Post on 04-Jun-2018
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Visualizing Cells
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Learning Objectives
History of Microscopy (Hooke, Zeiss, Perkin)Anatomy of Compound Microscope Capabilities & Limitations of Various Technologies Fluorescence Microscopy Laser Scanning Microscopy Fluorescent Proteins
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Early Cell Biologists
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Robert Hooke (1635 - 1703)
Built one of the first useful compound microscopesObserved structure of cork
Coined the term Cell.
Published Micrographia(1665)
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1665 Hooke publishes Micrographia
1678 van Leeuwenhoek observes protozoa (little animals)
1838-9 Schleiden & Schwann proposed Cell Theory
1857 Carl Zeiss produces the Stand 1microscope
1865 Perkins invents synthetic (aniline) dyes
A Brief History of Optical Microscopy
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Stand 1MicroscopeCarl Zeiss
(1816 1888)
Microscopy for the Rest of Us
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stem section mouse fibroblast
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1665 Hooke publishes Micrographia
1678 van Leeuwenhoek observes protozoa (little animals)
1838-9 Schleiden & Schwann proposed Cell Theory
1857 Carl Zeiss produces the Stand 1microscope
1865 Perkins invents synthetic (aniline) dyes
A Brief History of Optical Microscopy
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William Henry Perkin(1838 1907)
August Wilhelm von Hoffman(1818 1892)
Pioneers of Organic Synthesis
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Quinine
quina-quinaChichona calisaya
Early Anti-malarial Drugs
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3-amino-2,9-dimethyl-5-phenyl-7-(p-tolylamino)phenazinium acetate
Mauveine (Perkins Mauve)
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(spiny dye-murex snail)Justinian I(482 - 565)
Bolinus brandaris
Imperial (Tyrian) Purple
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Figure 1-5 Essential Cell Biology ( Garland Science 2010)
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Walther Flemming(1843 1905)
Zellsubstanz, Kern
und Zelltheilung, 1882
Discovery of Chromosomes (Colored Bodies) & Mitosis
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1931 Ruska invents electron microscope
1932 Zerniki develops phase contrast microscopy
1955 Minsky invents the laser scanning microscope (LSM)
1989 Webb, Denk & Strickler invent multiphoton LSM
1995 Stefan Hell invents Super Resolution Microscopy
A Brief History of Optical Microscopy
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Compound Microscope *
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Detection & Analysis
Magnification
Light Gathering
Illumination
*
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Olympus IX71
Inverted MicroscopeOlympus BX51
Upright Microscope
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Issac Newton
Albert Einstein Richard FeynmanJames C. Maxwell
George B. AiryAlhazen
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James C. Maxwell
Electromagnetic Theory of Light Propagation
*
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Wave-like Properties of Light
refraction diffraction
*
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Refraction at the Air - Water Interface
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Optical Convergence using a Thin Lens
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n1sin!= n2sin"
n2 sin!
=
n1 sin"
For n2> n1
nair = 1.0
n2 =sin!
sin"
water
air
nwater = 1.33
nglass = 1.51
Snells Law *
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Ripple Tank
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Diffraction is a characteristic of wave dynamics
Slit ~ wavelength ($)
barrier
slitba
rrier
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Slit ~ 4 $
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Interference
Positive
Interference
Negative
Interference
*
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*
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*
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Phase Contrast Microscopy
*
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Bright Field Phase Contrast
Differential Interference
Contrast (DIC)
Nomarski
Dark Field
Images of a Fibroblast *
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Objective Lens capturesonly a small portion of
the light rays that are
diffracted and refracted
by the specimen.
The optical imageis
always incomplete
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(0.61)$
n sin ( )
$= wavelength of lightn = refractive index
= angular aperture
Limit of resolution (D)
D =
Limit of Spatial Resolution of Optical Microscopy
NOT A FUNCTION OF MAGNIFYING POWER OF LENS
*
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(0.61) $n sin ( )
Limit of Spatial Resolution of Optical Microscopy
Limit of resolution (D)
D =
Numerical Aperture
(light gathering ability)
N.A. ~ 0.3 - 1.65
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(0.61) $n sin ( )
Limit of Spatial Resolution of Optical Microscopy
Limit of resolution (D)
D =
Numerical Aperture
(light gathering ability)
N.A. ~ 0.3 - 1.65
Higher is Better
*
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Objective Lens
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(0.61)$
n sin ( )
Limit of Spatial Resolution of Optical Microscopy
Limit of resolution (D)
D =
Wavelength of Light
Shorter is Better
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Visible Spectrum
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D =(0.61) $
n sin (
)
(0.61) (450 nm)
(1.5) sin (70o)D =
~ 200 nm
~ 0.2 m (~ /2)glass & oil
70o
Resolution is ultimately limited by the
wavelength of the illuminating light
$
Limit of Resolution approaches ~ !of the wavelength
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Electron Microscopy
Array of Au atoms
Wavelength ($) of an(accelerated) electron
= 0.004 nm
Resolution = $/ 2~ 0.002 nm
This is 100,000 times
better than optical
microscopy ~ 200 nm
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glass
glass
glass
retina
visible light
magnet
magnet
magnet
phosphor
electrons
Light Microscopy TEM
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Figure 9-54 Molecular Biology of the Cell( Garland Science 2008)
Actin Filaments
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Scanning electron microscopy (SEM)
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Figure 9-52 Molecular Biology of the Cell( Garland Science 2008)
*
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SEM of a metal cast
of a wheat flower.
Its not so muchabout size --
Its about resolution
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SEM
DIC
TEM
Stereocillia from bullfrog auditory hair cell
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Fluorescence Microscopy
mouse fibroblast mouse fibroblast
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Stuff Fluoresces
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Fluorescence Spectrum of FITC
blue red
absorbanceemission
{
Stokes Shift
*
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Fluorescence Spectrum of FITC
fluorescein isothiocyanate(FITC) blue red
absorbanceemission
{
Stokes Shift
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% %
Sigma (% vs Pi (&) Bonding
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Filter CubeEnables Fluorescence Microscopy
Filter Cube
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Filter CubeEnables Fluorescence Microscopy
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Filter
Cube
*
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Figure 9-18 Molecular Biology of the Cell( Garland Science 2008)
Indirect Immunofluorescence *
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All organic fluors undergo photobleaching
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Quantum Dots
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Quantum Dots
Highly resistant to photobleachingAll are excited by UV light
Emitted color is a function of size
*
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slit
Conventional Fluorescence Microscopy
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Conventional Fluorescence Microscopy
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Epifluorescence Image Confocal Image
All emitted light
under the objective
lens
Only light emitted
from within the focal
plane
More photons Better photons
*
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Confocal Imaging by laser scanning microscopy (LSM)
Confocal Microscope
Light
Source
Object plane
Objective Lens
Image plane
Tubus Lens
Light
Source
Object plane
Objective Lens
Image plane
Tubus Lens
Pinhole GalvanometerxScannerGalvanometer
yScanner
Laser
Objective
Lens
Object
Plane
Mirror
y
x
Galvanometer
xScannerGalvanometer
yScanner
Laser
Objective
Lens
Object
Plane
Mirror
y
x
Line Scan
laser
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y
z
x
z-Stack
50 mReconstructed
Neuron
Confocal Imaging
Confocal Microscope
Light
Source
Object plane
Objective Lens
Image plane
Tubus Lens
Light
Source
Object plane
Objective Lens
Image plane
Tubus Lens
Pinhole
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3D Reconstruction from Consecutive Optical Sections
Metaphase Spindle Complex
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Aequorea victoria
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Green Fluorescent Protein
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Green Fluorescent Protein
GFP Fluorophor
- Ser65 - Tyr66 - Gly67 -
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GFP transcriptional reporter
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GFP transcriptional reporter
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GFP variants produced by artificial selection in E coli.
Roger Tsien Lab
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Tsien, Shimomura, Chalfie2008 Nobel Prize in Chemistry
Roger Tsien
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Deconvolution Microscopy
Point Spread Function for 2 Fluorescent Dyes
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Deconvolution Microscopy
Acquired Image Deconvolved Image