Single-pixel imaging is an imaging technique that has recently attracted a lot of attention from several areas. This paper presents a study on the influence of the Hadamard basis ordering on the image reconstruction quality, using simulation and experimental methods. During this work, five different orderings, Natural, Walsh, Cake-cutting, High Frequency and Random orders, along with two different reconstruction algorithms, TVAL3 and NESTA, were tested. Also, three different noise levels and compression ratios from 0.1 to 1 were evaluated. A single-pixel camera was developed using a digital micromirror device for the experimental phase. For a compression ratio of 0.1, the Cake-cutting order achieved the best reconstruction quality, while the best contrast was achieved by Walsh order. For compression ratios of 0.5, the Walsh and Cake-cutting orders achieved similar results. Both Walsh and Cake-cutting orders reconstructed the images with good quality using compression ratios from 0.3. Finally, the TVAL3 algorithm showed better image reconstruction quality, in comparison with NESTA, when considering compression ratios from 0.1 to 0.5.

Diabetes is accompanied by an increased autofluorescence of the cornea, probably because of accumulation of advanced glycation end products (AGEs). The pathogenic mechanism is still unknown. This study aimed to quantify differences in corneal cell densities between diabetic patients and healthy controls. The left cornea of 15 patients with non-insulin-dependent diabetes mellitus (NIDDM) with level of retinopathy 20 according to the Early Treatment of Diabetic Retinopathy Study (ETDRS) and of 15 healthy controls were examined by noninvasive in vivo confocal microscopy in an observational prospective study. The cell densities in 6 corneal layers were determined along the optical axis of the cornea by using stereologic methods. The average cell density per unit area in the superficial and basal epithelium and the endothelial layer was 725 ± 171, 5950 ± 653, and 2690 ± 302 cells/mm2 in controls and 815 ± 260, 5060 ± 301, and 2660 ± 364 cells/mm2 in diabetic patients. The cell density per unit volume in the anterior, mid-, and posterior stroma was 26,300 ± 4090, 19,390 ± 3120, and 25,700 ± 3260 cells/mm3 in controls and 27,560 ± 3880, 21,930 ± 2110, and 25,790 ± 3090 cells/mm3 in patients with diabetes. In both groups, the density in the midstroma was significantly lower than in both the anterior stroma and the posterior stroma (P < 0.02). The cell density in the basal layer of diabetic patients was significantly lower than in healthy controls (−15.0%, P < 0.0004). In the other layers, no significant differences between both groups (P > 0.07) were observed. The lower basal cell density found in patients with diabetes may result from a combination of different mechanisms including decreased innervation at the subbasal nerve plexus, basement membrane alterations, and higher turnover rate in basal epithelial cells. The lower cell density in the midstroma of diabetic patients and healthy controls may be attributed in part to differences in oxygen concentration in the stromal layers (depths). Changes in cellular density did not seem to be responsible for the increased autofluorescence in diabetes.

Purpose: Method and validation of a technique to quantify cell density in vivo in 6 corneal layers with a scanning slit confocal microscope (SSCM). Method: A confocal image of a small volume in a corneal layer is registered on videotape. Cells or nuclei according to a layer classification are counted manually using an unbiased frame. Surface cell density is calculated from an image on the screen, and volumetric density is obtained using stereological methods. Results: Image distortion on the screen is less than 3%. The classification of a cell layer is verified by determining the position of the measurement volume in the cornea. Validation of density measurements is performed by comparing confocal results with those obtained by histology. The difference between the two methods varies from –24.1% (posterior stroma) to +16.4% (basal layer). Intersession and intrasession repeatability are 8.3 and 5.8%, respectively. The cell density (mean ± SD) in 20 healthy controls in the superficial, basal and endothelial layers was 759 ± 162, 5,817 ± 632 and 2,743 ± 285 cells·mm–2 (surface), and in the anterior, mid and posterior stroma 28,616 ± 3,081, 19,578 ± 4,421 and 26,073 ± 5,962 cells·mm–3 (volumetric). These results are in accordance with those of other investigators. Conclusions: The SSCM can produce repeatable quantitative measurements of corneal cell density in conscious humans.

Purpose: The purpose of this study was to evaluate the feasibility of using two-photon imaging (TPI) to assess the condition of human corneas for transplantation. Methods: Human corneas were imaged after different storage times: short-term (STS), medium-term (MTS), and long-term (LTS) storage. A high-resolution, custom-built 5-dimensional multiphoton microscope with 12-fs pulsed laser excitation was used for image acquisition. Results: Optical discrimination between different corneal layers and sublayers based on their morphologic characteristics revealed by two-photon autofluorescence (AF) is possible. Furthermore, all layers were characterized based on AF lifetimes to gain information on metabolic activities of cells. The NAD(P)H free to protein-bound ratio (a₁/a₂) of epithelial cells increased significantly in both MTS and LTS corneas compared with STS corneas. In endothelial cells, NAD(P)H a₁/a₂ was significantly increased in MTS samples. For keratocytes, the NAD(P)H a₁/a₂ decreased significantly with storage time. This could indicate that the metabolic activity of the epithelial and endothelial cells reduces, whereas the activity of keratocytes increases with storage time. The analysis of the stroma SHG images indicated that the organization of collagen fibers decreases with storage time. The feasibility of measuring the endothelial cell density (ECD) using TPI was demonstrated. An ECD of 1461 ± 190 cells/mm² was obtained for MTS samples based on TPI. Conclusions: TPI can provide information not accessible by current clinical methods, such as the cells' metabolic state and structural organization of the stroma, with subcellular resolution. Thus, it may improve the screening process of corneas prior to transplantation and might help to optimize the storage conditions.

Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.