Transcript Announcements - Central Michigan University

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. Hertzler’s?
1. Crossover revisited
2. Reflection imaging
3. TBA: Reflection imaging
and Sequential
Scanning
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
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
Posterization solutions
• Click the <LUT> 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.
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.
(a-c) AlexaFluor 488 and Cy3 simultaneous scanning: required for live samples
(d-f) AlexaFluor 488 and Cy3 sequential scanning: possible w/ fixed samples
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 multiplylabeled specimens, the brightest and most
photostable fluorophores should be reserved
for the least abundant cellular targets.
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
Emission only
Balancing emission intensities
reduces much crossover
Minimizing crossover: instrumental
approaches (Molecular Expressions)
1.
Absorption spectra are generally skewed towards
shorter wavelengths whereas emission spectra are
skewed towards longer wavelengths.
•
•
2.
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.
Setting up Fluoview for Sequential Scanning
to minimize crossover (Manual, 2-71, 72)
1.
Select the [Surface XY-Norm] option button in the
[Mode] group box in the [Acquire] panel and select
<Sequen> 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 <▲> and
<▼> buttons appear on the lower part of each [CH]
group box.
Set the group number according to the reagent in use
with the <▲> and <▼> buttons.
2.
3.
4.
•
5.
Set the longer wavelength first to avoid bleaching.
Click the <Seq. Once> button to acquire the image.
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:
1. Image green-labeled sample w/488 in Ch. 1, look for
cross-over in Ch. 2.
2. Image red-labeled sample w/543 in Ch. 2, look for
crossover in Ch. 1.
3. Using these settings, image double-labeled sample
(same stain concentrations as above).
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.
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.
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 insitu 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 outof-focus debris is not imaged in this mode.
Reflected mode can also provide context to fluorescence image, like
laser transmitted DIC
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)spontaneously arranged from suspension on a pattern which has been ionbeam 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/
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
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.
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.