Supporting information for animals.

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αB-R120G-crystallin has more amyloid-like secondary structure than wild-type αB-crystallin proteins <i>in vitro</i> by 2DIR and TEM.

(A, B) Overlay of diagonal slices for room temperature αB-crystallin (solid light blue) and heated sample (dashed light blue), or αB-R120G-crystallin (solid maroon) and heated sample (dashed maroon). The dotted vertical line indicates the amyloid-like β-sheet protein secondary structure peak frequency for αB-crystallin, as previously determined through TEM and 2DIR spectroscopy [10]. (C) Overlay of cross peaks for room temperature αB-crystallin (solid light blue) and heated sample (dashed light blue). (D) Overlay of cross peaks for room temperature αB-R120G-crystallin (solid maroon) and heated sample (dashed maroon). (E, F) TEM images for room temperature αB-crystallin (solid light blue) and heated sample (dashed light blue). White scale bars 100 nm. (G, H) TEM images for room temperature αB-R120G-crystallin (solid maroon) and heated sample (dashed maroon). White scale bar at 100 nm, dark blue scale bar at 200 nm. Room temperature samples were incubated at room temperature for 27 hours. Heated samples were heated for 2 hours at 43°C and then left at room temperature for 25 hours.</p

A 2DIR and TEM comparison of αB-crystallin and αB-R120G-crystallin with acid treatment.

(A) Overlay of diagonal slices for room temperature αB-crystallin (solid light blue, data in Fig 2A), heated sample (dashed light blue, data in Fig 2A), and acid treated sample (dotted light blue). Data has been normalized to 1639 cm−1 to match the room temperature sample. (B) Overlay of diagonal slices for room temperature αB-R120G-crystallin (solid maroon, data in Fig 2B), heated sample (dashed maroon, data in Fig 2B), and acid treated sample (dotted maroon). Data has been normalized to 1632 cm−1 to match the room temperature sample. (C) TEM image of acid treated αB-crystallin. Dark blue scale bar is 100 nm. (TIF)</p

<i>Cryab</i>-R120G mutant mouse lenses have more amyloid-like secondary structure features than wild- type mouse lenses by 2DIR image analysis.

(A, B) Ratio image of 1636/1641 cm-1 intensities for a wild type (A) and mutant (B) mouse lens tissue sections. (C) An overlay of two example diagonal slices for the wild type (blue, from white box in A, E) and Cryab-R120G mutant (red, from white box in B, F) mouse lens sections, normalized to 1641 cm-1. The vertical lines (green) indicate the frequencies used in determining the diagonal ratio values (1636 and 1641 cm-1). (D) An overlay of two example cross peaks for the wild type (blue, from white box in A, E) and Cryab-R120G mutant (red, from white box in B, F) mouse lens sections. (E, F) Cross peak intensity image at the Cryab-R120G mutant mouse cross peak frequency (ωpump = 1632 cm-1, ωprobe = 1701 cm-1) for the wild type (E) and mutant (F) mouse lens sections.</p

Comparison of frozen, dried, and fixed/paraffin embedded tissue for human lenses.

(A) Diagonal slice overlays show a shift to higher frequency and less intensity as the lens tissue goes from the most polar (frozen, rehydrated in buffer) to most nonpolar (paraffin wax) environment. Normalized to juvenile lens tissue peak at 1632 cm-1 for frozen, 1636 cm-1 for dried, and 1641 cm-1 for fixed lens tissue. (B) Peak frequency distributions are heterogeneous, with distinct shifts to higher frequencies as the lens tissue goes from a polar to nonpolar environment. Red: Cataract lens tissue (frozen, then rehydrated in buffer); Blue: Juvenile lens tissue (frozen, then rehydrated in buffer); Purple: Cataract lens tissue (frozen, then dried under nitrogen); Yellow: Juvenile lens tissue (frozen, then dried under nitrogen); Light Blue: Cataract lens tissue (fixed and paraffin embedded); Green: Juvenile lens tissue (fixed and paraffin embedded). (TIF)</p

An example of a 2DIR contour plot.

(A) A 2DIR contour plot showing data of a 100 μm region of a mutant mouse lens tissue slice. The plot exhibits a pair of positive (red) and negative (blue) peaks that have been normalized on the diagonal at ωpump = ωprobe = 1641 cm−1. The plot also exhibits a cross peak (grey box) with a maximum at ωpump = 1632 cm-1, ωprobe = 1701 cm−1. (B) A plot of the dashed diagonal line in (A) (grey dashed). The vertical lines (green) indicate the frequencies used in determining the diagonal ratio values (1636 and 1641 cm-1). (C) A plot of the solid horizontal line in (A) (grey). A baseline (green) is subtracted to give the cross peak intensity. When structures change from native β-sheets to amyloid-like β-sheets, the cross peak intensity increases, and its position shifts to a smaller pump frequency and larger probe frequency.</p

Comparison of fixed lens anharmonicity values.

Percent of locations versus anharmonicity values for juvenile human lens (yellow, top row), cataract human lens (purple, second row), three wild type mouse lenses combined (blue, third row), and three Cryab-R120G mutant mouse lenses (red, bottom row). (TIF)</p

Comparison of frozen and fixed tissue for mouse lenses.

(A) Diagonal slice overlays show a shift to higher frequency and less intensity as the lens tissue goes from the most polar (frozen, rehydrated in buffer) to most nonpolar (paraffin wax) environment. Normalized to wild type mouse lens tissue (peak at 1632 cm-1 for frozen, 1641 cm-1 for fixed). (B) Photographs of fixed, paraffin embedded lens slices shows a lens slice rejected from measurement because of large rips (top left, from Cryab-mutant lens sample 3) and a lens slice typical of those used in this study (bottom right, from Cryab-mutant lens sample 1). Maroon: Cryab-R120G mutant mouse lens tissue (frozen, then rehydrated in buffer); Green: wild type mouse lens tissue (frozen, then rehydrated in buffer); Red: Cryab-R120G mutant mouse lens tissue (fixed and paraffin embedded); Blue: wild type mouse lens tissue (fixed and paraffin embedded). (TIF)</p

2DIR contour plots for data shown in Fig 2.

(A) Contour plot for room temperature αB-crystallin (solid light blue). (B) Contour plot for heated αB-crystallin (dashed light blue). (C) Contour plot for room temperature αB-R120G-crystallin (solid maroon). (D) Contour plot for heated αB-R120G-crystallin (dashed maroon). (TIF)</p

An FFPE age-related cataract human lens tissue contains more amyloid-like secondary structure features than an FFPE juvenile human lens tissue by 2DIR image analysis.

(A, B) Ratio image of 1636/1641 cm-1 intensities for a juvenile (16 years old, A) and age-related cataract (63 years old, B) human lens tissue sections. (C) An overlay of two example diagonal slices for the juvenile (yellow, from green dot in A, E) and cataract (purple, from black dot in B, F) human lens sections, normalized to 1641 cm-1. Additionally, the wild type (blue) and Cryab-mutant (red) mouse lens sections from Fig 3 are also shown for comparison. The vertical lines (green) indicate the frequencies used in determining the diagonal ratio values (1636 and 1641 cm-1). (D) An overlay of two example cross peaks for the juvenile (yellow, from green dot in A, E) and cataract (purple, from black dot in B, F) human lens sections. (E, F) Cross peak intensity image at the human cataract cross peak frequency (ωpump = 1632 cm-1, ωprobe = 1695 cm-1) for the juvenile (E) and cataract (F) human lens sections.</p