Announcements/Outline

1
Announcements/Outline

• Handout Fluoview 300 manual (contents)
• Start up your computers for a Java Tutorial
  today.
• Paper for discussion next time Robb and Alvarado
  (2002) PDF available on web site.
• Come prepared to participate
• Lecture topic next time Digital Imaging
• Dinner at Dr. Hertzlers?

1. Crossover revisited
2. Reflection imaging
3. TBA Reflection imaging and Sequential Scanning

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Midterm Exam Results

• Average 67/99 (68)
• High 78/99 (79), 2 students
• Curved grades (90, 80, 70 of 78/99)
• A (70-78) 5
• B (62-69) 4
• C (54-61) 3
• Answer key will be posted in hall beside BR 179

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Confirmation Questions

• 17. The blue argon laser produces what
  wavelength?
• 18. The green helium neon laser produces what
  wavelength?
• A. 488 nm
• B. 514 nm
• C. 543 nm
• D. 568 nm
• E. 633 nm

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Posterization solutions

• Click the ltLUTgt button to display the Color Tool,
  and confirm that Intensity Mapping Low 0, High
  4095, Gamma 1.
• Save as 24-bit TIF files instead of 8-bit TIF.
• Save to the 2nd imaging computer.
• You can save every image in TILEd displays by
  selecting all images during save display.

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Comments on Reports and Project Proposals

• Probes for antibody labeling Mouse anti-tubulin
  or anti-engrailed primary antibodies, goat
  anti-mouse IgG secondary antibody, conjugated to
  AlexaFluor 546.
• Reflection artifacts in CH1 from BPAE cells are
  caused by not having the filters in on the scan
  head.
• Use Figure Legend format to describe images.
• Tardigrades, Daphnia, and planarians are ordered
  and should come in this week.
• Fertile chicken eggs have been ordered from two
  local farms.
• 22610 neuronal antibody has been ordered.
  Rhodamine-phalloidin and AlexaFluor 546-goat
  anti-mouse antibodies are available from me
  anytime.
• Rule of thumb for projects spend about 6-8 hours
  per week. Keep track of your time, along with
  what you do, in your lab book.

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(a-c) AlexaFluor 488 and Cy3 simultaneous
scanning required for live samples (d-f)
AlexaFluor 488 and Cy3 sequential scanning
possible w/ fixed samples
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Minimizing crossover specimen labeling
precautions (Molecular Expressions)

1. Choose fluorochromes with as widely separated
   spectra as possible.
2. Adjust concentrations of fluorescent stains so
   that intensities are close to equal
3. When selecting fluorescent probes for
   multiply-labeled specimens, the brightest and
   most photostable fluorophores should be reserved
   for the least abundant cellular targets.

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Java Tutorial Crossover compensation

• http//www.olympusconfocal.com/theory/bleedthrough
  .html
• Java tutorial http//www.olympusconfocal.com/java
  /crossoversimulator/index.html
• Start with the Swiss Mouse Embryo Cell Culture

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Balancing emission intensities reduces much
crossover
Emission only
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Minimizing crossover instrumental approaches
(Molecular Expressions)

• Absorption spectra are generally skewed towards
  shorter wavelengths whereas emission spectra are
  skewed towards longer wavelengths.
• For this reason, multicolor fluorescence imaging
  should be conducted with the reddest (longest
  wavelength peak emission) dye imaged first, using
  excitation wavelengths that are only minimally
  absorbed by the skewed spectral tails of the
  bluer dyes.
• This also minimizes photobleaching from
  higher-intensity argon laser.
• Utilize sequential scan instrument settings.
• More efficient than manually turning lasers on
  and off.

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Setting up Fluoview for Sequential Scanning to
minimize crossover (Manual, 2-71, 72)

• Select the Surface XY-Norm option button in the
  Mode group box in the Acquire panel and
  select ltSequengt from the list displayed below it.
• Select the observation mode with the option
  buttons in the Acquire panel.
• Group (which means each laser) and the lt?gt and
  lt?gt buttons appear on the lower part of each CH
  group box.
• Set the group number according to the reagent in
  use with the lt?gt and lt?gt buttons.
• Set the longer wavelength first to avoid
  bleaching.
• Click the ltSeq. Oncegt button to acquire the
  image.

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Controls for Double Labeling

• Background control specimen without secondary
  antibody or fluorochrome
• Bleed-through control specimen labeled with each
  fluorochrome separately. To determine maximum
  gain before bleed-through
• Image green-labeled sample w/488 in Ch. 1, look
  for cross-over in Ch. 2.
• Image red-labeled sample w/543 in Ch. 2, look for
  crossover in Ch. 1.
• Using these settings, image double-labeled sample
  (same stain concentrations as above).

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Quantum Dots

• Semi-conductor nanocrystals coated with inert
  polymer to which biomolecules can be attached,
  e.g. antibody.
• Advantages
• Less photobleaching, high quantum yield
• Narrow, symmetrical emission spectra means less
  spectral overlap.
• Various colors can be excited by same laser line
• Electron-dense, good for correlative LM-EM
• Diadvantages
• Toxic (Cadmium Selenide), so must be used with
  fixed cells.

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Reflection (or Reflectance) Imaging

• Used to image surface topography of sample,
  especially in materials science.
• Can also be used in labeled or unlabeled
  biological samples.
• Brain, skin, bone, teeth, eye
• Probes that can be used in reflected light mode
  for single label experiments include gold
  particles, peroxidase labels, and silver grains.
• Metal-coated SEM samples
• These are confocal images, unlike laser
  transmitted images.

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Illustrated in Figure 3 are several digital
images collected from confocal reflection
microscopy experiments with silver grains. The
specimen is peripheral blood cells from an
HIV-infected individual prepared for in-situ
hybridization and stained with Giemsa. Figure
3(a) illustrates the preparation under standard
brightfield illumination, while Figure 3(b) shows
the same field in darkfield illumination with a
significant amount of out-of-focus debris. The
results with confocal reflection microscopy (of
the same viewfield) are presented in Figure 3(c).
Note that the out-of-focus debris is not imaged
in this mode. Reflected mode can also provide
context to fluorescence image, like laser
transmitted DIC
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Colloidal Silica Crystal Seeded on Ion Beam
Milled Glass Substrate Leica SP-2 confocal
microscope reflection mode image of monodisperse
rhodamine core silica microspheres(1um)spontaneous
ly arranged from suspension on a pattern which
has been ion-beam milled into the surface of a
glass coverslip. This arrangement is designed to
form the first layer of a crystalline three
dimensional structure. Image Courtesy Michael
Bevan, Ph.D., Beckman Fellow, Beckman Institute
for Advanced Science and Technology, UIUC, 61801
http//www.itg.uiuc.edu/exhibits/iotw/2002-02-14/
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Diatoms imaged in reflection mode

• From the Laboratory of Research on the Structure
  of Matter (LRSM), University of Pennsylvania,
  http//glinda.lrsm.upenn.edu/resolution.html
• Note that the resolution approaches the
  theoretical limit

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TBA, Part 1 Reflection Imaging

• Use a coin supported by a slide, e.g. Lincoln
  seated in his memorial on a penny.
• Focus with 10X with room lights on.
• Set up the confocal for imaging with the HeNe
  laser and collecting all light in channel 2. No
  filters should be used in this case.
• You will need to turn the PMT and Gain way down.
• Collect a Z series, using an appropriate step
  size, and reconstruct a 3D image.
• Save extended focus (lastnameFig1A) and 3D
  (lastnameFig1B) images onto 2nd Imaging Computer
  and submit Figure legend with tech info next week.

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TBA, Part 2 Sequential scanning

• Go back to the BPAE cells using the 40X lens.
  Find a nicely-stained cell and zoom up to 3-4X.
• Optimize your levels as best you can, without
  changing the laser power, in normal simultaneous
  scan mode.
• Collect a green/red image, save as lastnameFig2A.
• Referring to 2-71 ff. from the manual, set up
  sequential scanning.
• Collect a green/red image, save as lastnameFig2B.
  You should note less yellow, indicating less
  crossover. Include Figure 2 legend with tech
  info.