Fluorescence Microscopy and Cell Imaging Shared Resource
The Power of Spectral Imaging
One challenge with using fluorescence for labeling proteins of interest is that cellular or tissue autofluorescence can be difficult to distinguish from the fluorescent label. In Figure 1, a tissue has been labeled with an antibody tagged with a green fluorescent marker. On the left is the image that can be seen through the green (FITC) filterset of a fluorescence microscope. Because this tissue has fairly strong intrinsic (auto) fluorescence that shows up through the green filterset, it is not possible to accurately tell where the labeled protein is. On the right is the composite image of the same field taken with a spectral camera. Individual spectra of the green fluorescent marker and the tissue autofluorescence are first obtained from spectral images of control slides. The camera and accompanying linear unmixing algorithm can then display the distinct locations of the labeled protein (green) and autofluorescence (grayscale).
The Fluorescence Microscopy Shared Resource has two spectral imaging options. The CRi Nuance spectral camera mounted on a Nikon TE2000 microscope acquires non-confocal spectral images. Confocal spectral imaging can be done using the spectral (Meta) detector on either of the Zeiss LSM 510 laser scanning confocal microscopes.
Figure 2. Spectral imaging clearly shows the location of labeled proteins as well as tissue autofluorescence. Section (4 μm thick) of cystic kidney from 12-week-old male rat stained with fluorescent antibodies to aquaporin 2 (collecting ducts- green) and hormone receptor (red). A spectral image was acquired with the Nuance camera, 10X objective and Nikon TE2000 microscope and unmixed to produce the composite images pictured. Autofluorescence (shown in grayscale) is displayed only in the image on the left. Images and analysis courtesy of Carol Deaton (IMSD undergraduate student) and Dr. Heather Ward.
Heather Ward, PhD, Research Assistant Professor in the Department of Internal Medicine Division of Nephrology, has made extensive use of the Nuance spectral camera in her work on polycystic kidney disease (PKD), an autosomal dominant disease which frequently results in end-stage renal disease. When mutant, the polycystin proteins cause PKD, however, the specific mechanisms that result in cyst formation, kidney enlargement, fibrosis, and loss of function are not well described. Dr. Ward and her colleagues have identified a hormone that, among other effects, increases renal filtration and reduces inflammation and is a promising candidate for PKD therapy. Figure 2 is a fluorescently labeled tissue section from a cystic rat kidney imaged with the Nuance spectral camera that shows the hormone receptor (red) is expressed on the luminal surface of renal cysts. The cysts are negative for a marker of collecting ducts (green). The image on the left includes autofluorescence (grayscale), which can be used to visualize overall tissue architecture; on the right is the same field displaying only the labeled proteins (hormone receptor and collecting ducts). These composite images, produced by linear unmixing of the original spectral image, demonstrate the utility of spectral imaging in clearly identifying and localizing labeled proteins, even in the presence of significant tissue autofluorescence.
Figure 3. Hep3B hepatic carcinoma cells simultaneously labeled with 8 fluorescent markers. Fifteen minutes following exposure to protocells loaded with 4 fluorescent "cargo" molecules (calcein, dsDNA, RFP, and quantum dots), the protocells are located in endosomes. The protocells themselves contain autofluorescent silica and red labeled lipid; cytosol and nucleus were labeled following cell fixation. Scale bar: 20 μm. Image courtesy of Dr. Carlee Ashley, Genevieve Phillips and Dr. Jeffrey Brinker.
In addition to removing autofluorescence, spectral imaging lets you see many more labels on a single sample. Using conventional fluorescence single channel imaging, it is possible to separate up to 4 fluorescent labels. With spectral imaging the individual spectra of each fluorescent label is identified and can be clearly distinguished from other labels, even with significant overlap in emission spectra.
In Figure 3, Hep3B hepatic carcinoma cells labeled with 8 fluorescent markers were imaged with the spectral detector of the resource's Zeiss LSM 510 Meta confocal microscope. This work, published in Nature Materials (Ashley, C.E., et al. 2011 Nat. Mater. 10:389-97), reports important advances in targeting and delivery of anti-cancer therapeutics. It would not have been possible to clearly separate the 8 fluorescent markers used for this work with conventional fluorescence imaging; spectral imaging was essential to the unequivocal identification of each component and the key conclusions.
What else can you do in the Fluorescence Microscopy Shared Resource?
We have 6 microscopes, 8 cameras/imaging systems and a gel scanner. You can do both fluorescence and transmission/brightfield imaging of a variety of different cells and tissues, labeled or unlabeled. Following are images and examples of what other users are doing in the Fluorescence Microscopy Shared Resource. They may provide ideas of what you too can do with some training and experience!
Confocal imaging of multiple fluorescence labels lets you localize and co-localize proteins.
This is a 3 channel fluorescent confocal image of living, activated RBL-2H3 mast cells. The location of Arf6 (red), dynamin (green) and the IgE receptor (grayscale) can be seen in the separate fluorescence channels of the image. Visualization of co-localization is made possible through a ‘Merge’ channel function, and can be more clearly demonstrated using the Line Profile feature of the confocal software. The graph plots fluorescence intensity vs. distance along the white line (Merge image) for each fluorescence channel. Black arrowheads indicate points where Arf6 and dynamin are co-localized; the red arrowhead indicates colocalization of all three labeled proteins. Scale bar: 10 μm.
Image acquired on the Zeiss LSM 510 meta confocal microscope; 63X objective. Line profile part of Zeiss Zen acquisition and analysis software.
Image courtesy of Dr. Cedric Cleyrat, Florian Dingreville and Dr. Bridget Wilson from Cleyrat, C. et al., 2013. The architectural relationship of components controlling mast cell endocytosis. J. Cell Sci.126(Pt 21):4913-25.
Confocal imaging using two-photon excitation lets you see what's going on inside of living tissues.
The movement of fluorescently labeled T lymphocytes can be tracked inside of living lymph nodes using two-photon confocal fluorescence microscopy. T lymphocytes labeled with either red (CMTMR) or green (CFSE) fluorescent labels are injected into a live mouse 20 hours prior to sacrifice and removal of lymph nodes. The nodes are maintained in a heated, perfused chamber on the microscope stage and confocal images are captured over time in 3 dimensions.
In this image, the positions of red and green labeled lymphocytes at a single time point can be seen in 3 spatial dimensions. More intense color indicates a cell that is closer to the viewer; less intense color indicates a cell that is deeper in the node. Play the time-compressed video to see lymphocyte movement within the lymph node (not labeled) during the 33 minute experiment. Analysis of these movies produces T cell trajectories and velocity information which are used by Dr. Judy Cannon and her group to study signaling pathways involved in T lymphocyte motility in secondary lymphoid organs.
Time series images acquired on the Zeiss LSM 510 two-photon confocal microscope using 25X objective; live cell incubation chamber; temperature maintenance at 37oC; 5% CO2, humidified environment, and constant perfusion with oxygenated media.
Images/movie courtesy of Dr. François Asperti-Boursin and Dr. Judy Cannon.
Montage imaging allows you to stitch together multiple images so that you can take pictures of features that are larger than one field of view.
This image montage is comprised of more than 400 individual bright field images acquired at 20X magnification and shows a 5 μm thick tissue section from a mouse tumor stained with hematoxylin and eosin. Scale bar = 1 mm
Montage image acquired with Olympus IX81 microscope; Q-Imaging Retiga 2000R color camera; 20X objective.
Image courtesy of Terisse Brocato, Dr. Vittorio Cristini, and Dr. Jeffrey Brinker.
You can acquire an image Z stack on a non-confocal fluorescence microscope and use image deconvolution (deblurring) software to produce a 3D image superior to one made by a laser scanning confocal scope.
On the left is a single image projection of a Z stack showing an RBL-2H3 mast cell labeled with a nuclear label (blue) and a fluorescent label binding mRNA for Il-4 (red). The image Z stack was acquired on a widefield (non-confocal) fluorescence microscope. The image on the right is a single image projection of the same Z stack after deconvolution, a mathematical process that can trace all of the fluorescent light in an image stack back to its original point source. After deconvolution, the individual mRNA molecules can be distinguished and quantified. The pictured cell is approximately 12 μm in diameter.
Image stack acquired on Olympus IX71 microscope: Andor iXon EMCCD camera; 150X objective. Image deconvolution done with Huygens Essential Deconvolution software, available on a computer workstation in the Microscopy Resource.
Images courtesy of Emanuel Salazar-Cavasos, Dr. Cedric Cleyrat, and Dr. Bridget Wilson.
You can do standard color imaging of slides labeled with high contrast labels (H&E or IHC).
5 μm section of normal rat kidney immunolabeled for Ki67, a marker of proliferating cells (DAB-brown) and counterstained with hematoxylin. Scale bar: 20 μm
Image acquired with Zeiss Axioskop 2 microscope; Zeiss Axiocam HR high resolution color camera; 63X objective.
Image courtesy of Shelley MacNeil and Dr. Heather Ward.
You can scan gels, blots and imaging plates to produce both images and quantitative data.
Scanned image of DNA labeled with 32P, treated with wild type or the indicated mutants of DNA polymerase δ, and separated electrophoretically. An imaging plate was exposed to the radioactive gel and was read by a Typhoon FLA 7000 scanner producing the image.
The plot shows exonuclease activity of the WT and mutant enzymes quantified by integrating the degraded DNA bands in each lane. Plot produced by the GE Multigauge analysis software, part of scanner system.
Image and plot courtesy of Dr. Yoshihiro Matsumoto.
Visit the Fluorescence Microscopy website http://hsc.unm.edu/crtc/microscopy/index.shtml for more information. Contact Becky Lee firstname.lastname@example.org to discuss how the resources available might benefit your research.