June
2010
Possible
Pitfalls
of
Using
Multiband
Emission
Filters
in
Fluorescence
Microscopy
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Possible Pitfalls of Using Multiband Emission Filters
in Fluorescence Microscopy
Introduction
This report shows some potentially serious problems that can result from using multiband
emission filters in multispectral fluorescent imaging. This is a timely topic as many
vendors are currently recommending the use of multiband emission filters to eliminate the
need of moving specific emission filters into the light path, particularly in high speed
illumination microscope designs using LEDs for excitation. It is not generally appreciated
that short wavelength light will excite to some degree all the fluorophores which have an
excitation peak at longer wavelengths. With the use of multiband emission filters this will
inevitably lead to crosstalk between fluorescence channels and image artifacts. Here we
demonstrate this phenomenon using a specialized colour CCD camera with 3 independent
CCD chips (Hamamatsu Model C7780-10) and beam splitters to generate red, green and
blue images during a single exposure time. Artifacts will be shown that arise when
imaging classic triple labelled specimens.
Results
Bovine pulmonary artery endothelial (BPAE) cells were triple labelled to stain the nuclei
(DAPI), actin filaments (Alexa Fluor® 488 phalloidin) and mitochondria (MitoTracker®
Red). Conventional fluorescence images were obtained using individual excitation filters
and single band pass emission filters specific for DAPI (Figure 1A), fluorescein (Figure 1B),
and Texas Red (Figure 1C).
When the DAPI staining alone was excited and imaged using a triple band pass emission
filter and the colour CCD (Figure 1D), it is evident that the actin and mitochondrial
staining are also excited and contribute to the resulting “DAPI” image (compare to Figures
1B and 1C). We note that the color balance for the camera is adjusted in Figure 1D (as
the DAPI signal is much stronger than the other two signals) such that the blue channel is
0.9 and the red and green channels are at 2.0. The whitish appearance of DAPI in Figure
1D is due to the fact that is has a very broad fluorescence emission spectra so it will
actually show up in all three channels, red, green and blue. If a standard monochrome
camera was used with this triple band pass emission filter all three fluorophores would
appear in the supposedly “DAPI” image. Similarly, when exciting fluorescein to image Factin with the triple band pass emission filter the mitochondrial stain is also seen in the
image as the MitoTracker® Red is also excited (Figure 1E). The color balance here is
adjusted so that the red channel is 2.0 and the green channel is 0.6. The contamination of
the green signal here with the mitochondrial staining is quite obvious, however, when
using a monochrome camera and working with a biological sample, where multiple colours
of probes may be labelling the same structures these kinds or artifacts will lead to
erroneous, non-quantitative fluorescent images.
Quorum
Technologies
Inc.
4673
Wellington
Road
#35
RR#6,
Guelph,
ON,
N1H
6J3
Tel:
519
824
0854
Fax:
519
824
5845
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Email:
info@quorumtechnologies.com
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These problems of crosstalk are further confounded when working with tissue sections.
When imaging a kidney cryostat section with the colour 3 chip CCD, with the multi band
pass emission filter, and exciting only for DAPI, there is a remarkable amount of signal
from the DAPI, the green (Alexa Fluor® 488 wheat germ agglutinin) and red (Alexa
Fluor® 568 phalloidin) fluorescent probes in what should be the “blue” image (Figure 1F).
Conclusions
The results presented here demonstrate important issues to be aware of when using
multiband emission filters to image multispectral fluorescence samples. Microscope users
should consider this information when outfitting microscope workstations and in designing
their experiments.
Our results show that incorrect labelling patterns are observed as a result of crosstalk
when using a multiband emission filter. The 3 chip color CCD camera from Hamamatsu
allows a more accurate use of triple band pass filters, and would be advantageous over
the use of single chip cameras if a triple band pass filter is required. Without a 3 chip
camera, we advise that the appropriate single band pass emission filters should be used
instead of a multiband emission filter.
Quorum
Technologies
Inc.
4673
Wellington
Road
#35
RR#6,
Guelph,
ON,
N1H
6J3
Tel:
519
824
0854
Fax:
519
824
5845
Web:www.quorumtechnologies.com
Email:
info@quorumtechnologies.com
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FIGURE 1 Single vs. Multiband Fluorescence Filter Performance
(A)- (E) Bovine pulmonary artery endothelial (BPAE) cells (FluoCells® prepared slide #1; Invitrogen
Catalogue # F36924) stained with blue-fluorescent DAPI to label the nuclei, green-fluorescent Alexa Fluor®
488 phalloidin to label F-actin and red-fluorescent MitoTracker® Red CMXRos to label mitochondria. (F)
Mouse kidney cryostat section (FluoCells® prepared slide #3; Invitogen Catalogue # F24630) stained with
blue-fluorescent DAPI to label nuclei, green-fluorescent Alexa Fluor® 488 wheat germ agglutinin to label
glomeruli and convoluted tubules, and red-fluorescent Alexa Fluor® 568 phalloidin to label F-actin.
FILTERS. Excitation and emission filter combinations are: (A) 405nm and 455nm DAPI combination (B)
488nm and 525nm Alexa Fluor® 488 combination (C) 555nm and 620nm MitoTracker® Red combination (D)
405nm and triple band pass ET DAPI/FITC/TexasRed (E) 488nm and triple band pass ET DAPI/FITC/TexasRed
(F) 405nm and triple band pass ET DAPI/FITC/TexasRed.
IMAGING. Cells were imaged using a Leica DMI3000 inverted microscope equipped with an EXFO X-Cite
Exacte light source, a SEDAT triple band set or individual excitation filters mounted in a Ludl filter wheel, and
an Orca 3 CCD camera from Hamamatsu (Model C7780-10). Please note that the presented images are not
full field images and were cropped. Image contrast was adjusted for printing quality. The scale bar in A
represents 10 µm.
Quorum
Technologies
Inc.
4673
Wellington
Road
#35
RR#6,
Guelph,
ON,
N1H
6J3
Tel:
519
824
0854
Fax:
519
824
5845
Web:www.quorumtechnologies.com
Email:
info@quorumtechnologies.com