**3. Multiphoton microscopy, image acquisition and processing**

A multiphoton microscope was used to obtain images of the retinal mosaic in 16 healthy (C) mice and sixteen mice with diabetic retinopathy (DR). The multiphoton microscope combines a fs-laser system (760 nm), a scanning unit, a Z-motor, and a detector (photon-counting) within an inverted microscope. The whole system was computer-controlled. Nonlinear microscopy images were acquired using two imaging modalities: regular *XY* images and fast 2-photon imaging tomography, **Figure 12**.

Eight Sprague-Dawley male rats, weighing 200 g were administered intraperitoneal via with 55 mg/kg from *streptozotocin* diluted in citrate buffer were housed in a temperature-and humidity-controlled room maintained on a 12-hour light/dark cycle (lights on: 07:00–19:00 hours) and had free access to food and water. Retinas were evaluated after 6 weeks from diabetic-induced.

The retinal mosaic was estimated for each subject (both eyes), at 270, 810, 1350 and 1890 μm of eccentricities from the optic nerve along the nasal, temporal, dorsal and ventral of both eyes. The photoreceptors arrangement was analyzed using Voronoi polygon and Delaunay triangulation analysis; estimations were done by using a sampling window S of 90 90 μm image sections, **Figure 13**.

Through the Voronoi partition, some contours of the tessellations should be modified if other points outside the analysis window S (90 90 μm) were acquired.

**Figure 12.** *Setup for scanning with a multiphoton microscope.*

## **Figure 13.**

*Data processing diagram in retinal tissues for healthy photoreceptors and with DR, starting from raw data and applying image processing and segmentation, feature extraction and classification, and finally polygonality measurements.*

These points are associated to the outline regions which are closer to the analysis window than to the considered point. The points which belong to such contour regions are considered to be at the border. All the marginal points are not noted in the subsequent calculations, because they cause statistical noise.
