluorescent light in just one small area of the
focal plane at a time – this excludes the out-of-focus light
A pinhole is used to collect the light before it reaches the detector
The whole object is scanned and the image is built up electronically
HOW DO THE TWO METHODS COVER THE IMAGE?
Figure 9.17 Light paths for two types of confocal microscopy.
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
3D confocal
Point scanning:
o A point laser light is
used, with the correct
excitation wavelength, to
scan the focal plane
Excitation/
/Emission
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
3D confocal
Point scanning:
o A point laser light is
used, with the correct
excitation wavelength, to
scan the focal plane
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
3D confocal
Point scanning:
o A point laser light is used, with the
correct excitation wavelength, to scan the
focal plane
o Emitted fluorescence is collected by a
photomultiplier tube
o Image is built up electronically
oTo create a 3D image - a series of images
at different depths are collected
+ = High resolution
- = 1. to scan each focal plane
can be time consuming
2. intense laser light can bleach
the fluorochrome
Dynamic images might not be
captured - Limited time to
expose to laser
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
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3D confocal
Spinning disk:
Tries to solve point
scanning problems – by
using a spread out laser
beam
o Two fast spinning disks
containing 20 000
microlenses and 20 000
pinholes respectively;
precisely aligned
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3D confocal
Spinning disk:
Tries to solve point scanning problems –
by using a spread out laser beam
o Two fast spinning disks containing 20
000 microlenses and 20 000 pinholes
respectively; precisely aligned
o With each spin the focal
plane is scanned several
times
o Light returning passes
through pinholes again
into digital camera
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
3D confocal
Spinning disk:
Tries to solve point scanning problems –
by using a spread out laser beam
o Two fast spinning disks containing 20
000 microlenses and 20 000 pinholes
respectively; precisely aligned
o With each spin the focal plane is
scanned several times
o Light returning passes through
pinholes again into digital camera
+ = Very fast
- = usually only configured
for 63X or 100X
magnification (tissue
samples need lower
magnifications)
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
Excitation/
/Emission
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3D confocal
Figure 9.18 Confocal microscopy produces an in-focus optical section through thick cells.
Experimental Figure 9.19 The dynamics of microtubules can be imaged on the spinning disk confocal
microscope.
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
7 examples of fluorescent microscopy
1. Ion Sensitive Fluorescent Dyes
2. Immunofluorescence and specific proteins in fixed cells
3. Fluorescent proteins tag specific proteins in living cells
4. Light Microscopy – 3D confocal and deconvolution
5. TIRF Microscopy
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TIRF Microscopy
Example no. 5
Experimental Figure 9.20 Fluorescent samples in a restricted focal plane can be imaged by total internal
reflection (TIRF) microscopy.
Confocal microscopy not ideal to see interaction
in thin focal plane close to cover slip (like
adhesion)
Total Internal Reflective Fluorescence (TIRF)
Microscopy
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BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
Example no. 5
TIRF Microscopy
Experimental Figure 9.20 Fluorescent samples in a restricted focal plane can be imaged by total internal
Confocal microscopy not ideal to see interaction in thin focal plane close
to cover slip (like adhesion)
Total Internal Reflective Fluorescence (TIRF) Microscopy
reflection (TIRF) microscopy.
oExcitation comes through the objective lens
oAdjust the angle at which the cover slip is
reached precisely at the correct angle to reflect
it back up the objective
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
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TIRF Microscopy
Example no. 5
Experimental Figure 9.20 Fluorescent samples in a restricted focal plane can be imaged by total internal
Confocal microscopy not ideal to see interaction in thin focal plane close to cover slip (like adhesion)
reflection (TIRF) microscopy.
Total Internal Reflective Fluorescence (TIRF) Microscopy
oExcitation comes through the objective lens
oAdjust the angle at which the cover slip is reached precisely at the
correct angle to reflect it back up the objective
oEvanescent wave created - narrow band that only
illuminate the 50 to 100 nm next to the cover slip
Excellent observations on the structures of microtubules and
actin filaments
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
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7 examples of fluorescent microscopy
1. Ion Sensitive Fluorescent Dyes
2. Immunofluorescence and specific proteins in fixed cells
3. Fluorescent proteins tag specific proteins in living cells
4. Light Microscopy – 3D confocal and deconvolution
5. TIRF Microscopy
6. FRAP
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Example no. 6
FRAP
How dynamic are certain cellular components
Ex GFP – does the green fluorescence represent a stable group of proteins, or proteins that are
dynamic and can move around?
Fluorescent recovery after photo bleaching
Experimental Figure 9.21 Fluorescence recovery
o Use a high intensity light to bleach an
already emitting fluorochrome in one
specific area
o if dynamic the dark, bleached area will
have fluorescent component returning
o can be observed with fluorescent
microscope
o rate of recovery measures the
dynamics of molecules
after photobleaching (FRAP) reveals the dynamics of
molecules.
Lateral movements of Specific
plasma membrane proteins
and lipids can be quantified by
FRAP
MDougM
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
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7 examples of fluorescent microscopy
1. Ion Sensitive Fluorescent Dyes
2. Immunofluorescence and specific proteins in fixed cells
3. Fluorescent proteins tag specific proteins in living cells
4. Light Microscopy – 3D confocal and deconvolution
5. TIRF Microscopy
6. FRAP
7. FRET
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Example no. 7
FRET
Do certain proteins interact in vivo?
Förster resonance energy transfer – two fluorescent proteins where the emission
wavelength of the first is equal to the excitation wavelength of the second
German physical chemist
Cyan fluorescent protein and yellow fluorescent protein as example
Excitation/
/Emission
critical
Figure 9.22 Protein-protein interactions can
be visualized by FRET.
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Example no. 7
FRET
German physical chemist
Do certain proteins interact in vivo?
Förster resonance energy transfer – two fluorescent proteins where the emission
wavelength of the first is equal to the excitation wavelength of the second
Cyan fluorescent protein and yellow fluorescent protein as example
YFP needs to be close by for fluorescent excitation
to take place Energy transfer efficiency proportional to R6
R is the distance between the fluorophores
Very sensitive and not detectable if R is >10 nm
R
Figure 9.22 Protein-protein interactions can
be visualized by FRET.
BCM367 THEME A: CULTURING, VISUALIZING AND PERTURBING CELLS – LIGHT MICROSCOPY
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FRET-sensor application
Chameleon sensor
for measuring intracellular levels of Ca2+
One polypeptide with two sensors – CFP and YFP (joined by Ca2+ binding polypeptide)
No Ca2+ no FRET – CFP and YFP to far apart
Ca2+ binds – conformational change brings CFP and YFP close enough for
FRET to occur
10nm > R
Experimental Figure 9.23 FRET biosensors can detect
local biochemical environments.
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