Chapter 4 - microscopy
BME 310
Lecture 20
Types of microscopy
1. Optical (Huygens, Galileo, Hooke,
Leeuwenhoek - early 17th cent.)
2. Fluorescence ( Stokes - 1852)
3. Confocal (Minsky - 1955)
4. Electron (Ruska - 1931, 1986 Nobel)
5. Scanning probe (Binnig & Rohrer 1981, 1986 Nobel)
1.1 Optical
•
•
•
•
Eyepiece
Objective lenses
Stage
Fine and coarse
adjustment knobs - move
stage up and down
www.leica-microsystems.com
1.2 Optical
• Close-up of objectives bulk of magnification
• Condenser - series of
lenses to illuminate
sample with parallel
beams of light to reduce
glare and ensure even
lighting
www.leica-microsystems.com
1.3 Optical
• Sample must transmit light
– Or reflect light in stereo optical microscope
• Staining to add contrast as most cells
are transparent (e.g., bacteria and
Gram stain)
• Resolution determined by light (0.3 m)
– Eye resolution <100 m
• Maximum magnification = 1000X
2.1 Fluorescence
• Adjunct to optical microscopy
• Sample must be thin
• Sample is stained with
fluorescent dye or marker
(fluorophor)
• Marker selectively attaches
to specific cell structures
• When light shines on marker,
it emits light
Triple fluorescence staining
of endothelium cells from a
pulmonary artery.
www.nobelprize.org
2.2 Fluorescence
1’
Energy
• Dye or marker
initially in ground
state, E0
• Photon hits marker,
sends it to excited
state, E1’
• Marker relaxes to E1
• Marker returns to E0
emitting photon
• E1 < E1’ thus
 emitted >  excited
0
1
2.3 Fluorescence
• Emitted light is low intensity
• Excitation light is at lower
wavelength and can be
filtered
• Multiple dyes may be used
on same sample
www.microscopyu.com
Triple fluorescence staining
of division of a Chinese
hamster ovary cell.
www.nobelprize.org
3.1 Confocal
• Laser shines through pinhole
• Light focused by objectives to a
point in sample
pinhole PMT
• Fluorescence from sample
apertures
passes filter
filter mirror
• Only light from focal point
Laser
passes pinhole aperture to
microscope
PMT
objectives
• Laser scanned in XYZ, process
Sample
repeats to build image
3.2 Confocal
• Image thick samples (laser may be focused
at depth)
• Combine with fluoroscopy
• Eliminate fluorescent glare from out-offocus sample
• Better resolution then optical
• Sloooooow (scanning, not widefield)
• Computer reconstruction of image
3.3 Confocal
www.leica-microsystems.com
3.4 Confocal
Human osteosarcoma (bone cancer) - nuclei are red, 60X
zoom, illuminated with laser light at 488 nm, 543nm, 633 nm
www.olympusconfocal.com
3.5 Confocal
Drosophila (fruit fly) brain - 3D rendering from multiple
optical sections, mushroom bodies (cerebral cortex of
insects) stained green
www.olympusconfocal.com
4.1 Electron
• Transmission electron microscopy
– Similar to optical microscope
– Electrons have much shorter  than light
– Resolution <1 nm (atomic scale),
magnification >100000X
– Sample under vacuum (electrons hit air)
– Source is tungsten wire cathode
– “Lenses” are magnetic coils
– Image created on phosphor screen
4.2 Electron
• Transmission electron microscopy
– No live samples
– Samples are fixed, washed, dehydrated,
infiltrated with resin, cured, and sectioned
– Thicker or denser parts of sample block
more electrons -> dark image
– Thinner or less dense parts of sample pass
more electrons -> light image
4.3 Electron
bacteriophage lambda
www.biochem.wisc.edu
4.4 Electron
• Scanning electron microscopy
–
–
–
–
–
Similar to confocal microscopy (1 point at a time)
Electrons focused to point (2 nm)
Samples made conductive w/ metal coating
Incident electrons displace secondary electrons
Secondary electrons detected by scintillator and
PMT
– More secondary electrons when beam more
parallel to surface
– More secondary electrons -> brighter
– Thus images have 3D appearance
4.4 Electron
Isolated mitochondria from cultured bovine lymphocytes. These mitochondria are either in the late stage of dividing,
or an early stage of fusing. White dots are 18 nm gold particles conjugated to an antibody.
www.ansci.wisc.edu
5.1 Scanning probe
• Many types in 2 main categories
– Scanning tunneling microscope
– Atomic force microscope
• Probe scans lines across sample surface
• Probe is displaced up/down as it is scanned
• Surface topography reconstructed from
many scanlines by computer
5.2 Scanning probe
• Sample moved nanometers at a
time by piezoelectric positioners
• Resolution ~1 pm possible
mrsec.wisc.edu
y7
scanlines
y6
y5
height (z) ->
brightness
y4
y3
y2
y1
z
y
x
x
5.3 Scanning probe
• Scanning tunneling microscope
– Sample must conduct
– Voltage between tip and sample
creates tunneling current (quantum)
– Current magnitude related to
distance between tip and surface
– Two methods • Keep current constant by moving tip
up/down
• Keep tip steady, measure changing
current
– Mainly used in materials research
– Tissue adhesion to biomaterials in
part dependent on nanoscale
topography
Pure silicon cut at 4 degree angle showing
individual silicon atoms in layers
mrgcvd.engr.wisc.edu
5.4 Scanning probe
• Atomic force microscopy
– Probe tip displaced
up/down physically
– Deflection measured by
movement of light reflected
off cantilever
– At initial position, equal
photons on photodetector
quadrants, no differential
current
– When light deflects, more
photons on one quadrant,
measurable differential
current
piezoelectric positioners
mrsec.wisc.edu
5.5 Scanning probe
• Atomic force microscopy in biology
– Can image living cells in solution
– Drag tip across cell to measure
friction/elasticity
– Move cell to measure adhesion to surface
– Try to pull ligand from receptor on cell surface
– Measure volume change of cell
Combined imaging
Phase contrast optical
Fluorescence
Atomic force height
Atomic force deflection (derivative of height)
Living fibroblast cell - image size is 80 x 60 m
www.jpk.com
Sources
• Bioinstrumentation, John Webster, Ed.
• www.microscopyu.com
(Nikon edu. site, excellent resource)
• www.olympusconfocal.com
• www.leica-microsystems.com
• www.nobelprize.org
• www.wisc.edu
• www.jpk.com