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 F- actin 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.

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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.

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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.

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