The anatomy of the foveola reinvestigated

Light and electron microscopy from monkey eyes

Twenty-four monkey eyes (Macaca fascicularis, 14 males, 10 females) were collected after sacrificing the animals under general anesthesia, i.e., intramuscular injection of ketamine hydrochloride followed by an intravenous sodium pentobarbitone (Lethabarb^®, Virbac, Australia) overdose. Monkeys were kept at Covance Laboratories GmbH (Münster, Germany study numbers 0382055, 8260977, 8274007) or SILABE-ADUEIS (Niederhausbergen, France). The Covance Laboratories GmbH test facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). This study was approved by the local Institutional Animal Care and Use Committee (IACUC), headed by Dr. Jörg Luft and the work was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). The monkeys from SILABE-ADUEIS were euthanized due to veterinarian reasons. Since they had not been included in a study before, they do not have a study number. The age of the monkeys varied between four to eight years. The eyes were enucleated 5 min post-mortem, cleaned of orbital tissue, and were slit carefully at the limbus without damaging the ora serata. Then, 200 µl of the fixative (5% glutaraldehyde) were carefully injected into the center of the vitreous. The intraocular pressure was balanced because vitreous could leak out from the opening thereby compensating the volume of the fixative. This protocol has been shown to minimize fixation artefacts according to our own experience. The eyes were then fixed at 4 °C by immersion into 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4; Sigma, St. Louis, MO, USA) overnight for electron microscopy. Glutaraldehyde fixed specimens were post-fixed with 1% OsO₄ at room temperature in 0.1 M cacodylate buffer (pH 7.4), stained with uranyl acetate, and the maculae were excised and embedded in Epon after dehydration in a graded series of ethanol and propylene oxide. Semi-thin sections were stained with toluidine blue and examined by light microscopy (Zeiss Axioplan 2 imaging; Zeiss, Jena, Germany). For electron microscopy, ultrathin sections were made and analyzed with a Zeiss 900 electron microscope (Zeiss, Jena, Germany). The foveae from 24 eyes were sectioned in a sagittal plane until the center of 21 foveolae was found. In three eyes, the foveal centers were missed. The center of the foveola was defined as the site where the cell fiber layers at the bottom of the foveal pit were free of nuclei and were 10 µm thin or less.

Light and electron microscopy from human eyes

Three human eyes from a 57 and 81-year-old male, and a 68-year-old female were fixed 8 and 12 h postmortem at 4 °C by immersion into 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4; Sigma, St. Louis, MO, USA) for half an hour. Then the cornea was removed and fixation continued overnight for electron microscopy. The human eyes were gifts from the Clinical Anatomy of the University of Tuebingen (ethical number for scientific issues 237/2007B01), and taken from full body donors, who had previously given informed consent. Embedding and sectioning was done as described for the monkey eyes.

Evaluation of serial sections through the fovea of monkey and humans

For 3D model reconstruction, semi-thin (700 nm) serial sections were performed from 6 monkey foveae.

Series 1 and 2 comprised 21 sections which correspond after mounting to a foveal tissue piece with a thickness of 14.7 µm. Unexpectedly, due to their curved shape, a foveal cone did not completely fit into such a tissue block. Therefore series 3 and 4 were sectioned in sagittal planes with 41 and 160 sections respectively. Additionally, series 5 and 6 were cut as transverse sections with 450 and 195 sections respectively. Sagittal sections run within or parallel to the optical axis, while transverse sections were made perpendicular to it.

Serial transverse sections were performed from two human foveae. Series 7 from a 68-year-old female and series 8 from an 81-year-old male contained 250 and 460 sections, respectively. From each section, the fovea was photographed at a 600-fold magnification.

3D modelling

The 3D reconstructions of the presented figures and measurements were performed with Amira^® software (version 5.6; FEI, Hillsboro, OR, USA). Using previous embedded fixed marker, the images of the sections were aligned manually following digitization by comparing superimposed slices, translating, and rotating adjacent slides with respect to one another. Additionally, the border of each slide and the regular structures and patterns were used as markers for alignment.

Once the aligned sections were imported into Amira, the structures of interest were labeled using the software segmentation tools. The structures of interest were cone nuclei, inner and outer segments, the outer limiting membrane, and Müller cells. The length of the inner and outer segments of cones from the parafovea and the central fovea were measured using the “Amira-3D length—measurement-tool”. For Fig. 2D and Video S1, areas of interest were selected by visually adjusting the grey value threshold of the volumetric dataset.

Focused ion beam/scanning electron microscopy

Focused ion beam/scanning electron microscopy (FIB/ SEM) tomography data were acquired using a Zeiss Auriga CrossBeam instrument at the Natural and Medical Sciences Institute at the University of Tuebingen (NMI, Reutlingen, Germany) as described (Steinmann et al., 2013). For FIB/SEM analysis, the block of the embedded sample was sputter coated with gold palladium and mounted on an appropriate SEM sample holder. A semi-thin section of the embedded sample was imaged with the light microscope and correlated with the SEM image of the ultramicrotome block face to define the region of interest for three-dimensional analysis. Using a Crossbeam instrument (Zeiss) equipped with a gallium FIB and a low voltage SEM, FIB/SEM serial sectioning tomography was accomplished. The gallium FIB produces thereby a series of cross-sections containing the region of interest. Each of these cross-sections is imaged by the low keV SEM using the energy-selected backscattered (EsB) electron detector for image acquisition. The following parameters were used for SEM: Primary energy of 1.8 keV with the aperture 60 µm: for image acquisition the EsB detector was used with a grid voltage of −1,500 V, i.e., only backscattered electrons with a maximum energy loss of 300 V were used for image acquisition. The resolution was 2,048 × 1,535 pixels with a pixel size of 42.41 nm.

For FIB, the following parameters were used: Primary energy of 30 keV, slicing was performed with a probe current of 2 nA, the slice thickness was 42 nm. In this way cubic voxels were obtained, i.e., the same resolution in x, y, and z, which is good for the reconstruction. The resulting stack of two-dimensional images was utilized for three-dimensional reconstruction using appropriate software.

Wholemount preparations from human retinae

The maculae of five eyes from three donors were excised using a trephine with 1 cm diameter. The donors were two females of 74 and 90 years old, and one male of age 18. Three maculae were fixed in formalin transferred into phosphate buffered saline and mounted on slides covered with a cover glass on wax feet to prevent squeezing of the retinae. Another two eyes were treated identically but fixation was omitted. Finally, the maculae were observed under the light microscope having the condenser replaced by a modification with adjustable mirror (Fig. 3C).

Illumination optics for measuring angle dependent light transmission through Müller cells

The optical fiber homogenizes the light of the light-emitting diode (LED). The light cone at the end of the fibre is collimated with an aspherical lens. With the focal length f_col, the core diameter D_fiber defines the divergence angle θ_div and the opening angle θ_NA defines the diameter D_spot of the light spot on the sample. The mirror can be tilted and slid. The angle of incidence of light on the sample is α_slide.

To homogenize the intensity distribution of the light spot to be projected, the light from a light emitting diode (high power white LED, type: OSLON SSL, LCW CP7P.PC) is coupled to an optical fibre with the core diameter D_core = 200 µm (Fig. 3C). Through the principle of total reflection, the incident light in this optical conductor is reflected on the wall several times. In this way, the light is mixed and emerges homogeneously from the conductor. The light cone emitted in the opening angle θ_NA is collimated by an aspheric lens of focal length f_col = 35 mm. Since the light is not a point source, the rays diverge slightly. The divergence angle is in paraxial approximation θ_div = D_core∕f_col = 0.2mm∕35mm = 5.7 mrad (0,3°). The diameter D_spot of the projected light spots is given as NA = 0.16 through the focal length f_col of the lens and the numerical aperture of the optical fibre. With NA =θ_NA∕2, D_spot = 2⋅f_col⋅NA = 2⋅35mm ⋅ 0,16=11,2 mm. The light striking the slide at an angle α_slides ispartially reflected according to the Fresnel equations and should be taken into account when measuring the intensity on the microscope. In normal incidence, α_slide = 0°, the transmitted light intensity is about 96% of the incident intensity, depending on the refractive index of the slide when n_slide = 1.5255 (λ = 546 nm, borosilicate glass/Schott). The intensity declines by about 4% when light incidence is under α_slide = 20°. The angle α_müller of a light beam to a Müller cell can be distinguished from the angle of incidence on the slide through the multiple refractions of previous optical layers (glass material etc.). This angle is determined by the refractive index n_front of the substrate before the Müller cell and the angle of incidence α_müller = α_slide∕n_front.

The maximum acceptance angle (half angle) under which a light beam can penetrate the cell is (1)${\displaystyle {\Theta }_{\max }={sin}^{-1}\left(\frac{1}{n_{\mathrm{front}}}\sqrt{n_{\mathrm{core}}^2-n_{\mathrm{gladding}}^2}\right),}$ with the refractive indices n_core of the substrate within the cell and n_gladding < n_core of the cell edge.

The acceptance angle of the Müller cells in vivo is estimated with the refractive indices according to Franze et al. (2007) whereby n_front = 1.358 (neuron), when the light coupling of a cell does not connect to the vitreous body of the eye, and n_core = 1.359 (end foot). The refractive index of the cell edge is not known and is set as n_gladding = n_front. According to Eq. (1), the acceptance angle in vivo is θ_max = 2,2°. When fixing the sample with phosphate buffered saline (PBS—buffer, n_PBS = 1.33) it can be assumed that the substrate surrounding the Müller cells has the index value n_gladding = n_PBS. The acceptance angle is then θ_max = 12,0° and is measured in the experiment with α_slide = n_front⋅θ_max = 16°.

Quantification of angle dependent foveal light transmission

Light micrographs with 100-fold magnification from the foveae and parafoveae of the three donors were taken under equal conditions with collimated light hitting the sample at a 0° , 5° , and 10° angle. The photos were processed using ImageJ 1.48v. The mean pixel intensity of the fovea or parafovea area was measured. The value of the image taken at the angle of 0° was set as 100% for each fovea. The mean loss of intensity ± standard deviation at an angle of 5° and 10° was calculated as a percentage of the 0°’s value.