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Confocal Microscopy Image Gallery
Mongolian Gerbil Lung Fibroblast Cells (GeLu Line)
Lung tissue of a rodent native to parts of Africa and Asia, the Mongolian gerbil (Meriones unguiculatus), served as the source of the initial cells from which the GeLu fibroblast line was established. The tissue sample was excised from a 403-day-old female.
The GeLu line demonstrates susceptibility to numerous viruses, including vesicular stomatitis (Indiana strain), Semliki forest, germiston, herpes simplex, and adenovirus 2. Studies indicate that the cells are resistant to poliovirus 1 and poliovirus 3. In culture, GeLu cells exhibit adherent growth in both glass and plastic dishes.
Lung fibroblasts, like those from which the GeLu cell line was initiated, are important for the process of healing in pulmonary tissue that has been injured. Indeed, the production of collagen and other substances by the cells is often considered an essential part of tissue repair. However, in some cases, the productive activity of fibroblasts may become excessive, and instead of simply healing the lungs, may cause additional damage. For example, the condition known as fibrosis is characterized by the fibroblast-related development of excess fibrous connective tissue in the lungs or other organs. A similar illness experienced by individuals that come into frequent contact with coal dust is called coal worker's pneumoconiosis. The augmented activity of fibroblasts in the lungs of those with this disease is instigated by an overwhelming of the body’s natural defenses and processing capabilities.
The microtubule network and cell nuclei are revealed in this confocal image of gerbil lung fibroblasts stained with Alexa Fluor 555 (tubulin) and TO-PRO-3 (DNA). The cells were fixed with 3.7 percent paraformaldehyde, permeabilized with Triton X-100, blocked with 10 percent normal goat serum and treated with mouse anti-alpha-tubulin primary antibodies. Goat anti-mouse secondary antibodies conjugated to Alexa Fluor 555 were mixed with the nucleic acid stain. Images were recorded with a 60x oil immersion objective using a zoom factor of 2.0 and sequential scanning with the 543-nanometer line from a green helium-neon laser and the 633-nanometer line of a red helium-neon laser. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles, with the exception of TO-PRO-3, which was pseudocolored cyan.
Additional Confocal Images of Mongolian Gerbil Lung Fibroblast (GeLu) Cells
Visualizing the Actin Cytoskeleton in Gerbil Lung Cells with BODIPY FL Conjugated to a Phallotoxin - Filamentous actin was targeted in a culture of GeLu fibroblasts with BODIPY FL conjugated to phallacidin, a bicyclic peptide derived from a toxic mushroom. In addition, the cells were treated with the mitochondrion-selective stain MitoTracker Red CMXRos and a nuclear counterstain, TO-PRO-3.
GeLu Fibroblasts Triple Labeled with MitoTracker Deep Red 633, Alexa Fluor 568, and YO-PRO-1 - The Mongolian gerbil lung cells appearing in this confocal image were resident in a culture labeled with Alexa Fluor 568 (pseudocolored blue) conjugated to phalloidin, targeting the F-actin cytoskeletal network, and MitoTracker Deep Red 633, which preferentially binds with intracellular mitochondria. DNA in the nucleus was counterstained with YO-PRO-1.
The Microtubule Network in Gerbil Lung Cells - Immunofluorescence with mouse anti-alpha-tubulin was employed to visualize details of the microtubule network in the log phase monolayer culture of gerbil lung cells presented in this section. The secondary antibody (goat anti-mouse IgG) was conjugated to Alexa Fluor 555 (pseudocolored green). Nuclei were counterstained with TO-PRO-3, a carbocyanine monomer with long-wavelength red fluorescence.
GeLu Cells with MitoTracker Red CMXRos, BODIPY FL, and TO-PRO-3 - A triple fluorophore combination of MitoTracker Red CMXRos, BODIPY FL conjugated to phallacidin, and TO-PRO-3 was used to label an adherent log phase culture of Mongolian gerbil lung cells for mitochondria, the F-actin network, and nuclear DNA, respectively. The cells were first treated with the MitoTracker probe in growth medium for one hour, washed and fixed with paraformaldehyde, permeabilized, and blocked with bovine serum albumen. The cells were subsequently labeled with the conjugated phallacidin and counterstained with the cyanine monomer (TO-PRO-3) reagent.
Revealing Details of the Mitochondrial Network in Mongolian Gerbil Lung Cell Cultures with MitoTracker Orange CMTMRos - The culture of GeLu fibroblast cells shown in this confocal image was labeled for the mitochondrial network with MitoTracker Orange CMTMRos (pseudocolored red), a derivative of tetramethylrosamine. The cells were also treated with the green-fluorescent nuclear and chromosome counterstain SYTOX Green.
Distribution of Filamentous Actin, Mitochondria, and DNA in GeLu Fibroblasts - An adherent monolayer GeLu cell culture was labeled for cytoskeletal filamentous actin and mitochondria with Alexa Fluor 488 conjugated to phalloidin and MitoTracker Red CMXRos, respectively. Nuclei present in the fibroblast cells were counterstained with the red-absorbing DNA probe DRAQ5.
Gerbil Lung Cells Labeled for Tubulin with Immunofluorescence - In order to visualize the microtubules present in the gerbil lung cells shown in this confocal image, a GeLu culture was immunofluorescently labeled with anti-tubulin mouse monoclonal primary antibodies followed by goat anti-mouse Fab fragments conjugated to Alexa Fluor 488. In addition, the cells were labeled for the cytoskeletal F-actin network with Alexa Fluor 546 (pseudocolored blue) conjugated to phalloidin, and for cell nuclei with TO-PRO-3.
Targeting the F-Actin Cytoskeleton in GeLu Cells with a Fluorescent Probe Conjugated to a Phallotoxin - The proximity between the F-actin and mitochondrial networks was revealed in Mongolian gerbil lung cells by staining a GeLu culture with Alexa Fluor 488 conjugated to a phallotoxin (phalloidin) and MitoTracker Red CMXRos (pseudocolored yellow). The red-absorbing dye TO-PRO-3 was used to counterstain the cells for DNA in the nucleus.
Microtubules Immunofluorescently Visualized in Mongolian Gerbil Lung Fibroblasts - The gerbil lung cells presented in this confocal image were resident in a culture fixed with 3.7 percent paraformaldehyde, permeabilized with Triton X-100, blocked with 10 percent normal goat serum and treated with mouse anti-alpha-tubulin primary antibodies. Goat anti-mouse secondary antibodies conjugated to Alexa Fluor 568 (pseudocolored green) were mixed with the DNA stain DRAQ5 in order to simultaneously visualize microtubules and cell nuclei.
Contributing Authors
Nathan S. Claxton, Shannon H. Neaves, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.