Announcements

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Announcements

• Artemia reports due today please put on front
  desk.
• Paper today Wongprasert et al. 2003 (Katie
  leads)
• Paper for next week Jud et al. 2007 (Molly
  leads)
• Start computers, open lecture PP for simulations
  again.
• Revised Fluoview manual (PDF file) is available
  on Fluoview computer.
• TBA and assignment this week Multi-channel
  imaging, including laser transmitted DIC
• Bright field contrast techniques
• Collect images of kidney slices and submit report
  (Fig. 1A channel 1, 1B channel 2, 1C merge of
  channels 1 and 2 1D channel 3 (DIC image).
• Using references, describe some structures in
  your images in your figure legend.

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Microcopy Facility Assistant Johns Hopkins University General Description A microscopy specialist is sought to provide user support in Johns Hopkins School of Medicine Microscope Facility. This facility provides light, fluorescence and electron microscopy services to more than 200 users throughout Johns Hopkins. In addition to assisting with routine maintenance of the facility equipment and supplies, the candidate will help assist in training and supervising new users, as well as help users troubleshoot experiments. The candidate will also be trained to help users analyze images and data. Because of the diversity of equipment within the facility, we will provide initial training as necessary. Consequently, the candidate must display resourceful independence, a willingness to learn and have strong analytical skills. The primary duties of the candidate, when trained, will be to provide training and supervision for new users when they use one of many confocal and wide-field light microscopes (Zeiss, Olympus, and Nikon). Basic knowledge of cell biology is critical for communicating with users. Helping users with software and specimen preparation and interpretation may be needed as part of user services on projects. Regular duties include cleaning these microscope work areas and maintaining relevant supplies. Secondary duties will include collecting and maintaining a library of protocols, manuals and tutorials for users. With all duties, timely record-keeping (electronic written) are required.
   
Qualifications https//hrnt.jhu.edu/jhujobs/job_view.cfm?view_req_id26703) BS/BA, or equivalent, in biological sciences, chemistry, or related field required. Comparable laboratory experience may be substituted for some education. At least one year of relevant work experience required. Previous experience with fluorescence microscope imaging, including basic image processing, is required. Experience with specimen preparation for light microscopy (immunostaining, histology/pathology), confocal microscopy, or interpretation of cellular physiology in images is useful, but not required. Strong organizational skills coupled with strong interpersonal and communication skills (both oral and written) are essential.
NOTE The successful candidate(s) for this position will be subject to a pre-employment background check
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Paper Discussion Schedule

• Today, Jan. 22 (Hertzler) Zucker 2006
• Jan. 29 (Katie) Wongprasert et al. 2003
• Feb. 5 (Molly) Jud et al. 2007
• Feb. 12 (Becky) Anders 1988
• Feb. 19 (Rachel) Tan et al. 2005
• Feb. 26 (Ellen)
• March 12 (Emily)
• March 19 (Amy)
• March 26 (Amanda)
• April 2 (Andrea)
• April 9 (Brittaney)
• April 16 (Lauren)
• April 23 (Joe and Molly)

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Outline Contrast Enhancement, Confocal Hardware

• Resolution and sampling frequency XY and Z
• Kohler Illumination and microscope setup
• Contrast Enhancement
• Brightfield
• Interference of light
• Phase contrast
• Polarization
• DIC
• Components of LSCM
• Scan Head
• Lasers
• Light Detectors
• Week 4 TBA

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Nyquist Sampling Theorem XY

• Hibbs, p. 126 When a continuous, analogue image
  is digitised, the information content of the
  signal will be retained only if the diameter of
  the area represented by each pixel is 2.3x
  smaller than the optical resolution limit of the
  microscope.
• So an objective with a theoretical resolution of
  0.2 µm requires a pixel size of 0.08 µm.
• How do you determine the pixel size (sampling
  frequency)?
• Measure it with your scale bar at different zoom
  factors

From Pawley, 2006. Handbook of Biological
Confocal Microscopy. Springer New York.
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Nyquist sampling of an image of two points
separated by the Rayleigh resolution(Pawley 2006)
Sampling interval d/2.3
From Pawley, 2006. Handbook of Biological
Confocal Microscopy. Springer New York.
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5. Axial Resolution (Z or raxial)

• Minimum distance between the 3D diffraction
  patterns (PSFs) of two points along the Z axis
  that can still be seen as two.
• From Pawley, 2006. Handbook of Biological
  Confocal Microscopy. Springer New York.

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5. Axial Resolution (Z or raxial)

• So with ? 1.5 for methyl salicylate

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Ideal step sizes
Ideal step size (lower Z resolution, e.g. NA0.7)
Undersampled (lower Z resolution, e.g. NA0.7)
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XY and Z resolutions (µm), XY Zoom andZ step
sizes (1024 X 1024 box size)
Dye ?ex/?em 10X 0.4 NA 20X 0.7 NA 40X 0.75 NA 60X 1.4 NA
rlateral Zoom fluorescein 488/518 0.790 4X 0.451 3X 0.421 1.5X 0.226 none
rlateral Zoom rhodamine 543/580 0.885 4X 0.505 3X 0.471 1.5X 0.253 none
raxial Step fluorescein 488/518 9.71 3.2 3.17 1.1 2.76 0.9 0.793 0.26
raxial Step rhodamine 543/580 10.9 3.7 3.55 1.2 3.09 1.0 0.888 0.30
Nyquist sample frequency of 2.3
Nyquist sample frequency of 3.0
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B. Kohler Illumination

• Purpose Bright, even illumination without
  illuminating unnecessary areas or excess flare.
• Steps
• Focus on the sample.
• Close field diaphragm until it can be seen, focus
  and center the condenser.
• Open field diaphragm until it disappears from
  view.

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Upright Scope
Epi- illumination Source
Brightfield Source
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Olympus BX50 Upright scope
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Inverted Microscope
Brightfield Source
Epi- illumination Source
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C. Enhancing contrast in LMFibroblast in
Culture Four Types of Light Microscopy
Bright-Field Phase-Contrast
Differential Interference Contrast Dark-Field
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1. Bright-field microscopy

• Is the simplest, but object must be colored to be
  seen. Histological staining usually requires
  killing the sample.
• Staining utilizes absorption e.g. red stain
  absorbs green and blue light, passing only red
  light. The specimen is now an amplitude object,
  where contrast is seen by reducing the amplitude
  of certain wavelengths of light.
• Microscopy of living cells, which are usually
  transparent, are limited by contrast, or the
  difference between light and dark.
• How can we see them without staining them?
• By exploiting the fact that samples are phase
  objects, which slow light down relative to other
  parts of specimen or to background.

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2. Interference
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3. Phase-Contrast Microscopy

• Annular Ring in Phase Condenser focuses cone of
  light onto sample.
• Specimen light is shifted -1/4 wavelength
• In Phase ring of Objective
• Direct light (background) passes through thin,
  dark part.
• Diffracted light (specimen) passes through thick,
  light part, shifted -1/4 wavelength.
• Specimen light shifted by ½ wavelength total.
• Rings must be aligned to get phase effect.

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Alignment of phase rings

• Jave tutorial
• http//micro.magnet.fsu.edu/primer/java/phasecont
  rast/phasemicroscope/index.html

ALIGNED
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Phase Contrast Microscope

• Surround wave (red) is undiffracted light that
  passes around and through the sample.
• Diffracted wave (blue) interacts with sample, is
  retarded by ¼ wavelength relative to S wave.
• Particle wave (green) results from interference
  between S and D waves. Amplitude difference
  between S and P determines the level of contrast
• PC scope shifts diffracted beam from specimen an
  additional ¼ wavelength to ½ ?, creating maximal
  destructive interference between S and D.
• Causes decrease in amplitude (brightness) in P,
  which can be seen against brighter background.

Object dimmer Background bright
Object brighter Background dimmer
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Limitations of Phase Contrast

• Phase images are usually surrounded by halos
  around the outlines of details. Such halos are
  optical artifacts, which sometimes obscure the
  boundaries of details.
• The phase annuli do limit the working numerical
  aperture of the optical system to a certain
  degree, thus reducing resolution.
• 20X PlanApo 0.7 NA compared with 20X Phase 0.4
  NA.
• Phase contrast does not work well with thick
  specimens because shifts in phase occur from
  areas slightly below or slightly above the plane
  that is in focus. Such phase shifts confuse the
  image and distort image detail.

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4. Polarization Microscopy

• Useful for crystalline materials or oriented
  structures in biological materials, e.g.
• Mitotic spindle fibers
• Microfilament bundles
• Striated muscle fibers
• These structures are said to be birefringent
  (having double refraction), meaning that they
  have at least two refractive indices.

Birefringent skeleton in sea urchin larva
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Polarization of Light
AKA Analyzer
Note laser light is already polarized
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Polarizing Sunglasses

• Human eye cant detect difference in randomly
  oriented versus polarized light.
• When polarizing sunglasses filter out parallel
  waves, eye detects less glare, lower amplitude.

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Isotropic versus anisotropic materials
Light slowed equally vibrating in any direction.
Light slowed less when vibrating N-S
n1
n1
n2
n1
Light slowed more when vibrating E-W
n1
Isotropic Glass, salt
Anisotropic (birefringent with two Refractive
indices) Sugar, muscle, gout crystals
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Birefringence

• First clue to explanation of polarization came
  from observation of calcite crystals by Erasmus
  Bartholin in 1669.
• http//www.microscopy.fsu.edu/primer/java/polarize
  dlight/icelandspar/index.html
• One of the light rays emerging from a
  birefringent crystal is termed the ordinary ray,
  while the other is called the extraordinary ray.

Split, both Polarized perpendicular
Incident ray oblique to optical axis of crystal
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Incident light perpendicular to optical axis of
specimen same trajectory, different path length
causes interference when recombined.
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Birefringent samples oriented 45o to crossed
polarizers are maximally bright

• Java tutorial http//micro.magnet.fsu.edu/primer/
  java/polarizedlight/crystal/index.html

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5. Differential Interference Contrast

• Also called Nomarski optics uses plane polarized
  light.
• Similar to Phase Contrast in that light from low
  contrast sample is caused to interfere
  destructively to produce amplitude changes.
• Produces contrast where changes in thickness,
  slope, or refractive index occur in cell,
  especially along edges, to give a pseudo three
  dimensional appearance.

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Cheek cells
Filamentous alga
Red blood cells
DIC images have no halos.
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DIC produces superior axial resolution, optical
sectioning.
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DIC PathwayComponents

• Light from lamp passes through Polarizer, is
  separated into O and E waves by Wollaston Prism
  (specific to each lens), then to Condenser.
• Phase specimen creates different optical path
  lengths for O and E, shifting their phase.
• After passing through specimen, light passes
  through Objective, and is recombined, resulting
  in interference, by second Wollaston Prism, then
  to Analyzer (second Polarizer) to Eyepiece.

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Effect of Bias Retardation in Analyzer

• Controls how O and E waves are recombined.
• Affects brightness, contrast, and color (optical
  staining) of specimen.
• Java tutorial http//micro.magnet.fsu.edu/primer/
  java/dic/lightpaths/index.html

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Advantages, Disadvantages of DIC

• Advantages
• Uses full NA of the lens, achieving optimal
  resolution and some optical sectioning ability.
• Provides optical color staining.
• No phase halos as with phase contrast.
• Main Disadvantage
• Tissue culture plastic or birefringent sample
  features can produce confusing effects.

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D. Confocal Hardware
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1. Fluoview 300 Scan Head Anatomy
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1
10
4
3
9
5
7
8 Beam splitter
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2. Lasers available for Olympus Fluoview Confocal
Microscopes

• Blue argon-ion (488 nanometer) laser (WE HAVE)
• Multi-line argon-ion (457, 488, and 514
  nanometers) laser
• Green helium-neon (543 nanometer) laser (WE HAVE)
• Red helium-neon (633 nanometer) laser (WE MAY
  GET)
• Yellow krypton-ion (568 nanometer) laser
• Blue-violet helium-cadmium (442 nanometer) laser
• Violet and blue-violet diode (405 and 440
  nanometer) lasers
• Ultraviolet argon-ion (351 nanometer) laser
• Infrared (750 nanometer) laser

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Adjusting Offset and PMT/Gain to maximize range
of grey levels collected
Pixel intensity 256 (4047) 0
Figure 5(a) illustrates the raw confocal image
along with the signal from the photomultiplier.
After first applying a negative offset voltage to
the photomultiplier, the signal and image appear
in Figure 5(b). Note that as the signal is
shifted to lower intensity values, the image
becomes darker (upper frame in Figure 5(b)). When
the gain is next adjusted to the full intensity
range (Figure 5(c)), the image exhibits a
significant amount of detail with good contrast
and high resolution.
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Note on Building stereo images

• Stereo Factor Sets the deviation between the
  left and right eyes when building a pair of
  stereo 3D images or a 3D image to be viewed
  through color (red/green) eyeglasses.
• You can change this.
• Z Stretch Factor Provides each section with a
  feeling of thickness. Usually this value does not
  need to be changed.

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Week 4 TBA

• Kohler illumination
• Phase contrast
• Polarization
• DIC
• Laser transmitted DIC
• Assignment Collect multi-channel fluorescence
  and laser transmitted images of prepared kidney
  slide, save, submit report as before.

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Rotation Scan (Manual, 2-64)
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Rotation of Z series
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Refraction
He sees the fish here.
But it is really here!!
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Refraction Bending of light as it passes, at an
angle, from one material to another
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Brewsters Angle and Reflection Polarization

• Light reflected off opaque surface (like a
  highway) is often polarized in a plane parallel
  to the surface of the material.
• Jave tutorial http//micro.magnet.fsu.edu/primer/
  java/polarizedlight/brewster/index.html