Announcements, Week 6
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Happy Darwin Day! (b. Feb. 12, 1809)
BPAE cell reports due today.
Paper discussion (Becky): Garcia-Pichel et al. 2001
TBA this week: Drosophila embryos, mouse intestine cryostat
sections
– Reports due Feb. 19.
– TBA for Group 4 following lecture.
• Week 7, Feb. 19: Fluorescent probes, live cell labeling
– TBA: onion epithelium, report due Feb. 26
– Paper discussion next week (Rachel): Tan et al. 2005.
• Week 8, Feb. 26: Midterm exam on lecture, lab material; microscope
checkout (change from end of semester) during TBA time – allow 30
minutes each
– Project descriptions also due.
Lecture Outline
A. Immunolabeling (cont’d)
1. General considerations
2. Autofluorescence and background
3. Controls
B. Fluorescence
1.
2.
3.
4.
Defined
Absorption and emission spectra
Filters
Cross-over (bleed-through) compensation
C. TBA: Drosophila and mouse intestine samples
Detection Methods
Method
Advantages
Disadvantages
Recommended
for
Fluorescence
High resolution
Doubling labeling
possible
Staining live cells
possible
Requires special
microscope
High resolution
studies
Double labeling
Enzyme
High sensitivity
Only need bright
field light
microscopy
Permanent
Low resolution
Endogenous
enzyme activities
Double staining
difficult
Substrate toxicity
Low resolution
studies
Rapid antigen
screens
From: Harlow and Lane, 1999
Antibody Choice
Polyclonal
antibodies
Monoclonal
antibodies
Pooled
monoclonal
antibodies
Signal strength
Excellent
Fair
Excellent
Specificity
Good, but some
background
Excellent, but
some crossreactions
Excellent, by
avoiding any
antibodies with
cross reactions
Good features
Signal strength
Specificity
Signal strength
and specificity
Bad features
Background
Often need to titre
Lower signal
strength
Availability
From: Harlow and Lane, 1999
Immunolabeling of live cells
• Commonly used for surface antigens.
• Cellular endocytosis can be used to take
up antibody.
• Streptolysin-O (SLO) can be used to
permeabilize cells and retain viability.
Troubleshooting
• Problem 1: No specific staining with single
labeling
• Possible causes:
– Antigen not cross reactive, destroyed or inaccessible
• Perform positive control, obtain fresh material, permeabilize
– Primary antibody dead or not concentrated enough
• Obtain fresh or increase concentration
– Secondary antibody dead or not concentrated
enough
• Obtain fresh or increase concentration
– Protocol errors
• Review protocol, modify
• Problem 2: Artifactual staining: do neg. controls.
Autofluorescence
• Autofluorescence is caused by the intrinsic properties of
some structures, independent of antibody labeling.
– Aromatic amino acids and other molecules containing ring
structures
– Chitin, chlorophyll, collagen, elastin
– Often worse with shorter wavelength excitation that with longer.
– Aldehyde cross-linking (especially gluteraldehyde), methanol
fixation
• Low level of autofluorescence may be helpful to see limit
of cells or tissues.
• Negative control with no fluorescent probe determines
location and limits of autofluorescence.
Background staining
• Caused by binding of antibody.
• Nonspecific background: Binding of antibodies by parts
other than antigen-binding site
– Spin down prior to use to remove large particles
– Titrate concentrations to minimum
– Use blocking reagents, e.g. BSA, nonfat dry milk, normal serum
from same species as labeled antibody
– Use detergent in all solutions
– Reduce incubation times, increase wash times and numbers
• Specific background: Caused by antigen binding in side
reactions
– Dilute primary antibody in 1% normal serum from same species
as labeled antibody
Controls for immunofluorescence
labeling
• Spectral properties of the available dyes limit the experimental
freedom.
• Often it is even difficult to clearly separate two fluorescence markers.
• With more markers, the problem grows increasingly complex.
Crossover
Controls for Double Labeling
(Indirect Immunofluorescence)
• Experimental:
– Mouse Primary 1 + anti-mouse IgG secondary-FL 1,
e.g. mouse anti-tubulin + goat anti-mouse-rhodamine.
– Rabbit Primary 2 + anti-rabbit IgG secondary-FL 2,
e.g. rabbit anti-actin + goat anti-rabbit-fluorescein.
• Negative Controls for Primary Specificity:
– Pre-Immune Serum or non-immune serum from
mouse + Anti-mouse IgG secondary antibodyrhodamine.
– Pre-immune Serum or non-immune serum from rabbit
+ Anti-rabbit IgG secondary antibody-fluorescein.
Controls for Double Labeling, Indirect
Immunofluorescence (cont’d)
• Negative Controls for Secondary Specificity:
– Primary 1 + Secondary 2, Primary 2 + Secondary 1
– Look for secondaries that say “no cross-reactivity”
• Staining with one antibody and another non-antibody probe (e.g.
rhodamine-phalloidin or Sytox Green) is a simpler matter.
• If you want to use two monoclonal antibodies, one solution is to
conjugate fluorochromes to them directly, e.g.
– mouse anti-tubulin-rhodamine
– mouse anti-actin-fluorescein
• Sequential staining is also possible, fixing between steps.
• Or another way is:
Fab Fragments for Blocking and Double Labeling of
Primary Antibodies from the Same Host Species
(Jackson ImmunoResearch Laboratories, Inc.)
Key:
Rabbit anti-Antigen X
Rabbit anti-Antigen Y
Fluorescein (FITC)
Fab fragment Goat anti-Rabbit IgG (H+L)
Rhodamine Red-X (RRX)
Goat anti-Rabbit IgG (H+L)
See alternative methods at www.jacksonimmuno.com
Imaging Double-Labeled
Samples
• Select dyes that that are well-separated in
their absorption and emission spectra.
– E.g. use Texas Red (596-620) instead of
rhodamine (550-580) with fluorescein (490520).
• Be careful about turning up the gain: you
can make almost anything “fluorescent.”
• Compensate for fluorescence cross-over
(discussed below).
What is fluorescence?
Single-photon excitation
• Where a molecule emits light at a specific wavelength
when irradiated by light of a shorter wavelength.
• Jablonski diagram depicts molecular events of singlephoton fluorescence:
2. excited lifetime
3. emission
1. absorption
Fluorescence: Multi-photon
excitation
• 2 longer (infrared)
wavelength photons
absorbed simultaneously,
emitting a shorter
wavelength photon.
• Advantage of multiphoton confocal
microscope is that thicker
samples can be
penetrated, compared to
UV or visible light.
Absorption and Emission Spectra:
Spectral overlap and Stokes shift
• Spectral overlap must be eliminated by
filters, otherwise brighter excitation light
will overwelm dimmer emission light.
• The bigger the Stokes shift, the easier it
is to separate excitation from emission.
Reduced quantum yield using suboptimal excitation wavelength
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Quantum yield is a measure of the efficiency of conversion of absorbed light
into emitted fluorescence.
Same green light is emitted from purple versus blue excitation, just dimmer
with purple excitation.
Local environment, e.g. protein conjugation, pH, can also affect absorption
spectrum and therefore quantum yield.
Filters and Dichroic Mirrors
• Dichroic mirrors
– Reflect some wavelengths
– Transmit other wavelengths
• Excitation and Emission
(Barrier) filters
– Absorbs some wavelengths,
transmits others
• short pass allows transmission below
cutoff
• long pass allows transmission above
cutoff
• narrow band pass allows a range to
be transmitted
Excitation filters, dichroic mirrors
and emission filters
Absorption and emission spectra,
FITC and rhodamine, with long
pass 510, 565 filters
490 520
550 580
BA510IF, BA530RIF – CH 1
FITC only
Rhodamine only or CH 2
2-channel imaging, using long pass
510 + short pass 530 (= narrow
band pass), long pass 565 filters
Typical Crossover Problem: FITC and
rhodamine (TRITC) emission spectra
570 cutoff:
Ch. 1 Ch. 2
Solution 1:
Cut off FITC
emission tail using
OFFSET in
Acquire panel or
attenuate laser
power
Solution 2: Collect 2
channels sequentially
“Bleed-through” of
FITC into Ch. 2
“Bleed-through” of
TRITC into Ch. 1
400
500
600
700
Fluorescence Tutorials from
Invitrogen/Molecular Probes
• Basic Fluorescence:
http://probes.invitrogen.com/resources/educatio
n/tutorials/1Introduction/player.html
• Spectra:
http://probes.invitrogen.com/resources/educatio
n/tutorials/2Spectra/player.html
• Filters:
http://probes.invitrogen.com/resources/educatio
n/tutorials/3Light_Sources_Filters/player.html
(a-c) AlexaFluor 488 and Cy3 simultaneous scanning: live samples require
(d-f) AlexaFluor 488 and Cy3 sequential scanning: possible w/ fixed samples
Sequential Scanning
Java Tutorial: Crossover
compensation
• http://www.olympusconfocal.com/theory/bleedthrough.html
• Java tutorial:
http://www.olympusconfocal.com/java/crossoversimulator/index.html
Minimizing crossover: specimen labeling
precautions (Molecular Expressions)
• Choose fluorochromes with as widely separated
spectra as possible.
• Adjust concentrations of fluorescent stains so
that intensities are close to equal
• When selecting fluorescent probes for multiplylabeled specimens, the brightest and most
photostable fluorophores should be reserved for
the least abundant cellular targets.
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.
Emission only
Balancing emission intensities
reduces much crossover
Controls for Double Labeling
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Background control: specimen without
secondary antibody or fluorochrome
– Controls for autofluorescence
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Bleed-through controls: specimens 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) using
sequential scan.
Quantum Dots
• Quantum dots are semi-conductor nanocrystals coated with
inert polymer to which biomolecules can be attached, e.g.
antibody.
• Advantages:
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Less photobleaching
High quantum yield
Narrow, symmetrical emission spectra means less spectral overlap.
Various colors can be excited by same laser line
TBA this week
1.
Use your stained fly slides to collect Z-series of (a)
lower mag overview (lastname 4A), and (b) high mag
detail (lastname 4B), (c) negative control (low mag,
lastname 4C).
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2.
Use references on reserve in library to identify stages and
structures stained.
Save images in Week 6 folder and turn in a report next
Monday (see new format).
Mouse intestine cryostat section (16 um)
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Collect 2-channel image using (a) simultaneous imaging
(lastname 5A) and (b) sequential imaging (lastname 5B).
Compare dual-labeled samples to controls
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Submit two reports, one for each sample.