Photochemistry and Photobiology, 2007, 83: 1325–1329
Imaging Mitochondria in Living Corneal Endothelial Cells Using Autofluorescence Microscopy Nicholas A. Ramey†1, Choul Yong Park†1,2, Peter L. Gehlbach1 and Roy S. Chuck*1 1 2
Department of Ophthalmology, Johns Hopkins University, Baltimore, MD Department of Ophthalmology, Dongguk University School of Medicine, Ilsan, Korea
Received 2 March 2007; accepted 26 April 2007; DOI: 10.1111/j.1751-1097.2007.00162.x
ABSTRACT We report noninvasive autofluorescence mitochondrial imaging in cultured human corneal endothelial cells (HCECs). HCECs harvested from eye bank corneas were cultured in thin glassbottom plates. Mitochondria were imaged with an autofluorescence microscope using a DAPI filter set (excitation: G365, emission: band pass 445 ⁄ 50) and then, after fixation with 4% paraformaldehyde, cells were stained with MitoTrackerTM Green FM (MTG). Both images were aligned using a linear conformal algorithm for image mapping based on manually selected corresponding feature points, and then mathematically compared using two-dimensional spatial image correlation coefficients. Autofluorescence imaging provided highly resolved mitochondrial signals from living HCECs, comparable to those taken with MTG. Both techniques yielded very similar images at high magnification and high resolution, demonstrating the tubular morphology and cytoplasmic distribution that are characteristic of mitochondria. Image registration using a linear conformal mapping technique and cross-correlations showed high correlation of overlapping autofluorescence and MTG images. This study validates the novel use of autofluorescence vital imaging as a noninvasive, inexpensive and functional alternative to the mitochondria-specific dyes in cultured HCEC. This noninvasive mitochondrial imaging technique can be useful in future applications studying mitochondrial biology of ocular cells.
INTRODUCTION Mitochondria are essential cellular organelles participating in oxidative metabolism, intracellular signaling and apoptosis (1–3). Mitochondrial dysfunction is linked extensively to ocular pathophysiology, as in Leber’s hereditary optic neuropathy, chronic progressive external ophthalmoplegia, retinal dysplasia, neurogenic ataxia retinitis pigmentosa and diabetic retinopathy (5–7). In addition to its essential role in ATP generation, the mitochondrion has been linked to a growing number of cellular homeostatic mechanisms involving redox regulation (8–10). Better understanding of these mechanisms of mitochondrial biology requires both structural and functional imaging †These authors contributed equally to this work. *Corresponding author email:
[email protected] (Roy S. Chuck) 2007 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/07
studies. Conventionally, efforts to study mitochondrial structure and function have employed specific commercial dyes like MitoTrackerTM Green FM (MTG) to gain images with adequate spatial resolution (11). However, the dimethyl sulfoxide (DMSO) solution that is used to increase the solubility of MTG may provoke various biologic effects on cells and unpredictably interfere with functional assay of these cells (12–15). Recently, efforts to correlate mitochondrial structure with function have popularized noninvasive, live cell imaging (16,17). Mitochondria may be imaged in living cells using a noninvasive technique known as autofluorescence imaging, without any damage to cells (17). Endogenous cellular molecules such as reduced pyridine nucleotides can be detected using an autofluorescence microscope, and have been colocalized to the mitochondrial compartment (18–20). This noninvasive imaging technique enables serial imaging of same cells and also subsequent functional assays. Applying autofluorescence imaging to the subcellular level is a relatively new technique. Although one report correlates the mitochondrial autofluorescence signal with functional parameters of the cell (17), the underlying signal has not been directly quantified as an accurate structural one. In this study, we use high-resolution photomicroscopy to demonstrate that autofluorescence mitochondrial imaging closely correlates with commercial mitochondrial staining (MTG) in cultured human corneal endothelial cells (HCECs). We examine the autofluorescence images for mitochondrial architecture and morphology found in stain-based images. A linear conformal algorithm is applied for intermodality image mapping based on manually selected corresponding feature points. To validate this novel use of high-resolution autofluorescence imaging, registered autofluorescence and mitochondria-stained images are then mathematically and graphically compared using one- and two-dimensional cross-correlation coefficients and graphical image-intensity analyses.
MATERIALS AND METHODS Culture of human corneal endothelial cells. Endothelial cells were cultured according to previously published methods (21). Corneal endothelial cells from freshly banked human donor tissue not suitable for transplantation (Central Florida Lions Eye and Tissue Bank, Tampa, FL; Tissue Banks International, Baltimore, MD) were harvested attached to Descemet’s membrane on or before the seventh day after death. After overnight incubation of the Descemet membrane ⁄ endothelial cell complex in OptiMem-I (Gibco
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1326 Nicholas A. Ramey et al. Invitrogen Corp., Carlsbad, CA) containing 8% fetal bovine serum (FBS), the complex was centrifuged and washed in Hank’s balanced salt solution (Mediatech, Inc., Herndon, VA). Next, the endothelial cells and Descemet’s membrane complex was incubated for 1 h in 0.02% of EDTA solution, triturated vigorously with a flame-polished pipette to disrupt cell junctions, centrifuged for 5 min at 3000 g and seeded onto culture plates coated with fibronectin-collugen (FNC) coating mix (Athena Enzyme System, Baltimore, MD) containing bovine fibronectin (10 lg mL)1) and bovine Type I collagen (35 lg mL)1). The cells were cultured in OptiMem-I media supplemented with FBS (8%), calcium chloride (200 mg L)1), chondroitin sulfate (0.08%), ascorbic acid (20 lg mL)1), pituitary extract (100 lg mL)1), epidermal growth factor (5 ng mL)1), nerve growth factor (20 ng mL)1), gentamicin (1:200), penicillin (1:100), streptomycin (1:100) and amphotericin (1:100) under 10% CO2. The medium was changed every 3 days. At confluence, the cells were split 1:3, and passage 4 cells were used for experiments. Autofluorescence microscopy. One hundred cells were seeded onto thin glass-bottom plates (MatTek Corp., Ashland, MA). After overnight incubation to subconfluence, cellular autofluorescence images were obtained using a Zeiss inverted microscope (Axiovert 200M; Thornwood, NY). The microscope was equipped with a mercury lamp (HB 103) and a cooled CCD camera (Axiocam MR5) for taking images using the Zeiss AxioVision 4.3 software package. Cells were focused under transmitted brightfield illumination. A 4,6-diamidino-2phenylindole (DAPI) filter set (excitation: G365 nm, emission: band pass 450 nm) was used to detect intrinsic reduced pyridine nucleotides. High-resolution images were taken at high magnification (1000·) and 0.26 lm per pixel using reflected light, a Zeiss EC Plan-Neofluar 100· oil immersion objective (1.3 NA), and exposure times of 500 ms. Experiments were conducted in a dark room, and potentially autofluorescing debris was removed from optical elements in the light path, including the buffered salt solution bathing cells for imaging, glassbottom plates, lenses and mirrors. Filter sliders were used to control exposure and block illumination between acquisitions. Mitochondria staining. After undergoing autofluorescence imaging, cells on thin glass-bottom plates were immediately fixed with 4% paraformaldehyde solution for 10 min. A 30 nM MitoTrackerTM Green FM (MTG) solution (Molecular Probes, Eugene, OR) was prepared in complete culture media and incubated with the cells for 30 min at 37C. After samples were rinsed twice with PBS, images were taken with a Zeiss FITC filter set (excitation: band pass 450 nm, emission band pass 550 nm). Image analysis. Images were processed with AxioVision 4.3 Software (Zeiss) to reduce anti-aliasing and preserve edges using the following sequence of standard image filters: Gaussian low pass, high pass and then low pass again. Images were displayed over their full pixel intensity ranges, with minimal and uniform brightness adjustment. Image overlays were computed with custom imaging software, using a linear conformal algorithm for image mapping. For this technique, corresponding feature points were manually selected in both autofluorescence and MTG images. These points were used to calculate a rigid-body transformation map used to align the two images. These linear conformal mappings preserved image feature orientations and distances by constraining the transformations to rotations, translations and scale (factor of 1.0·) in the plane of the image. Image statistics were calculated to compare autofluorescence with MTG images. Whole image similarity was assessed in Matlab using the following standard formula for the two-dimensional cross-correlation (R) of images:
RESULTS Autofluorescence microscopy and mitochondrial staining Autofluorescence and mitochondria-specific staining (MTG) images were obtained for multiple sets of corneal endothelial cells, and are shown in Fig. 1. Autofluorescence imaging (Fig. 1a,b) provided highly resolved cellular reduced pyridine nucleotide signals demonstrating the tubular morphology and cytoplasmic distribution that are characteristic of mitochondria. These features are qualitatively consistent with those in images taken with mitochondria-specific staining (Fig. 1c,d). Both techniques yield very similar images at high magnification (1000·) and high resolution. However, without fixation, mitochondria are highly dynamic and undergo fusion and fission events as well as changes in membrane potential. This migration is apparent in the cell imaged in Fig. 2a–c. To avoid this migration, we applied immediate fixation after taking brief autofluorescence images and achieved images with close correspondence between autofluorescence signals and mitochondria-specific staining (Fig. 2d–f). Nonetheless, some differences between autofluorescence and MTG images are apparent. Several perinuclear bright spots can be seen in autofluorescence images and may be attributed to the appearance of more metabolically active
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50 µm (c)
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P P i j ðAij AÞðBij BÞ R ¼ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P P P P ffi 2 2 i j ðAij AÞ i j ðBij BÞ where A and B are the fluorescence intensity values of autofluorescence and MTG images to be correlated. A and B are the mean values over all pixels in each image, i and j are the pixel row and column locations, respectively. One-dimensional correlations (r) were also evaluated graphically and analytically for arrays of 25 radially sampled scan-lines per image, intersecting cell nuclei and bodies.
Figure 1. MitoTrackerTM Green FM (MTG) and reduced pyridine nucleotide autofluorescence. Human corneal endothelial cells on uncoated glass plates. (a, b) Cells in balanced salt solution (BSS) demonstrating autofluorescence under a Zeiss DAPI filter set with excitation at 365 nm, and emission detection at 450 nm. (c, d) Cells in BSS after 30 min incubation with MTG, imaged under the Zeiss FITC filter set with excitation at 450 nm, and emission detection at 550 nm.
Photochemistry and Photobiology, 2007, 83 (a)
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50 um Figure 2. Time-elapsed same-cell imaging vs. fixation imaging. Two human corneal epithelium cells are displayed (top and bottom rows), first imaged for their autofluorescence (a, d) with the DAPI filter set, then incubated with MitoTrackerTM Green (MTG) for 30 min and re-imaged (b, e) with the FITC filter set. Only the cell in the bottom row was fixed (4% formaldehyde for 10 min) before incubation with MTG. Note the discrepancy from mitochondrial migration in the overlay (c) of autofluorescence (green) and MTG (red) signals of the nonfixed cell. Also note the close correspondence (f) between red and green signals of the fixed cell. The autofluorescence and MTG images were registered and overlaid using linear conformal image mappings (c, f).
single or grain mitochondria, or long-thread mitochondria imaged on end. As autofluorescence measures relative signal intensities that arise from variations in mitochondrial activity, it also demonstrates more perinuclear clouding and brightness variation than MTG. MTG staining lacks this functional sensitivity, contributing to the more binary appearance of these images. Finally, while the DAPI filter passes NAD(P)H autofluorescence at 450 nm, its relatively wide emission bandwidth of 50 nm was designed for more efficient fluorophores and contribute to the background fluorescence and reduced contrast of the autofluorescence images. Image analysis To further support the finding that the reduced pyridine autofluorescence emanates from mitochondria (18–20), autofluorescence images were quantitatively compared with MTG images. Because it is difficult to obtain these images simultaneously, and to ensure the autofluorescence signal was not influenced by the addition of MTG or by its fluorescence, it was necessary to acquire these images at separate times. The significant mitochondrial migration seen above was suspended with fixation, which served the goal of synchronizing the autofluorescence and MTG responses. With this technique, cells imaged with fixation would be expected to exhibit higher intermodality correlation than those without fixation. Registered autofluorescence and MTG images were analyzed quantitatively using one- and two-dimensional intensity correlations (Fig. 3). These methods of comparison provide both analytical and graphical evidence for whole-image (two-
dimensional) and scan-line-based (one-dimensional) comparison. Without fixation or accommodating for mitochondrial migration, image correlation between autofluorescence and MTG reached R = 0.44 for the cell pictured in Fig. 2a,b. Twenty-five radial scan-lines, each spanning 500 pixel units or 130 lm, and subsampled every 10 pixels, were evaluated in this cell to an average correlation of r = 0.46 ± 0.2. Data from these scan-lines are compared in the scatter plot shown in Fig. 3a. Another nonfixed cell, which is not shown, yielded R = 0.55 and r = 0.62 ± 0.2 (Fig. 3b). Conversely, in the cell fixed with paraformaldehyde shown in Fig. 2d, correlations were R = 0.86 and r = 0.87 ± 0.08 (Fig. 3c). Values of R = 0.86 and r = 0.90 ± 0.08 were obtained for another cell with fixation, not pictured (Fig. 3d).
DISCUSSION In this study, we validated the use of reduced pyridine nucleotide autofluorescence in high-resolution functional mitochondrial imaging. Using autofluorescence, we achieved high correlation with a commercial mitochondria-specific dye (MTG). We verified that appropriate technique and highresolution photomicroscopy can yield mitochondrial autofluorescence images with a relatively strong signal that is comparable to conventional staining methods. Cellular reduced pyridine nucleotides (NADH and NADPH) and oxidized flavoproteins (flavin, FAD and FMN) are widely used targets for autofluorescence imaging of cells and tissues (22–24). With the development of multiphoton spectroscopy techniques, these cellular molecules have been used as sources
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Figure 3. Scatter plots of registered radial scan-lines for pre- and postMitoTrackerTM Green (MTG) stained cells. Single human corneal endothelial cells were imaged before and after MTG staining. Without fixation, mitochondria migrate in the 30 min period required for MTG incubation, yielding averaged one-dimensional correlations of r = 0.46 ± 0.2 (a) and r = 0.62 ± 0.2 (b). Using 4% formaldehyde fixation for 5 min (c) and 30 s (d), mitochondrial migration was effectively halted, allowing accurate pre- and post-stain registration and correlation of autofluorescence ⁄ MTG signals. The average onedimensional correlation for cell (c) was r = 0.87 ± 0.08 and for cell (d) was r = 0.90 ± 0.08. Fluorescence intensities in pixel units (pix) have been normalized by the maximum pixel intensity (pixmax) in each respective image.
for tissue autofluorescence in donor cornea and animal eyes, with adequate resolution to discriminate different collagen fiber layers and tissue viability (25,26). However, applying this autofluorescence technique to the subcellular level of mitochondria is a relatively new approach. Although several papers have been published using this technique, the resolution of previous studies has been insufficient to visualize detailed mitochondrial structures (27–30). By specifically concentrating on the weaker signal inherent in autofluorescence mitochondrial imaging, it is possible to achieve an image resolution equal to that of MTG. To optimize imaging conditions for autofluorescence, experimental factors were carefully controlled. Reflected light was used in conjunction with a high numerical aperture oil immersion objective lens to maintain brightness and image detail at high magnification and low exposure times. Further, to avoid photobleaching and increase the signal-to-noise ratio of images, ambient illumination of the room was minimized, filter sliders were used, the optical train was cleaned of any potentially autofluorescing particles and a cooled CCD camera was employed. This technique requires no additives and the autofluorescence signal will not dilute with cell division. It is therefore highly comparable from one time point to the next—giving it potential as a high-throughput screening technique, identifying subcellular aberrations before they manifest at the tissue level.
Noninvasive, autofluorescence mitochondrial imaging has several advantages over conventional staining methods. Although the conventional methods successfully identified mitochondria and proved insensitive to mitochondrial membrane potential, they precluded further functional cellular assay, as the DMSO used to dissolve the powder form of MTG could alter cellular metabolism (12–15). In contrast, the noninvasive autofluorescence imaging technique enables expeditious serial follow-up of mitochondrial structure using the same cell population with minimal damage to cells. In addition, the autofluorescence signal from cellular reduced pyridine nucleotides can provide information about the cellular redox state (19,25,31,32). Combined with noninvasive structural imaging, this information can be valuable in the evaluation of cellular responses to various pharmacologic stimuli or therapeutic efforts. Various etiologies of visual impairment have been associated with mitochondrial abnormalities (4–7). Variations of mitochondrial size and quantity have been detected in some of these conditions (6). Recently, oxidative damage has been implicated as an important causal factor in many ocular diseases (33,34); mitochondrial biology has become central in the investigation of cellular oxidative stress, given the organelle’s role in protection from excessive oxidation via superoxide dismutase (35,36). In addition, several studies have indicated that NAD+ ⁄ NADH play critical roles not only in energy metabolism, but also in cell death and various cellular functions including regulation of calcium homeostasis and gene expression (37,38). Mitochondrial distribution has been reported as a stem cell indicator (19). In this setting, mitochondrial autofluorescence imaging has very promising applications. While allowing visualization of living cells, there are aspects of this technique which require additional consideration. Cellular fixation with 4% paraformaldehyde caused mitochondrial ballooning, as in Fig. 2e, even with a 30 s exposure. Nonetheless, MTG staining revealed mitochondrial distribution that closely matched the autofluorescence signal. There have been concerns relating to potential apoptotic cell death and photochemical damage when exposing cells to the UV range of excitation light in the DAPI filter set (34,39). To reduce this risk, we have limited exposure with the DAPI filter set to 500 ms and avoided repetitive imaging to maintain cellular viability and homeostasis. In summary, mitochondrial autofluorescence provides high-resolution functional images with accurate structural detail in HCECs. In future studies where expense, disruption of normal cell activity by stain additives, autofluorophore suppression or long incubation times are of concern, functional reduced pyridine nucleotide autofluorescence may yield the sought-after data. Although basic autofluorescence techniques are not new, technological advances in image acquisition, image processing and novel application hold promise for an increasingly important role for this evolving technique. Acknowledgements—Supported by NIH Grant EY000412-04, StarkMosher Center for Cataract and Diseases, Research to Prevent Blindness Inc. unrestricted grant and Career Development Award (P.L.G.). The authors thank Stephen P. Hersh, MD, and the Marriott Foundation unrestricted grant for their support of this work.
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