Avoiding bleedthrough artifacts on the confocal microscope

1
Avoiding bleed-through artifacts on the confocal
microscope

• Fluorescent dyes are molecules that, when exposed
  to light of a specific range of wavelengths, will
  emit light in a longer range of wavelengths.
• These ranges of wavelengths are usually presented
  as excitation and emission curves (often
  normalized to 100).

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Avoiding bleed-through artifacts on the confocal
microscope

• Product inserts will frequently provide
  excitation and emission maxima as single
  wavelengths for simplicitys sake, but the
  responses of the dyes to light are much more
  complicated.

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This graph shows the excitation curves of two
popular fluorescent dyes that have emission
curves that are similar to the well-known dyes
Fluorescein and Texas Red.
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Overlaid on the excitation curves are the
wavelengths of two of the confocal microscopes
lasers, 488nm and 543nm.
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You will note that the laser wavelengths do not
actually coincide with the peak excitation of the
dyes. This will be important later.
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This graph shows the emission curves of the two
fluorescent dyes. Since they are commonly
portrayed as green and red fluorescence, weve
used those colors here.
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Note how much the emission curve from the green
dye overlaps that of the red dye. This is
portrayed in yellow because green red yellow.
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This overlap of the two emission curves is what
can create the appearance of co-localization if
the dyes are imaged simultaneously on the
confocal.
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Since the green dye was excited at approximately
60 of maximum and the red dye was excited at 35
of maximum, the emission curves actually look
more like this
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You can see that this makes the yellow overlap
region an even more significant issue.
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Confocal microscopes use photomultiplier tubes
(PMT) to detect light. Because PMTs only detect
photons (regardless of color), the confocal uses
filters to select the range of light that each
detector sees.
PMT 1
PMT 2
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The gray overlay shows the light that is blocked
by the filters. If the confocal is set up to
simultaneously excite and image these two dyes, a
problem occurs.
PMT 1
PMT 2
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In this example, PMT 1 will see the green
fluorescence. PMT 2 will see the red
fluorescence and a portion of the spectra from
the green fluorescence (shown as yellow).
PMT 1
PMT 2
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When the two images are overlaid, it will appear
as if the green and some of the red staining are
co-localized. This is due to the artifact of the
green fluorescence being detected in the red
channel (PMT 2).
PMT 1
PMT 2
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There is a simple way to avoid this artifact
PMT 1
PMT 2
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Acquire the images sequentially, using the 488nm
laser and PMT 1 to acquire the image of the
green fluorescence.
PMT 1
PMT 2
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Then acquire the image of the red fluorescence,
using the 543nm laser and PMT 2. It is very
important that the 488nm laser be turned off
during this step.
PMT 1
PMT 2
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Without the 488nm laser, there is no excitation
of the green dye and there is no bleed-through
into PMT 2. This means that the only light that
PMT 2 sees is from the red dye.
PMT 1
PMT 2
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• Confocal microscopes make solving this problem
  fairly easy, since the lasers can be turned on
  and off as needed.
• If you are using a standard epifluorescence
  microscope with a CCD camera you will also need
  to be careful about bleed-through. Multiple
  wavelength filter cubes can have significant
  bleed-through artifacts. Acquiring images of
  each of the fluorescent dyes with a single
  wavelength cube will lessen this problem, but may
  not completely eliminate it. This is because the
  light source is continuously emitting all
  wavelengths and fluorescence filters are not
  perfect.
• An alternative is to use two dyes that do not
  overlap, such as fluorescein (or a similar green
  dye) and CY5 (a far-red emitting dye).

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Example
CH 2 red
CH 1 green
Using the 488 nm laser only, but looking at both
the green and red channels, here is what youd
see Remember that the image is of light
intensities (greyscale), the colors are added for
our benefit. See the ghost image of the green
channel appearing in the red channel, thats
bleedthrough.
Using the 543 nm laser only, but looking at both
the green and red channels, here is what youd
see Note that there is no bleedthrough from the
red channel into the green channel. Bleedthrough
almost always is from the shorter wavelength
(blue end of the spectrum) into the longer
wavelength (redder end of the spectrum).
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Example
CH 2 red
CH 1 green
Here would be an example of what you might see if
you acquired both channels simultaneously. The
red channel shows some bleedthrough of the green
channel image (contrast it with the image below).
Again, using the 543 nm laser only, but looking
at both the green and red channels, here is what
youd see Contrast this red image with the one
above. No bleedthrough here
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Example
Both channels combined
This image is an example of a simultaneous
acquisition. The green image is bleeding through
into the red, so the two images combined shows a
greenish-yellow color in the mitochondria.
This image is an example of a sequential
acquisition, one channel, then the other, so that
bleedthrough doesnt happen. Notice that the
mitochondria are a true green color.