Microscopy 101

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Microscopy 101
Dr. Gisèle Giorgi Merritt College
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• Topics

• Properties of light
• Contrast DIC
• Airy disk, N.A. and resolution
• Choice of techniques
• Merritt Microscopy Program

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Traveling light
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Properties of light
transmitted
reflected
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Microscopy techniques
light source/objective
Transmitted brightfield phase DIC
Incident/epi-illumination fluorescence
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Refraction

• Refraction (bending of light) occurs when light
  passes from a medium with one n to a medium with
  another n.
• n refractive index
• Note
• denser materials slow light down more, so they
  have a higher n.
• n is wavelength (?) dependant
• there are no units for n

refraction
n Cvacuum/Cmedium Cspeed of light
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Refractive indexes

Cells are mostly water.
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Objects and transmitted light
light wave
amplitude object
seen as color
phase object
not seen
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Objects and transmitted light
amplitude object - pigmented or stained samples
- e.g. histology specimens - seen with
brightfield microscopy
phase object - most biological samples! - seen
with phase or DIC microscopy
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Objects and transmitted light
We cant measure phase directly. However, we
can measure phase differences. Several types
of transmitted light microscopes, including DIC,
can capture phase differences.
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• Contrast or
• youre so transparent

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CONTRAST!
Cells typically are - transparent (not
amplitude objects) - phase objects - low in
contrast

• Contrast-generating techniques such as DIC
  turn phase differences into intensity differences
  so we can see unstained cells using transmitted
  light!

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Contrast
We need contrast to be able to see an
object. Contrast can come from variations in -
intensity (DIC, phase) - color (brightfield,
fluorescence)
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DIC (a.k.a. Nomarski)
DIC turns gradients in optical path into
gradients in intensity, thus generating contrast.
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Optical path of light
OP (optical path) nd n refractive index d
distance traveled Differences in optical path
will readout as contrast.
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DIC

• Looks 3-D, but be careful when interpreting the
  images!
• When you see contrast, you are seeing some
  combination of differences in n and/or
  differences in thickness.
• Best for regions with gradients in n and
  thickness, e.g. egde of a cell or organelle.
• - Also note cant use plastic dishes.

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Koehler - align and center condensor (using
field diapraghm in front of light source)- open
condensor diapraghm (using back focal plane of
oculars).Always Koehler again when you switch
to another objective! Fluorescence -
objective is condensor. - still need to consider
how much to open diapraghm
Remember to Koehler
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Tradeoff contrast vs. resolution. Open
condensor diapraghm for - more resolution -
less contrast - less depth of focus Viceversa
when you close it down more.General starting
point 2/3 open.
Tradeoff
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DIC
5) a polar selects components so interference
can occur
4) recombines light
3) phase shifts/optical path differences occur
2) splits the light
1) polarizes light
Image from http//micro.magnet.fsu.edu/primer/inde
x.html
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When a spot is not a spot
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Diffraction of light

• Light from a point source passing
• through an aperture diffracts.
• Aperture can be eye, or objective.
• Pattern of diffraction is known as an Airy disk.

Images from http//micro.magnet.fsu.edu/primer/ind
ex.html
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NA affects Airy disk

• The higher the NA (bigger aperture), the narrower
• the Airy disk

Images from http//micro.magnet.fsu.edu/primer/ind
ex.html
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NAor why size matters
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Numerical Aperture (NA)

• NA nsin?
• ? 1/2 angle of maximum cone of light that can
  enter the objective
• Note that the refractive index plays a role here.
  The NA on the objective is a max, not a
  guarantee!

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NA affects Airy disk
NA1.3
NA0.2
Images from http//micro.magnet.fsu.edu/primer/ind
ex.html
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N.A. affects resolution

• The higher the N.A., the better the resolution!

Images from http//micro.magnet.fsu.edu/primer/ind
ex.html
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The power of N.A.

• ? Rule of thumb magnification should be
  500-1000x the NA (more than that is empty
  magnification)
• ? Depth of focus is also a function of NA
• Depth of focus n?/NA2
• Magnification and NA are both important for
  resolution.
• Example
• 63x 1.4 NA objective resolution of .24
• 100x 1.3 NA objective resolution of .26

an objective
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A tool for every need
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technique strengths

• Brightfield histology
• Phase unstained, very thin specimens
• DIC unstained, thin specimens
• Polarized light mitotic spindles, DNA
• Widefield fluorescence thin specimens
• Confocal fixed, thicker specimens, Z sections
• Spinning disk live, Z sections
• Multi-photon live, even thicker specimens, Z
  sections
• Deconvolution dim, live, at limits of
  resolution

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Want more info? See www.merritt.edu/microscopy
for a list of books, websites, and microscopy
tips.
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Merritt Microscopy Program
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• Merritt College
• in nearby Oakland Hills
• only 20 unit
• part of Peralta Community
• College District (26,000 students)
• access and diversity!
• www.merritt.edu/microscopy

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• Merritt Resources
• Measure A funding for equipment.
• Science building renovation.
• Faculty expertise in microscopy and genomics.
• GenomXY Institute genomics facility with 28
  capillary sequencers, robotics, microarray
  capability.
• Collaborations with UC extension, SFSU, Broad
  Institute, JGI.
• Students!

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• Merritt Microscopy Program
• Students
• Re-training for employment
• Academic path
• Enrichment
• Curriculum
• 1 year certificate with summer internship
• A.S. degree
• Research

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• Merritt Microscopy Program
• Equipment
• Zeiss, Olympus, Nikon and Leica microscopes
• DIC, phase
• Widefield epifluorescence (several fully
  motorized)
• Confocals
• EM/SEM
• Tissue culture
• Tele-microscopy

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• Merritt Microscopy Program
• Students will be able to
• Operate a wide variety of microscopes
• Design experiments, analyze data, communicate
  results
• Prepare specimens
• Troubleshoot optical issues
• Assess and adapt to new technologies
• Employment in
• Microscopy imaging core facilities
• Biotech
• Research

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Advisory Board
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• Where do you come in?!
• Collaborations
• Equipment
• Guest lectures
• Teaching assistants (in return for scope time)
• Employment!

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www.merritt.edu/microscopy