May 2015
Volume 56, Issue 5
Free
Cornea  |   May 2015
Quantum Dot Labeling and Tracking of Cultured Limbal Epithelial Cell Transplants In Vitro
Author Affiliations & Notes
  • Nuria Genicio
    Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, United Kingdom
    National Institute for Health Research Biomedical Research Centre, Moorfields Eye Hospital and UCL Institute of Ophthalmology, London, United Kingdom
  • Juan Gallo Paramo
    Comprehensive Cancer Imaging Centre, Imperial College, South Kensington, London, London, United Kingdom
    Department of Chemistry, Imperial College London, South Kensington, London, United Kingdom
  • Alex J. Shortt
    Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, United Kingdom
    National Institute for Health Research Biomedical Research Centre, Moorfields Eye Hospital and UCL Institute of Ophthalmology, London, United Kingdom
    Cornea and External Disease Service, Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom
  • Correspondence: Alex J. Shortt, Cornea and External Disease Service, NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, Bath Street, London, EC1V 9EL, UK; a.shortt@ucl.ac.uk
Investigative Ophthalmology & Visual Science May 2015, Vol.56, 3051-3059. doi:10.1167/iovs.14-15973
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      Nuria Genicio, Juan Gallo Paramo, Alex J. Shortt; Quantum Dot Labeling and Tracking of Cultured Limbal Epithelial Cell Transplants In Vitro. Invest. Ophthalmol. Vis. Sci. 2015;56(5):3051-3059. doi: 10.1167/iovs.14-15973.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Cultured human limbal epithelial cells (HLECs) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behavior, and long-term fate after transplantation. The aim of this research was to evaluate, in vitro, quantum dot (Qdot) technology as a tool for tracking transplanted HLECs.

Methods.: In vitro cultured HLECs were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony-forming efficiency assays and expression of CK3, P63alpha, and ABCG2. Sheets of cultured HLECs labeled with Qdot nanocrystals were transplanted onto decellularized human corneoscleral rims in an organ culture model and observed to investigate the behavior of transplanted cells.

Results.: Quantum dot labeling had no detrimental effect on HLEC viability or function in vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks after transplantation in our organ culture model. In addition, it was possible to label and observe epithelial cells in intact human corneas by using the Rostock corneal module adapted for use with the Heidelberg HRA.

Conclusions.: This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks after transplantation; however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterization in in vivo models are required.

Transplantation of ex vivo cultured limbal epithelial stem cells (LESCs) is an established treatment for human patients with corneal limbal stem cell deficiency (LSCD).13 Studies reporting outcomes of this treatment use a variety of different measures and means of assessment.2,4,5 These include grading of clinical outcomes, impression cytology sampling of the ocular surface with cytokeratin profiling, in vivo confocal microscopy, and examination of excised penetrating keratoplasty buttons. These outcome measures provide the evidence base for the effectiveness of this therapy and for its increasing clinical use.69 
One aspect of this cell therapy that cannot be assessed with certainty is the survival and fate of transplanted cells in patients after treatment. Genotyping using PCR technology has been used to determine the origin of the cells populating the ocular surface after treatment, but this approach is limited by the fact that it is an invasive procedure that can be used only in allogeneic transplants to detect differences in donor and recipient DNA profiles.7 Another drawback is that the presence of donor DNA on the ocular surface is not necessarily evidence of successful engraftment of functioning donor stem cells. To date, no study has demonstrated direct evidence that the improved clinical appearance of the cornea after this therapy is directly related to the survival and function of transplanted cells. The fate of transplanted cells is unknown and can be truly understood only by tracking and monitoring transplanted cells in real time in vivo. 
The tracking of cells in vivo is a vibrant research area and new tools are emerging.10 One such tool is quantum dot nanotechnology.11 Quantum dots (Qdots) are tuneable fluorophores, particles that can be efficiently excited at any wavelength shorter than their emission wavelength then re-emit photons at a longer wavelength. Variation of material and size of these semiconductors results in different emission colors (spanning from the UV to the infrared) but all of which are excitable with a single wavelength.12 Quantum dots have been estimated to be up to 20 times brighter and 100 times more stable than standard organic dyes.12 These unique properties enable them to be used in biomedical applications, such as imaging and tracking. The high photostability and emission intensity make them an ideal tool for ultrasensitive detection and long-term imaging.13 The surface chemistry of Qdots dictates how they behave in cell cultures. Biofunctionalization, the coating of quantum dots with biologically active agents, can render Qdots functional, biocompatible, water soluble, and safe when interacting with biological systems. Most common and suitable surface coatings are biomolecules, such as peptides, sugars, and biopolymers. Although several groups have applied Qdots in vivo in animal models, they have yet to be used in humans.14 
The aim of this work was to investigate the safety and efficacy of Qdots as a noninvasive method of labeling and tracking mixed populations of human limbal epithelial cells (HLECs) in an organ culture model of this cell therapy. 
Methods
Tissue for In Vitro Imaging, Immunofluorescence, and Cell Culture Studies
Cadaveric human corneas with research consent were obtained from the Moorfields Eye Hospital Lions Eye Bank (London, UK), North Carolina Eye Bank (Winston-Salem, NC, USA), and San Diego Eye Bank (San Diego, CA, USA). Experiments on human tissue were approved by the local research ethics committee. Cadaveric rabbit corneas were obtained from an abattoir. 
Labeling of Primary HLECs in Culture
Primary HLECs were isolated from cadaveric human donor corneas and cultured on a mitomycin-C (MMC) growth-arrested 3T3 feeder layer in corneal epithelial culture medium as previously described.15 Cells were seeded and cultured in Permanox 4-well chamber slides (Nunc Lab-Tek Chamber Slide system; Sigma-Aldrich Company Ltd., Dorset, England) for 7 to 10 days following cell isolation until macroscopically visible colonies had grown. They were then labeled using a commercially available kit, the Qtracker 655 Cell Labeling Kit from Molecular Probes/Invitrogen (Q25021MP; Molecular Probes/Invitrogen, Paisley, UK). This kit uses a custom cell-penetrating peptide bound to the surface of Qdots to induce their cellular uptake. The manufacturers' protocol was followed: 4 μL each of Qtracker component A, which comprised the nanocrystals, and component B, which contained the cell-penetrating peptide, were mixed in a 2.0-mL microcentrifuge tube. This solution was left to stand at room temperature for 5 minutes. Serum containing cell culture medium (800 μL) was added and the solution was vortexed for 30 seconds to produce the final labeling solution of which 200 μL was added to each well of the 4-well chamber slide containing HLECs. Slides containing cells that had been through the labeling process with the exception of the Qdot reagent were used as negative controls The cells were incubated at 37°C for 60 minutes, after which they were washed twice with culture medium. 
Imaging of Qdot-labeled Cells
Cells were imaged using both fluorescence and confocal microscopy. An Olympus digital light/fluorescence microscopy system (Olympus Corporation, Tokyo, Japan) was used to capture fluorescence images. An HBO lamp (Olympus Corporation) with a 450- to 550-nm band pass filter (Olympus Corporation) was used for excitation, and a 650-nm long-pass filter for detection. These images were analyzed using image analysis software (Soft Imaging System GmbH, Munster, Germany). Confocal images were captured using a Zeiss LSM 510 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). A 548-nm Argon laser (Olympus Corporation) was used for excitation and a 650-nm long-pass filter (Olympus Corporation) for detection. 
Electron Microscopy to Confirm the Cellular Location of Qdots
Samples of HLECs labeled with Qdots were fixed for 2 to 12 hours with a mixture of 1% paraformaldehyde and 3% glutaraldehyde in 0.1 M sodium cacodylate buffered to pH 7.4 with 0.1 M HCl at 4°C. Samples were rinsed with three changes of 0.1 M sodium cacodylate (pH 7.4) and transferred to 1.0% aqueous osmium tetroxide for a further 2 hours. Following osmication, samples were dehydrated in ascending alcohols and propylene oxide and infiltrated overnight in a 1:1 mixture of propylene oxide and araldite. Following two 3-hour changes of araldite, specimens were embedded and cured overnight at 60°C. Semi- and ultrathin sections were cut with an ultracut S microtome (Leica Microsystems GmBH, Wetzlar, Germany) fitted with a diamond knife. Sections for electron microscopy were collected on copper grids and examined either stained in uranyl acetate and lead citrate or unstained in a Jeol 1200 EX transmission electron microscope (Jeol, Tokyo, Japan) operating at 80 kV. 
Toxicity of Qdot in Labeled HLECs
The HLECs were cultured in Permanox four-well chamber slides and then labeled with 200 μL Qdot nanocrystal labeling solution. Slides containing cells that had been through the labeling process, with the exception of the Qdot reagent, were used as negative controls. The ratio of living to dead cells was assessed at 24 hours after labeling and again at 72 hours after labeling using a live/dead assay kit (LIVE/DEAD Viability Cytotoxicity Assay Kit; Invitrogen/Molecular Probes). Images were captured using the Olympus digital light/fluorescence microscopy system, and the proportion of live and dead cells was counted. This was performed three times, a different cadaveric human donor cornea being used each time (n = 3). 
Labeling Efficiency and Duration of Labeling in Proliferating HLECs
Colonies of HLECs were established from four different cadaveric human donor corneas. Cells from each cornea were seeded into two wells of a six-well plate, yielding eight cultures in total. Once macroscopically visible colonies had formed, the cells were incubated with 1 mL Qdot labeling solution (as previously described) for 60 minutes. The cultures were washed three times with culture medium, then incubated as normal. At 24 hours after labeling, images were collected using the Olympus digital light/fluorescence microscopy system. Ten images were collected from different HLEC colonies in each of eight replicate wells. The number of Qdot-containing cells was counted and this was expressed as a percentage of the total number of cells. The cultures were then passaged and reseeded onto fresh feeder layers at a density of 2000 cells per well of a six-well plate. Six days later (day 7 following the initial labeling of cells), the percentage of cells containing Qdots was again evaluated. The cells were then passaged for a third and final time and the percentage of cells containing Qdots was counted again 14 days after the initial labeling. 
Effect of Qdots on Differentiation of HLECs
The differentiation state of HLECs was determined using immunofluorescence to assess expression of the putative LESC markers ABCG2 and p63alpha and the corneal epithelium differentiation marker CK3 (cytokeratin 3). Human limbal epithelial cells were cultured in four-well chamber slides and labeled with Qdots. Slides containing unlabeled cells were used as negative controls. Five days after labeling, cells were washed, fixed with 3.2% paraformaldehyde (ABCG2/CK3) or methanol (p63alpha) for 5 minutes at room temperature, washed with PBS containing 0.5% Triton X-100 (Sigma-Aldrich Company Ltd.), and blocking of nonspecific primary antibody binding was performed using 10% goat serum in PBS for 30 minutes. Incubation with primary antibodies to ABCG2 (Chemicon/Merck Milllipore Ltd., Billerica, MA, USA), p63alpha (Cell Signaling Technologies, Danvers, MA, USA), and CK3 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and CK3 (Santa Cruz Biotechnologies) was carried out overnight at 4°C. The slides were washed and incubation with the secondary antibody was performed at room temperature for 2 hours. Slides were then washed and mounted using Vectashield containing 4′,6-diamidino-2-phenylindole (Vector Labs). Slides were examined and images collected using a Zeiss LSM 510 confocal microscope. 
Effect of Qdot Labeling on Colony-Forming Efficiency of HLECs
Human limbal epithelial cells were isolated and 200 cells were seeded into each well of a six-well plate containing an MMC growth-arrested 3T3 feeder layer. On day 1 after seeding, cells in half of the wells were labeled with 1 mL Qdot nanocrystal labeling solution for 60 minutes followed by three washes with culture medium. The remaining unlabeled HLECs served as controls. Human limbal epithelial cells were cultured until macroscopically visible colonies had grown. This took between 7 and 10 days following cell isolation. The presence of Qdots within colonies was evaluated by fluorescence microscopy. Cells were then fixed with methanol at −20°C for 30 minutes, rehydrated with PBS, and stained with 1% Rhodamine B for 30 minutes at 37°C, washed with copious amounts of water, and photographed. The number of colonies larger than 2 mm in diameter was counted and expressed as a proportion of cells seeded (colony-forming efficiency).16,17 Clonal analysis was not performed. 
Tracking of Transplanted Cells in an Organ Culture Model
Human corneoscleral rims were obtained from the Moorfields Lions Eye Bank. They were decellularized by incubation in 100 mM EDTA (Sigma-Aldrich Company Ltd.) for 2 hours at 37°C followed by gentle mechanical abrasion of the epithelium. This protocol was effective in removing virtually all the native epithelial cells, as previously described (Supplementary Fig. S1).15 Human limbal epithelial cells were isolated as above and cultured in a 12-well plate until they formed confluent epithelial sheets. Once confluent, they were incubated with 500 μL Qdot nanocrystal labeling solution for 60 minutes, then rinsed twice with culture medium. Dispase II (Roche Diagnostics, Ltd., East Sussex, UK) was used to separate the intact sheet of cells from the culture plate. The cell sheet was transplanted onto the limbus of a decellularized corneal scleral rim and cultured for 2 weeks. At 7 days and 14 days after transplantation, the sample was examined using a Zeiss LSM 510 confocal microscope. 
Development of Method of Tracking Qdots Ex Vivo Using Existing Clinical Technology
To determine whether existing confocal microscopes in clinical practice could be used to detect Qdots, the Rostock corneal module (Heidelberg Engineering GMbH, Heidelberg, Germany) for the Heidelberg Retinal Tomograph II (HRT II) was modified to enable its attachment to the Heidelberg Spectralis (HRA-II/OCT). This modification required the fabrication of a plastic sleeve that fitted around the imaging head of the HRA-II that enabled the Rostock corneal module to be securely fitted onto it and the position of the Rostock module to be adjusted. 
Fresh whole cadaveric rabbit eyes were obtained from an abattoir. Rabbit corneas were chosen because their size made handling easy and because a ready supply was available. The globes were sterilized by washing with 5% povidone iodine for 1 minute then rinsing three times in antibiotic and antimycotic solution (100 IU/mL each penicillin/streptomycin and 0.25 μg/mL amphotericin B; Gibco Life Technologies, Invitrogen, Paisley, UK). The corneoscleral disc was removed from the globe and placed epithelium down in the well of a six-well plate containing 3 mL Qdot nanocrystal labeling solution so that the entire corneal epithelium was submerged. The tissue was placed in a tissue culture incubator for 4 hours at 37°C. The corneas were then removed, washed twice with culture medium, and incubated in moist chambers. At 12 hours after labeling, the corneal epithelium was imaged using the Rostock corneal module attached to the HRA-II. 
Results
Labeling of HLECs in Culture
Intracellular Qdots demonstrated a broad excitation range between 405 and 615 nm. In contrast, they had narrow range of emission wavelengths with a peak at 655 nm. The pattern and intensity of signal from labeled cells was very homogeneous. The appearance of these particles on fluorescence microscopy and the morphology of labeled HLECs are demonstrated in Figure 1
Figure 1
 
Quantum dot labeling of primary HLECs. Phase-contrast microscopy (A, C) and fluorescence microscopy (B, D) of Qdot-labeled primary HLECs using ×20 objective (A, B) and ×40 objective (C, D). These images demonstrate that Qdots are not visible with phase-contrast microscopy using white light. Images (C) and (D) demonstrate the corresponding area in (A) and (C) when viewed with a green wavelength light (450–550 nm) and viewed using a red filter. The Qdots (red) are seen to fluoresce brightly and appear to be aggregated in clumps within individual cells.
Figure 1
 
Quantum dot labeling of primary HLECs. Phase-contrast microscopy (A, C) and fluorescence microscopy (B, D) of Qdot-labeled primary HLECs using ×20 objective (A, B) and ×40 objective (C, D). These images demonstrate that Qdots are not visible with phase-contrast microscopy using white light. Images (C) and (D) demonstrate the corresponding area in (A) and (C) when viewed with a green wavelength light (450–550 nm) and viewed using a red filter. The Qdots (red) are seen to fluoresce brightly and appear to be aggregated in clumps within individual cells.
Confocal and Electron Microscopy Confirmed the Intracellular Location of Qdots
Confocal and electron microscopy demonstrated that at 24 hours after labeling, Qdots were present in membrane-less aggregates freely distributed throughout the cytoplasm of labeled cells (Fig. 2). At 1 week after labeling, Qdots were located primarily in the nucleus but some cytoplasmic aggregates were present, which on electron microscopy were found to be encapsulated by a membrane and associated with the Golgi apparatus. At 2 weeks after labeling, Qdots were located primarily in the nucleus (Fig. 2). 
Figure 2
 
Intracellular localization of Qdots. Confocal microscopy ([A], merged phase and fluorescence; [B], fluorescence image) and electron microscopy (C, D) of Qdot-labeled primary HLECs taken 24 hours after labeling showing the widespread distribution of Qdot aggregates throughout the cytoplasm. The cytoplasmic aggregates are not encapsulated by a cellular membrane, but rather are free distributed. At 1 week after labeling, confocal microscopy ([E], merged phase and fluorescence image; [F], fluorescence image) and electron microscopy (F, G) show that Qdots are primarily located in the nucleus. Some cytoplasmic aggregates are present at this time point, which on electron microscopy were found to be encapsulated by a membrane and associated with the Golgi apparatus (F). At 2 weeks after labeling, Qdots were located primarily in the nucleus with few cytoplasmic aggregates ([H], merged phase and fluorescence image; [I], fluorescence image).
Figure 2
 
Intracellular localization of Qdots. Confocal microscopy ([A], merged phase and fluorescence; [B], fluorescence image) and electron microscopy (C, D) of Qdot-labeled primary HLECs taken 24 hours after labeling showing the widespread distribution of Qdot aggregates throughout the cytoplasm. The cytoplasmic aggregates are not encapsulated by a cellular membrane, but rather are free distributed. At 1 week after labeling, confocal microscopy ([E], merged phase and fluorescence image; [F], fluorescence image) and electron microscopy (F, G) show that Qdots are primarily located in the nucleus. Some cytoplasmic aggregates are present at this time point, which on electron microscopy were found to be encapsulated by a membrane and associated with the Golgi apparatus (F). At 2 weeks after labeling, Qdots were located primarily in the nucleus with few cytoplasmic aggregates ([H], merged phase and fluorescence image; [I], fluorescence image).
Toxicity of Qdots in Labeled HLECs
There were no differences in the number of living or dead cells between labeled and unlabeled cells at 24 hours or 72 hours after labeling (Fig. 3). 
Figure 3
 
Results of live-dead assay to evaluate the toxicity of Qdots. Unlabeled control cells (A) and cells labeled with Qdots (B) demonstrate a similar ratio of live to dead cells. Scale bars: 50 μm. (C) Quantification of the number of live (green bars) and dead cells (red bars) in both groups reveals no difference in the percentage of live or dead cells in either group. Error bars represent the SEM (n = 3).
Figure 3
 
Results of live-dead assay to evaluate the toxicity of Qdots. Unlabeled control cells (A) and cells labeled with Qdots (B) demonstrate a similar ratio of live to dead cells. Scale bars: 50 μm. (C) Quantification of the number of live (green bars) and dead cells (red bars) in both groups reveals no difference in the percentage of live or dead cells in either group. Error bars represent the SEM (n = 3).
Labeling Efficiency and Duration of Labeling in Proliferating HLECs
The labeling efficiency at 24 hours after labeling was 94.6% ± 3.3% (mean ± SEM, n = 8). Continued proliferation resulted in a gradual reduction in percentage of cells containing Qdots to 63.0% ± 4.6% at 7 days and 22.4% ± 6.3% at 14 days after labeling. 
Effect of Qdots on Colony-Forming Efficiency and Differentiation of HLECs
The colony-forming efficiency of labeled HLECs (8.5% ± 1.8%) (mean ± SEM) was similar to that of nonlabeled HLECs (10.0% ± 2.9%) (Fig. 4). There was no significant difference between groups (t-test). This indicates that neither the labeling process, nor the presence of Qdots within cells interferes with the capacity of epithelial cells to form colonies in culture. 
Figure 4
 
Effect of Qdot labeling of primary HLECs on colony-forming efficiency (CFE). (A) Photograph of CFE assay for HLECs labeled with Qdots demonstrating the presence of numerous colonies with classic holoclone morphology. (B) Corresponding photograph for nonlabeled HLECs demonstrating a similar number and size of holoclone colonies. n = 3. (C) Colony-forming efficiency of Qdot-labeled cells versus nonlabeled cells.
Figure 4
 
Effect of Qdot labeling of primary HLECs on colony-forming efficiency (CFE). (A) Photograph of CFE assay for HLECs labeled with Qdots demonstrating the presence of numerous colonies with classic holoclone morphology. (B) Corresponding photograph for nonlabeled HLECs demonstrating a similar number and size of holoclone colonies. n = 3. (C) Colony-forming efficiency of Qdot-labeled cells versus nonlabeled cells.
The degree of differentiation of HLECs was determined using immunofluorescence to assess the expression of the putative LESC “markers” ABCG2 and p63alpha and the corneal epithelium differentiation marker CK3 (Fig. 5). The pattern and distribution of expression of these ligands was identical between groups. 
Figure 5
 
Effect of Qdots on differentiation of HLECs. Confocal microscopic images of immunofluorescence for the differentiation marker CK3 and putative stem cell “markers” p63alpha and ABCG2 at day 5 after labeling of HLECs. Scale bars: 50 μm.
Figure 5
 
Effect of Qdots on differentiation of HLECs. Confocal microscopic images of immunofluorescence for the differentiation marker CK3 and putative stem cell “markers” p63alpha and ABCG2 at day 5 after labeling of HLECs. Scale bars: 50 μm.
Tracking of Transplanted Cells in an Organ Culture Model
Sheets of cultured HLECs labeled with Qdots were successfully transplanted onto decellularized human corneoscleral rims in an organ culture model and observed for up to 14 days to investigate the behavior of transplanted cells (Fig. 6). To ensure that the decellularization protocol was successful in removing all of the native limbal epithelium, decellularized rims that did not receive transplants of cultured HLECs served as a control. These were cultured for up to 14 days alongside those rims that did receive transplants of labeled HLECs. No viable cells were found on the surface of the control sample at any time point, indicating the effectiveness of the decellularization process (data not shown). 
Figure 6
 
Method of culture and transplantation of a labeled sheet of HLECs in an organ culture model. (A) Fluorescence microscopy image of sheet of HLECs that has been labeled with Qdot. Scale bar: 50 μm. (B) Sheet of Qdot-labeled HLECs that has been separated from the culture dish using dispase. (C) Decellularized human corneoscleral rims in an organ culture chamber. (D) Image obtained from recombined corneoscleral rim and HLEC sheet 7 days after transplantation using a Zeiss LSM 510 confocal microscope. Scale bar: 10 μm.
Figure 6
 
Method of culture and transplantation of a labeled sheet of HLECs in an organ culture model. (A) Fluorescence microscopy image of sheet of HLECs that has been labeled with Qdot. Scale bar: 50 μm. (B) Sheet of Qdot-labeled HLECs that has been separated from the culture dish using dispase. (C) Decellularized human corneoscleral rims in an organ culture chamber. (D) Image obtained from recombined corneoscleral rim and HLEC sheet 7 days after transplantation using a Zeiss LSM 510 confocal microscope. Scale bar: 10 μm.
Results of Transplantation at 7 Days.
At 7 days after transplantation, an intact epithelial sheet covered the entire cornea and limbus. A substantial proportion of these cells contained Qdots, indicating that the transplanted cells had survived for the first 7 days. It was noticeable that in the limbal region, more cells still contained Qdots than those on the peripheral cornea. Also, the limbal area contained clusters of very Qdot-bright cells. These were not present on the cornea (Fig. 7). 
Figure 7
 
Results of transplanting labeled HLEC sheets onto decellularized corneoscleral rims. Columns (A) and (D) indicate the location from which the adjacent confocal microscopic images were collected. Columns (B) and (C) demonstrated the presence of labeled HLECs on the surface of the cornea at 7 days and 14 days post transplantation. Columns (E) and (F) demonstrate the appearance of labeled HLECs on the surface of the limbus at 7 days and 14 days post transplantation. At 7 days post transplantation, an intact epithelial sheet covered the entire cornea (B) and limbus (E). A substantial proportion of these cells contained Qdots, indicating that the transplanted cells had survived for the first 7 days. It was noticeable that in the limbal region, more cells still contained Qdots (E) than those on the peripheral cornea (B). Also, the limbal area contained clusters of very Qdot-bright cells (E). These were not present on the cornea (B). At day 14, the number of Qdot-labeled cells had decreased but there was still an intact layer of epithelium covering the cornea (C) and limbus (F). The decline in Qdot signal was greatest in the cornea (C) and less in the limbus (F). The clusters of Qdot-bright cells were still present in the limbal region at 2 weeks, but they were less raised and prominent than initially (F).
Figure 7
 
Results of transplanting labeled HLEC sheets onto decellularized corneoscleral rims. Columns (A) and (D) indicate the location from which the adjacent confocal microscopic images were collected. Columns (B) and (C) demonstrated the presence of labeled HLECs on the surface of the cornea at 7 days and 14 days post transplantation. Columns (E) and (F) demonstrate the appearance of labeled HLECs on the surface of the limbus at 7 days and 14 days post transplantation. At 7 days post transplantation, an intact epithelial sheet covered the entire cornea (B) and limbus (E). A substantial proportion of these cells contained Qdots, indicating that the transplanted cells had survived for the first 7 days. It was noticeable that in the limbal region, more cells still contained Qdots (E) than those on the peripheral cornea (B). Also, the limbal area contained clusters of very Qdot-bright cells (E). These were not present on the cornea (B). At day 14, the number of Qdot-labeled cells had decreased but there was still an intact layer of epithelium covering the cornea (C) and limbus (F). The decline in Qdot signal was greatest in the cornea (C) and less in the limbus (F). The clusters of Qdot-bright cells were still present in the limbal region at 2 weeks, but they were less raised and prominent than initially (F).
Results of Transplantation at 14 Days.
At day 14, the number of Qdot-labeled cells had decreased but there was still an intact layer of epithelium covering the corneal and limbus. The decline in Qdot signal was greatest in the cornea and less in the limbus. The clusters of Qdot-bright cells were still present in the limbal region at 2 weeks, but they were less raised and prominent than initially (Fig. 7). 
Development of Method of Tracking Qdots In Vivo Using Existing Clinical Technology
Using the Rostock corneal module attached to the Heidelberg HRA-II, it was possible to label and observe epithelial cells in rabbit corneas ex vivo. The HRA-II contains three different wavelength lasers for excitation and a range of emission filters. The optimal configuration for detecting Qdots was found to be the same as that for performing fundus autofluorescence scanning (i.e., the 488-nm solid-state laser was used for excitation and emitted light detected above 500 nm using a long-pass filter). The appearance of Qdot-labeled rabbit corneal epithelial cells is demonstrated in Figure 8. Although fluorescence from Qdots could be detected with ease, the quality of images of individual rabbit epithelial cells was poor. 
Figure 8
 
Ex vivo confocal microscopy of rabbit corneal epithelium labeled with Qdots. Images were acquired using the Rostock corneal (Heidelberg) module modified to attach onto the Spectralis OCT clinical imaging equipment. (A, B) Unlabeled control rabbit cornea. Individual corneal epithelial cells can be distinguished. (C, D) Quantum dot–labeled rabbit corneal epithelium. The white dots scattered throughout the field of view are the Qdot aggregates within the cytoplasm of labeled cells. Scale bar: 50 μm.
Figure 8
 
Ex vivo confocal microscopy of rabbit corneal epithelium labeled with Qdots. Images were acquired using the Rostock corneal (Heidelberg) module modified to attach onto the Spectralis OCT clinical imaging equipment. (A, B) Unlabeled control rabbit cornea. Individual corneal epithelial cells can be distinguished. (C, D) Quantum dot–labeled rabbit corneal epithelium. The white dots scattered throughout the field of view are the Qdot aggregates within the cytoplasm of labeled cells. Scale bar: 50 μm.
Discussion
The clinical use of ex vivo expanded HLECs to treat patients with LSCD has preceded a clear scientific understanding of the mechanism of action of this therapy. Specifically, it is unclear whether ex vivo cultured HLEC cell transplants reintegrate into the recipient's LESC niche and continue to function in the long term to maintain a healthy corneal epithelium. An alternative hypothesis is that the temporary restoration of a healthy corneal epithelium by the transplanted cells acts as a biological bandage and promotes regeneration of the recipient's own LESC population. This question could most definitively be answered by tracking transplanted cells in vivo. This has not been performed to date, not least due to the lack of laboratory data on labeling efficiency, duration, toxicity, and effects on transplanted HLEC function. The aim of this study was to determine whether Qdots could be used to label and track ex vivo cultured limbal epithelial cells without impacting on cell viability and function. We have shown that Qdots have potential in this regard and have demonstrated proof of principle for their use in tracking transplanted cells in the cornea. Last, we demonstrated how existing clinical equipment could be modified to enable in vivo detection of Qdot-labeled cells in animal or human studies. 
The ability to follow the distribution and migration of biologically active cells in living organisms is crucial for both the development of cell-based therapies and for the elucidation of biological mechanisms in stem cell research. Two key components are required to achieve this: a method of selectively labeling transplanted cells and a technique for detecting and visualizing the transplanted cells in vivo. 
There are two distinct approaches to cell labeling: indirect and direct labeling of cells. Indirect labeling methods use a reporter gene that is introduced into a cell and translated into enzymes, receptors, or fluorescent or bioluminescent proteins. If the expression of the reporter gene is stable, labeled cells can be observed over the entire lifetime of cells. Indirect labeling has proved an immensely valuable tool in terms of basic research, but there are significant obstacles to the translation of this technology into clinical practice, not least the ethical and regulatory issues that result from the use of genetically modified cells. Direct cell labeling refers to attachment of exogenous detectable labels (fluorophores, magnetic particles, or radioisotopes) to the cell membrane or their intracellular incorporation using a variety of techniques. Direct labeling is technically straightforward and does not involve genetic modification of the cell. It is therefore a safer and more clinically useful approach and the one we chose to use. 
By virtue of its transparency, the cornea is an ideal tissue in which to directly observe transplanted stem cells using a fluorophore label and in vivo microscopy. This combination can achieve a level of resolution that other detection systems, such as magnetic resonance imaging, single-photon emission computed tomography, and positron emission tomography, cannot.10 When selecting a fluorophore, we set the criteria that it should be nontoxic, photostable (i.e., resistant to photobleaching), and sufficiently bright to generate a signal detectable through living tissues. We chose to use Qdots because they exhibit some important differences as compared with traditional fluorophores, such as organic fluorescent dyes and naturally fluorescent proteins. 
Structurally, Qdots are inorganic composites consisting of nanometer-scale (roughly protein-sized) clusters of atoms (containing from a few hundred to a few thousand atoms) of a semiconductor material (cadmium mixed with selenium or tellurium has been the most common for biological applications), which has been coated with an additional semiconductor shell (ZnS) to improve the stability and optical properties of the material. Quantum dots fluoresce in a completely different way than do traditional fluorophores. In Qdots, fluorescence is achieved via the formation of excitons, or Coulomb-correlated electron-hole pairs that are created between the two semiconductor materials. The exciton can be thought of as analogous to the excited state of traditional fluorophores; however, excitons typically have much longer lifetimes (>10 ns, up to ∼200 nanoseconds, versus <5 ns for organic fluorophores).18 This structural and functional difference engenders two key advantages of Qdots over conventional fluorophores. First, photostability of Qdot nanocrystals is many orders of magnitude greater than that associated with traditional fluorescent molecules and hence photo-induced deterioration of the emitted signal (known as bleaching) is substantially less than that seen with other types of fluorophores. Second, the optical efficiency of Qdots (quantum yield, in general terms the amount of light emitted relative to that absorbed) is many times that observed for other classes of fluorophores. As a result, less illumination is required to achieve a detectable signal from Qdots than from other classes of fluorophores. One more general advantage over classic fluorophores comes from multiplexing. Different color Qdots can be excited with a single wavelength and the narrow emission band of Qdots makes deconvolution straightforward. 
We used a commercial Qdot kit that uses a custom cell-penetrating peptide bound to the surface of Qdots to induce their cellular uptake.19,20 These peptides are a beta-amino acid analogue of Tat 47-57, the portion of the human immunodeficiency virus (HIV) Tat DNA-binding protein that is responsible for the ability of the HIV Tat protein to readily cross the cell membrane.21 Tat 47-75 analogues have negligible cytotoxicity in HeLa, Chinese hamster ovary (CHO-K1), and human umbilical vein endothelial cells.22 The corneal toxicity of cell-penetrating peptides has been assessed in a rabbit drug-delivery model and found to be negligible.23 The intracellular target of Tat 47-57 and its analogues is the nucleus.19,20 In the initial 24 hours after labeling, Qdots formed membrane-less aggregates throughout the cytoplasm, presumably reflecting the fact that they are trafficking to the nucleus. By 1 week after labeling, Qdots were primarily located in the nucleus, as expected. Some cytoplasmic aggregates were still present at this time point and on electron microscopy these were found to be encapsulated by a membrane and associated with the Golgi apparatus. These findings suggest that some metabolism of and possibly exocytosis of Qdots were occurring at this stage. By 2 weeks after labeling, Qdots were located primarily in the nucleus, with few cytoplasmic aggregates, suggesting that by this time point the labeling was stable. There is evidence that mouse embryonic stem cells can both degrade and exocytose the same commercial Qdot system that we have used, resulting in loss of Qdot signal within 48 hours of labeling.24 In contrast, the same study found that mouse embryonic fibroblasts did not degrade or exocytose Qdots, and that the reduction in signal seen over time in this cell type was due to the diluting effect of cell division. Our results show that Qdots can result in a labeling efficiency of more than 94% immediately after labeling, which gradually reduced to 22% after 14 days as the cells proliferated. This is remarkably similar to the findings in mouse embryonic fibroblasts24 and we therefore hypothesize that although limited metabolism and exocytosis do occur in HLECs, the primary reason for the reduction in Qdot signal over time is dilution due to cell division. 
Another significant limitation of our organ culture model is that we have been unsuccessful in maintaining limbal epithelial cell sheets (both labeled and unlabeled) in culture on stromal explants for longer than 2 weeks. This is because the transplanted epithelium differentiates rapidly between day 14 and 21 post transplantation. Although our model does not permit us to track cells beyond 2 weeks, this does not mean that quantum dot–labeled cells will be undetectable beyond this time point. In vivo studies will be necessary to determine the duration of labeling with certainty. Two-photon imaging has been used to demonstrate that Qdots can be retained within the cornea for up to 26 days in an in vivo mouse model, albeit this was a model of toxicity and not a model of cell tracking.25 
For Qdots to be used clinically, it is essential for their toxicity to be investigated. It was encouraging to find that a high level of labeling efficiency could be achieved and that neither the labeling process nor the ongoing presence of Qdots within cells caused toxicity or promoted differentiation. This is in contrast with one previous study by Kuo et al.25 in which two-photon microscopic imaging was used to study the distribution of chemically similar (CdSe/ZnS core/shell) Qdots throughout the cornea. Kuo et al.25 found evidence of cytotoxicity in bovine corneal stromal cells. Cell viability decreased significantly as the Qdot concentration and incubation period increased. The reasons for the discrepancy between this study and our results could be that different cell types or even different species handle Qdots differently. Alternatively, the different concentrations of Qdots used (50 nM versus 10 μg/mL in our study) and exposure periods (24–48 hours versus 60 minutes in our study) reflect the fact that Kuo et al.25 were seeking to define limits of toxicity, whereas we were using an optimized and validated protocol for cell labeling. 
When assessing the effect of Qdots on proliferation and differentiation of HLECs, we did not perform clonal analysis. We used a mixed population of cells that will have included holoclones, meroclones, and possibly some paraclones. We cannot therefore be certain whether these results apply specifically to LESCs (holoclones), transient amplifying cells (meroclones), or both.16,17 
Our organ culture transplantation model showed that we could still detect transplanted cells at 14 days after transplantation. At present, there is a reliable method of directly visualizing transplanted ex vivo cultured HLECs transplanted in human patients. Our techniques could be used to monitor cells for up 2 weeks and perhaps longer, depending on the rate of proliferation of transplanted cells. Although it would be desirable to be able to track cells for their entire lifetime, the technology to do this safely in vivo is not currently available. The ability to track cells even for 1 month after transplantation would lead to significant advances in understanding the biological mechanism of action and posttreatment behavior of such transplants. 
Based on the observations that LESCs divide infrequently in vivo and therefore retain bromodeoxyuridine longer than their differentiated progeny26 and that the rate of proliferation determines the rate of label dilution and therefore duration of labeling, we hypothesize that post transplantation cells that divide rapidly, such as transient amplifying cells, will see the number of Qdots in their cytoplasm rapidly reduce to undetectable levels, whereas cells that divide infrequently, as stem cells do, may retain a high number of Qdots. In our organ culture model of HLEC transplantation, it was observed that there was a marked heterogeneity of Qdot signal with the brightest cells containing most Qdots being found in clusters at the limbus. This suggests that the limbus has an effect on the transplanted cells, which the cornea does not. It also suggests that some of these slow-cycling cells may reintegrate into the limbus and remain in a slow-cycling or quiescent state. From our data it is not possible to confirm that these cells possess a limbal stem cell phenotype but further work will aim to investigate whether this is indeed the case. 
In vivo confocal microscopy has been increasingly used to investigate the cornea in health and disease. In particular, the Rostock corneal module for the HRT II has enabled cellular-level investigation of corneal structure and the limbal stem cell niche.15,27,28 Our results from ex vivo rabbit corneas demonstrate that the Rostock corneal module can be modified to work with the Heidelberg Spectralis HRA-OCT to enable in vivo detection of cells labeled with Qdots. There is undoubtedly room for a more precisely engineered method of combining in vivo confocal microscopy with fluorescence detection, but the data we present demonstrate that this is technically possible. 
Conclusions
This work demonstrates that Qdots can be used to label HLECs for up to 2 weeks in culture without causing toxicity or differentiation and that the technology exists to image Qdots in vivo in humans. However, there are a number of obstacles that need to be overcome before Qdots can be realistically used clinically. The main limitation of this study is that our organ culture transplantation model permits tracking of cells for only 2 weeks at present. It is possible that higher-resolution imaging, prevention of Qdot aggregation within cells, and modifications to the designs of the Qdots themselves may prolong the duration of labeling. 
Acknowledgments
Provisional data from this manuscript was presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, United States, May 3, 2007. 
Supported by a research training fellowship from the Medical Research Council, UK (London, UK; AJS), and by research grants from Fight for Sight UK (London, UK) and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology (London, UK). The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health. No author has a proprietary interest in this work. The authors alone are responsible for the content and writing of the article. 
Disclosure: N. Genicio, None; J. Gallo Paramo, None; A.J. Shortt, None 
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Figure 1
 
Quantum dot labeling of primary HLECs. Phase-contrast microscopy (A, C) and fluorescence microscopy (B, D) of Qdot-labeled primary HLECs using ×20 objective (A, B) and ×40 objective (C, D). These images demonstrate that Qdots are not visible with phase-contrast microscopy using white light. Images (C) and (D) demonstrate the corresponding area in (A) and (C) when viewed with a green wavelength light (450–550 nm) and viewed using a red filter. The Qdots (red) are seen to fluoresce brightly and appear to be aggregated in clumps within individual cells.
Figure 1
 
Quantum dot labeling of primary HLECs. Phase-contrast microscopy (A, C) and fluorescence microscopy (B, D) of Qdot-labeled primary HLECs using ×20 objective (A, B) and ×40 objective (C, D). These images demonstrate that Qdots are not visible with phase-contrast microscopy using white light. Images (C) and (D) demonstrate the corresponding area in (A) and (C) when viewed with a green wavelength light (450–550 nm) and viewed using a red filter. The Qdots (red) are seen to fluoresce brightly and appear to be aggregated in clumps within individual cells.
Figure 2
 
Intracellular localization of Qdots. Confocal microscopy ([A], merged phase and fluorescence; [B], fluorescence image) and electron microscopy (C, D) of Qdot-labeled primary HLECs taken 24 hours after labeling showing the widespread distribution of Qdot aggregates throughout the cytoplasm. The cytoplasmic aggregates are not encapsulated by a cellular membrane, but rather are free distributed. At 1 week after labeling, confocal microscopy ([E], merged phase and fluorescence image; [F], fluorescence image) and electron microscopy (F, G) show that Qdots are primarily located in the nucleus. Some cytoplasmic aggregates are present at this time point, which on electron microscopy were found to be encapsulated by a membrane and associated with the Golgi apparatus (F). At 2 weeks after labeling, Qdots were located primarily in the nucleus with few cytoplasmic aggregates ([H], merged phase and fluorescence image; [I], fluorescence image).
Figure 2
 
Intracellular localization of Qdots. Confocal microscopy ([A], merged phase and fluorescence; [B], fluorescence image) and electron microscopy (C, D) of Qdot-labeled primary HLECs taken 24 hours after labeling showing the widespread distribution of Qdot aggregates throughout the cytoplasm. The cytoplasmic aggregates are not encapsulated by a cellular membrane, but rather are free distributed. At 1 week after labeling, confocal microscopy ([E], merged phase and fluorescence image; [F], fluorescence image) and electron microscopy (F, G) show that Qdots are primarily located in the nucleus. Some cytoplasmic aggregates are present at this time point, which on electron microscopy were found to be encapsulated by a membrane and associated with the Golgi apparatus (F). At 2 weeks after labeling, Qdots were located primarily in the nucleus with few cytoplasmic aggregates ([H], merged phase and fluorescence image; [I], fluorescence image).
Figure 3
 
Results of live-dead assay to evaluate the toxicity of Qdots. Unlabeled control cells (A) and cells labeled with Qdots (B) demonstrate a similar ratio of live to dead cells. Scale bars: 50 μm. (C) Quantification of the number of live (green bars) and dead cells (red bars) in both groups reveals no difference in the percentage of live or dead cells in either group. Error bars represent the SEM (n = 3).
Figure 3
 
Results of live-dead assay to evaluate the toxicity of Qdots. Unlabeled control cells (A) and cells labeled with Qdots (B) demonstrate a similar ratio of live to dead cells. Scale bars: 50 μm. (C) Quantification of the number of live (green bars) and dead cells (red bars) in both groups reveals no difference in the percentage of live or dead cells in either group. Error bars represent the SEM (n = 3).
Figure 4
 
Effect of Qdot labeling of primary HLECs on colony-forming efficiency (CFE). (A) Photograph of CFE assay for HLECs labeled with Qdots demonstrating the presence of numerous colonies with classic holoclone morphology. (B) Corresponding photograph for nonlabeled HLECs demonstrating a similar number and size of holoclone colonies. n = 3. (C) Colony-forming efficiency of Qdot-labeled cells versus nonlabeled cells.
Figure 4
 
Effect of Qdot labeling of primary HLECs on colony-forming efficiency (CFE). (A) Photograph of CFE assay for HLECs labeled with Qdots demonstrating the presence of numerous colonies with classic holoclone morphology. (B) Corresponding photograph for nonlabeled HLECs demonstrating a similar number and size of holoclone colonies. n = 3. (C) Colony-forming efficiency of Qdot-labeled cells versus nonlabeled cells.
Figure 5
 
Effect of Qdots on differentiation of HLECs. Confocal microscopic images of immunofluorescence for the differentiation marker CK3 and putative stem cell “markers” p63alpha and ABCG2 at day 5 after labeling of HLECs. Scale bars: 50 μm.
Figure 5
 
Effect of Qdots on differentiation of HLECs. Confocal microscopic images of immunofluorescence for the differentiation marker CK3 and putative stem cell “markers” p63alpha and ABCG2 at day 5 after labeling of HLECs. Scale bars: 50 μm.
Figure 6
 
Method of culture and transplantation of a labeled sheet of HLECs in an organ culture model. (A) Fluorescence microscopy image of sheet of HLECs that has been labeled with Qdot. Scale bar: 50 μm. (B) Sheet of Qdot-labeled HLECs that has been separated from the culture dish using dispase. (C) Decellularized human corneoscleral rims in an organ culture chamber. (D) Image obtained from recombined corneoscleral rim and HLEC sheet 7 days after transplantation using a Zeiss LSM 510 confocal microscope. Scale bar: 10 μm.
Figure 6
 
Method of culture and transplantation of a labeled sheet of HLECs in an organ culture model. (A) Fluorescence microscopy image of sheet of HLECs that has been labeled with Qdot. Scale bar: 50 μm. (B) Sheet of Qdot-labeled HLECs that has been separated from the culture dish using dispase. (C) Decellularized human corneoscleral rims in an organ culture chamber. (D) Image obtained from recombined corneoscleral rim and HLEC sheet 7 days after transplantation using a Zeiss LSM 510 confocal microscope. Scale bar: 10 μm.
Figure 7
 
Results of transplanting labeled HLEC sheets onto decellularized corneoscleral rims. Columns (A) and (D) indicate the location from which the adjacent confocal microscopic images were collected. Columns (B) and (C) demonstrated the presence of labeled HLECs on the surface of the cornea at 7 days and 14 days post transplantation. Columns (E) and (F) demonstrate the appearance of labeled HLECs on the surface of the limbus at 7 days and 14 days post transplantation. At 7 days post transplantation, an intact epithelial sheet covered the entire cornea (B) and limbus (E). A substantial proportion of these cells contained Qdots, indicating that the transplanted cells had survived for the first 7 days. It was noticeable that in the limbal region, more cells still contained Qdots (E) than those on the peripheral cornea (B). Also, the limbal area contained clusters of very Qdot-bright cells (E). These were not present on the cornea (B). At day 14, the number of Qdot-labeled cells had decreased but there was still an intact layer of epithelium covering the cornea (C) and limbus (F). The decline in Qdot signal was greatest in the cornea (C) and less in the limbus (F). The clusters of Qdot-bright cells were still present in the limbal region at 2 weeks, but they were less raised and prominent than initially (F).
Figure 7
 
Results of transplanting labeled HLEC sheets onto decellularized corneoscleral rims. Columns (A) and (D) indicate the location from which the adjacent confocal microscopic images were collected. Columns (B) and (C) demonstrated the presence of labeled HLECs on the surface of the cornea at 7 days and 14 days post transplantation. Columns (E) and (F) demonstrate the appearance of labeled HLECs on the surface of the limbus at 7 days and 14 days post transplantation. At 7 days post transplantation, an intact epithelial sheet covered the entire cornea (B) and limbus (E). A substantial proportion of these cells contained Qdots, indicating that the transplanted cells had survived for the first 7 days. It was noticeable that in the limbal region, more cells still contained Qdots (E) than those on the peripheral cornea (B). Also, the limbal area contained clusters of very Qdot-bright cells (E). These were not present on the cornea (B). At day 14, the number of Qdot-labeled cells had decreased but there was still an intact layer of epithelium covering the cornea (C) and limbus (F). The decline in Qdot signal was greatest in the cornea (C) and less in the limbus (F). The clusters of Qdot-bright cells were still present in the limbal region at 2 weeks, but they were less raised and prominent than initially (F).
Figure 8
 
Ex vivo confocal microscopy of rabbit corneal epithelium labeled with Qdots. Images were acquired using the Rostock corneal (Heidelberg) module modified to attach onto the Spectralis OCT clinical imaging equipment. (A, B) Unlabeled control rabbit cornea. Individual corneal epithelial cells can be distinguished. (C, D) Quantum dot–labeled rabbit corneal epithelium. The white dots scattered throughout the field of view are the Qdot aggregates within the cytoplasm of labeled cells. Scale bar: 50 μm.
Figure 8
 
Ex vivo confocal microscopy of rabbit corneal epithelium labeled with Qdots. Images were acquired using the Rostock corneal (Heidelberg) module modified to attach onto the Spectralis OCT clinical imaging equipment. (A, B) Unlabeled control rabbit cornea. Individual corneal epithelial cells can be distinguished. (C, D) Quantum dot–labeled rabbit corneal epithelium. The white dots scattered throughout the field of view are the Qdot aggregates within the cytoplasm of labeled cells. Scale bar: 50 μm.
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