Figure 1.
Methodology to compare the different imaging techniques on rat and human corneas.
Cornea preparation, orientation of the different histological and numerical sections and imaging geometry are indicated for each imaging technique. Histological sections are unstained for SHG microscopy, stained with toluidine blue for transmitted light microscopy and stained with uranyl and lead citrate solutions for transmission electron microscopy.
Figure 2.
Confocal microscopy (CM) and SHG microscopy imaging of the DM from control and diabetic rat corneas.
Unstained intact rat cornea observed by (a and c) in vivo CM and (b, d–i, j) ex vivo SHG microscopy detected in the (e) backward direction and in the (b,d,i–j) forward direction : (b-e) frontal optical sections, (i) 3D view and (j) transverse numerical reconstruction of the DM (image size: 108×108×7 µm3). Intensity profiles from (f) CM, (g) forward-detected and (h) backward-detected SHG microscopy along 100 µm are plotted under the corresponding images. The number of detected photons has been corrected from the channel sensitivity. St: stroma, DM: Descemet’s membrane. White arrows indicate collagen abnormal deposits in the DM. The look-up-table (LUT) used for SHG images is indicated near (b).
Table 1.
Diabetic and control rat corneas: clinical data and observations by SHG microscopy and other imaging techniques.
Figure 3.
Multimodal imaging of histological sections from the same diabetic rat cornea.
(a–c) Frontal and (d–f) transverse histological sections of the cornea observed (a, d) by transmitted light microcopy, (b, c and e) by TEM, where insets show long-spacing collagen, and (f) by SHG microscopy. St: stroma, DM: Descemet’s membrane. White arrows indicate collagen abnormal deposits in the DM.
Figure 4.
Multimodal imaging of the DM from the same diabetic human cornea.
(a–d) Frontal and (e–h) transverse sections. (a, e) SHG microscopy of intact cornea: (a) frontal optical section, (e) transverse numerical reconstruction. (b, f) Transmitted light microcopy of stained histological sections, where the abnormalities are visible with few contrast. (c, d, g) TEM views of (c, g) the entire DM and (d) its posterior part and the endothelium: long-spacing collagen appears to be synthesized by the endothelial cell. (h) SHG imaging of the same transverse histological section, where fibrillar collagen is clearly identified in the DM. St: stroma, ABL: anterior banded layer, PNBL: posterior nonbanded layer, En: endothelium. White arrows indicate collagen abnormal deposits in the DM.
Table 2.
Human corneas: clinical data and observations by SHG microscopy and other imaging techniques.
Figure 5.
SHG imaging of the DM from human corneas with different clinical data.
Transverse optical section in the DM (a) from a diabetic donor with unbalanced type 2 diabetes, (b) from a diabetic donor with balanced type 2 diabetes, (c) from a donor with hypertension and (d) from a donor without clinical data related to hyperglycemia or hypertension.
Figure 6.
In vivo SHG imaging of the DM from a rat cornea.
(a–b) experimental setup for in vivo imaging of the anesthetized rat (backward detection). (c–d) In vivo SHG observation of DM (between the dashed lines) (c) without any SHG signal in the control rat and (d) with SHG signals from fibrillar collagen in the diabetic rat. (e) TEM observation of DM collagen deposits after sacrifice of the diabetic rat.