Light microscopes

To understand light microscopy, you need to

understand three concepts:
Light is a wave
Refraction
Diffraction
Three concepts
Light is a wave
Ray of light
wavelength ()
Refraction is the change in direction of a wave due
to a change in velocity at the interface between two
transmission media.
Refraction
Refraction
http://www.youtube.com/watch?v=ybn-qr-Pvnw
Magnification
Magnification is accomplished by refraction.
Diffraction refers to various phenomena that occur
when a wave encounters an obstacle. The diffraction
phenomenon is described as the apparent bending of
waves around small obstacles and the spreading out
of waves past small openings.
Diffraction
Diffraction
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When the aperture is comparable or smaller than the
wavelength, the distortion of the wave is large. This
limits the ability to resolve nearby objects.
Diffraction
d ~
(d is the minimum distance that can be resolved)
( is the wavelength)
(~ means roughly)
Resolution
d ~
d ~ 100 nm 1000 nm
~ 100 nm 1000 nm
Resolution
d = 0.5/NA
(NA is the numerical aperture of the objective
lens)
(NA is a measure of the objective lens ability
to gather light and depends on material and
shape of the lens)
(requires more physics to derive)
Resolution
Parts of the microscope
Parts of the microscope
Fixation and staining of
specimen

1. Spread culture on slide
2. Air dry
3. Fix with heat (attach cells to
slide)
4. Apply stain, rinse, dry
5. Microscopy

Simple staining - use of a single
dye
Differential staining - use of >1
dye to differentiate between
different types of cells
1)
2)
3)
4)
5)
Staining to improve contrast
Staining to improve contrast
Time-lapse microscopy
Take many photographs and concatenate the
still images into a movie
Time-lapse microscopy

Used to visualize specimens
that fluoresce
Emit light of one color when
illuminated with another color of
light
Can be used with live cells
Some cells fluoresce naturally
(autofluorescence)
Stain cells with a fluorescent
dye
Highlight particular cell
structures
Express a fluorescent protein

FM4-64
(membrane)
DAPI
(nucleic acid)
YFP
(cytoplasm)
overlay
Fluorescence microscopy
Time-lapse fluorescent microscopy
http://www.youtube.com/watch?v=rBYYpPisjEs
Electron microscopes
Electron microscopy (EM)
Involves the use of:
electromagnets instead of glass
lenses
electrons instead of light

Electron microscopy (EM)
Involves the use of:
electromagnets instead of glass
lenses
electrons instead of light

Provides much higher resolution than a
light microscope
Light microscope scope ~ 200 nm (0.2
m)
EM ~ 0.5 nm

Resolution (d) = 0.5l/NA
visible light: l ~ 450 nm
electrons: l ~ 0.005 nm
Electron microscopy (EM)
Involves the use of:
electromagnets instead of glass
lenses
electrons instead of light

Provides much higher resolution than a
light microscope
Light microscope scope ~ 200 nm (0.2
m)
EM ~ 0.5 nm

Electrons behave as waves with very
short wavelengths hence the
difference in resolution between LM's
and EM's

Electron microscopy (EM)
Involves the use of:
electromagnets instead of glass
lenses
electrons instead of light

Provides much higher resolution than a
light microscope
Light microscope scope ~ 200 nm (0.2
m)
EM ~ 0.5 nm

Electrons behave as waves with very
short wavelengths, hence the
difference in resolution between LM's
and EM's

Two main types of EM's
Transmission EM - reveals great detail
of internal structure of cells; 2D image
Scanning EM - reveals great detail of
external structure of cells; 3D image
Figure 2.9
The history of the microscope