Transcript Lecture 3 Techniques II

Microscopy
Resolution= Resolving Power
-the smallest distance (d) at which two
objects can be successfully distinguished.
Resolution (d): d = (0.61 x )/ NA
= wave length
NA= numerical aperture
NA= n sin 
Microscopy
Resolution= Resolving Power
-the smallest distance (d) at which two
objects can be successfully distinguished.
Resolution (d): d = (0.61 x )/ NA
= wave length
n = refractive index of medium
NA= numerical aperture = ½ of angular aperture
NA= n sin 
NA= n sin 
Refractive index (η) of different media
Air=1.0003
Water=1.33
Immersion Oil=1.515
Resolution versus Wavelength
Resolution: d= 0.61 x 
NA
Wavelength (nanometers)
360
400
450
500
550
600
650
700
Resolution (micrometers)
.19
.21
.24
.26
.29
.32
.34
.37
Resolving Distance
(d)
Human eye
Light Microscope
Scanning Electron Microscope
Transmission Electron Microscope
0.2 mm
0.2 um
2.5 nm
1.0 nm
Resolution: d= (0.61 x )/ NA
HistoTip: Avoid confusion when discussing
resolution. Increased resolution or resolving power
usually means a SMALLER value of d (distance).
PROBLEM:
Objective lens A:
Magnification = 40X
N.A. = 0.45
Objective lens B:
Magnification = 40X
N.A. = 0.80
-->Which objective lens would give the
sharper image and why?
PROBLEM:
You photograph some liquid crystalline DNA using
objective D and objective E. You then enlarge the
images to the same size using Photoshop in the
manner described below.
Image D : 20X objective, NA= 0.40, enlarged 10X
Image E : 4X objective, NA= 0.10, enlarged 50X
Which image would be sharper and why?
Empty Magnification: an image is enlarged, but no additional
detail is resolved.
A : 20X objective, NA= 0.40, enlarged 10X. Magnified 200
B : 4X objective, NA= 0.10, enlarged 50X. Magnified 200
HistoTip: Maximum useful magnification=1000 X N.A.
Empty Magnification: an image is enlarged, but no additional
detail is resolved.
A : 20X objective, NA= 0.40, enlarged 10X.
B : 4X objective, NA= 0.10, enlarged 50X.
HistoTip: Maximum useful magnification=1000 X N.A.
Image of specimen:
- made of points appearing as Airy patterns with center disk.
- result of light diffracted as it passes through specimen.
- size influenced by NA:
NAa<NAb<NAc
http://www.microscopy.fsu.edu/primer/anatomy/numaperture.html
Resolution determined by overlap of Airy disks.
http://www.microscopy.fsu.edu/primer/anatomy/numaperture.html
“Criterion for resolution: the central ring in the diffraction
pattern of one image should fall on the first dark interval
between the Airy disk of the other and its first diffraction
ring.”
Point sources
of light appear
as Airy
diffraction
patterns
(disks) in the
microscope.
http://www.microscopyu.com/articles/formulas/formulasresolution.html
Compound microscope
Optical Components
- Light source
- Diaphragm
- Condenser
- Lenses
- objectives
- oculars
Nikon E200
Condenser Aperture Setting and Image Quality
Contrast increases as less light passes through condenser
(a) 90% (b) 60 % (c) 20%
Microscopy: Phase contrast
- excellent for living tissue: unstained specimens with little
contrast
- utilizes differences in refractive index or thickness to
create contrast (manipulation of phase of light)
Living Cells in Brightfield
Living Cells in Phase Contrast
a.
b.
c.
d.
Organelles have different refractive indexes but appear invisible in bright field
microscopy.
Light is refracted & slowed by objects proportional to their refractive index & thickness.
A phase plate allows barely refracted light to pass undisturbed while highly refracted
light passes is slowed further (1/4 wavelength).
Organelles with higher refractive indices appear darker in phase contrast microscopy.
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/phase.html
Phase Contrast Microscopy: Phase plate and annulus
Alignment required
Objective
Condenser
http://microscopy.berkeley.edu/Resources/instruction/images/phase_contrast_img_0.jpg
Differential Interference Contrast (DIC)
(aka Nomarski) - shows phase differences in the specimen
in a relief-like fashion.
Phase contrast
DIC
Differential Interference Contrast (DIC)
DIC
Phase
Contrast
Epithelial cell
Kidney tubule
Stem
Fluorescence Microscopy
Fluorescent molecule = fluorochrome
- absorbs light of specific wavelength
- when excited by absorption, the
fluorochrome emits light of longer
wavelength
Every fluorochrome has an absorption
and emission spectra.
Fluorescence Microscopy
Fluorochrome
Structurally unstable when excited
Fluorescence Microscopy
EXCITATION SPECTRA
EMISSION SPECTRA
WAVELENGTH
Fluorochrome
DAPI
FITC
Rhodamine
Fluorescence Microscopy
SPECIMEN
Components:
Light source
Excitation filter
Emission filter
Dichroic mirror
-reflects short 
-passes long 
EYE PIECE
Frog Neuromuscular Junction
Texas RedAcetylated
TUBULIN
FITC
ACTIN
By Stephanie Moeckel-Cole
BrainBow
Labeled neurons
in brain with
different
combination of
fluorochromes.
Fluorchromes:
DiO, DiI, DiD
Gan WB, Grutzendler
J, Wong WT, Wong
RO, Lichtman JW.
FLUORESCENCE MICROSCOPY
PROBLEM: Photobleaching (fading)
Photobeaching: Fluorochrome loses ability
to fluoresce, absorb and emit light, due to
damage or covalent modification.
http://microscopyu.com/articles/fluorescence/fluorescenceintro.html
Fluorochrome
PHOTOBLEACHING
(a-f) Images collected at 2 minute intervals.
http://microscopyu.com/articles/fluorescence/fluorescenceintro.html
Quantum Dots : semiconductor nanoparticles, such as
cadmium selenide, that emitted light after light excitation.
Advantages: brighter, no photobleaching, broad excitation 
Disadvantages: potential toxicity for in vivo imaging
Alivisatos et al.; Quantum dots as cellular probes.; Annu
Rev Biomed Eng. 2005;7:55-76.