Visualizing Cells
Molecular Biology of the Cell
Resolution, Detection Magnification
Interaction of Light with matter:
Absorbtion, Refraction, Reflection, Fluorescence
◼ Light Microscopy
Absorbtion of Light (dyes, stains)
Refraction methods
Fluorescence Microscopy
◼ Green Fluorescent Protein
◼ Confocal Microscopy
◼ Total internal reflection fluorescence
Electron microscopy
Staining methods
Scanning Electron Microscopy
Size
Typical cell is 20
> 2 meters (2x100 micrometers (um) = 20x10-
meters) 6 meters
1.5 centimeter (1.5x10-2
meters) Large organelles, 2
micrometers (um) = 2x10-6
meters
Macromolecular
complexes, 0.2
Average human hair (diameter) .1 micrometers (um) = 2x10-7
mm = 100 micrometers (um) = meters
100x10-6 meters
Figure 9-1
Resolution vs Magnification
Resolution
Resolution is determined by the wavelength of
light and the numerical aperture of the objective
lens. Resolution IMPROVES if D gets SMALLER
Resolution is directly proportional to the lens
numerical aperture
Resolution is inversely proportional to the
wavelength used for imaging
Visible light wavelength are between about 360
and 780 nm
The resolution limit of the light microscope (using
near UV illumination) is about .2µm (200 nm). (A
red blood cell is 7µm across)
Long Wavelength Light
Long wavelength light
is less damaging and
less easily scattered
than short wavelength
light and can
illuminate deep
structures
Cy5 dyes emit near-
infrared light
Resolution vs Detection
Objects smaller than the resolution limit of a
microscope can be detected
Microtubules24 nm diameter
(DIC image)
Objects smaller than the resolution
limit can be detected but are not
resolved
What is magnification good for?
Imaging devices have discrete detector elements
Rods and Cones in Eye
Pixels in Digital Camera
Magnification
The minimal adequate magnification is one that
allows the smallest object you want to resolve to fall
on 3 discrete elements of the imaging device.
Properties of Interaction
Four properties of the interaction between light and
matter influence the design of microscopes used to
produce contrast.
Absorption methods: Stained tissue
sections
Stains are compounds that absorb light or electrons.
Black stain absorbs all colors of light
Colored stains absorb some colors of light, others to pass
Kidney Collecting Adipose Tissue Purkinje neuron
Duct Fig 9-11
Different components of a cell can be
selectively stained
Chemical compounds of some stains bind
preferentially to specific cellular components
Hematoxylin (blue) stains nucleic
acids
Hematoxylin & eosin (proteins stain Periodic acid Shiff’s stains
Osmium stains lipids in neuron sheath pink) carbohydrates
Antibodies are used to detect specific
cell components
Antibodies can be linked to enzymes
that produce colored products
Craniofacial neurons in E10.5 mouse Delta I gene expression in developing
(horseradish peroxidase labeling), Sahay et somites (beta-gal staining)
al., J. Neurosci 2003. 23:6671-80
Refraction Techniques in Light
Microscopy
Very little incident light is absorbed, reflected or
refracted by a living biological specimen
Brightfield Imaging: refracted light from the specimen is poorly detected
Methods for imaging refracted light I.
Darkfield Imaging
Living Cells
Living cells are seen clearly using phase contrast microscopy to
image the difference between refracted and non-refracted
light.
Phase Contrast
Imaging
Features of living cells can be seen clearly using phase contrast
microscopy.
Shelden
Fluorescence microscopy:
Fluorescent molecules absorb high energy light and then emit less energetic,
longer wavelength light. The shift in wavelength between absorbed and
emitted light is called the Stokes shift.
maxima
Excitation Emission spectrum
spectrum
Rhodamine anti-MAP in cultured neurons
Fluorescence Microscopes
Fluorescence microscopes use excitation, emission and dichroic
filters to take advantage of the Stokes shift
Fig 9-11
Fluorescent Stains
Chemical compounds of some fluorescent stains bind
preferentially to specific cellular components
Fluorescent
phalloidin (red)
stains actin
DAPI (blue)
stains nucleic
acids
Shelden
Fluorophores
Antibody linked fluorophores detect specific cellular
antigens
Anti-tubulin (green)
Anti-neurofilaments
(green)
FISH
Fluorescence in situ hybridization (FISH) uses synthetic fluorescent RNA
probes to detect compatible mRNA in cells and tissue
Fluorescent RNA
mRNA probe
Figure 9-12
Light emitting dyes reveal changes in ion concentrations
Figure 9.32
Calcium changes during fertilization
visualized with aequorin
Fluorescent Protiens
Green fluorescent proteins can be expressed in
living organisms
Aequorea victoria
GFP
Gene Promoters
Cell-type specific gene promoters can be used to express GFP
in specific cells or tissues
Neuron specific
promoter
GFP Coding
Sequence
Figure 9-25 (transgenic fruit fly larva)
Fluorescent Fusion Protiens
Green fluorescent fusion proteins can be used to label proteins
in living cells
Recombinant DNA
Relocation of GFP-tagged proteins in
muscle cells
Fluorescent Probes
Dynamic studies of fluorescent probes can be conducted using
Fluorescence Recovery After Photobleaching (FRAP)
Fig 9-31
Fluorescent Probes
Dynamic studies of fluorescent probes can be
conducted using photo-activatable probes
Fig 9-30
Electron Microscope
The electron microscope uses electrons to resolve fine structure of the cell
The wavelength of an electron can be .004 nanometers, so the theoretical
limit of resolution of an electron microscope is 1/20 angstoms, or 1/20 the
diameter of a hydrogen atom.
Electrons pass
through the
specimen in TEM
Fig 9-42
EM Stains
Stains used for electron microscopy (EM) are very dense (metals) so they absorb electrons
EM stains are generally soluble, reactive forms of metals such as lead, uranium, gold, silver
and tungsten
Water
Osmium
tetroxide
Uranyl
acetate
Shelden
Gaseous metals can be
directionally applied (shadowing)
Sputter coatter
Fig 9-52
Metal Shadowing
Metal shadowing can be applies to surface structures or
interior structures using freeze fracture and freeze etching
methods
Cryoelectron microscopy of freeze-
Cryoelectron microscopy of freeze-
fractured intestinal microvilli
etched skeletal muscle fibers
Specimens are frozen, then split using a microtome and exposed surfaces prepared and imaged.
EM Negative Staining
EM Negative staining allows isolated macromolecules
to be seen Actin filaments
Macromolecules on
substrate
Stain in
excess
Fig 9-54
Bacteriophage virus
Gene Meyer
University of S. Carolina
Excess stain removed and
dried
Shelden
Computational Methods
Computational methods can produce three
dimensional structural inormation from multiple
views of an object
Figure 9-55 reconstruction of the Hepatitis B virus
Antibodies
Antibodies can be attached to metal particles to detect
specific cellular components
Immuno-gold labeling of microtubules in an Immuno-gold labeling of centromere proteins.
interphase cells Fig 9-46
Pulse Chase Radio-Labeling
Dynamic studies of molecules can be conduced at the EM level
by pulse chase radio-labeling.
Fig 9-38
Reflection techniques in microscopy:
Scanning EM:
Fig 9-49 Fig 9-50