Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy
- Department of Physics, University of California, United States
- Department of Molecular and Cell Biology, University of California, United States
- Helen Wills Neuroscience Institute, University of California, United States
- School of Optometry, University of California, United States
- Vision Science Program, University of California, United States
- Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, United States
Figures
Figure 1 with 3 supplements
AO-2PFM for diffraction-limited imaging of the mouse retina in vivo.
(A) Schematics of AO-2PFM. Inset 1: direct wavefront measurement by a Shack-Hartmann (SH) sensor composed of a lenslet array and a camera. Inset 2: wavefront correction with a deformable mirror …
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Figure 1—source data 1
Source image stacks of retinal axons (Figure 1B).
‘1-NoAO_8fAvg_stack.tif’ (No AO) and ‘2-AO_8fAvg_stack.tif’ (AO) Figure 1—source data 2 Source image stacks of retinal dendrites (Figure 1C): ‘1-NoAO_8fAvg_160_100_stack.tif’ (No AO) and ‘2-AO_8fAvg_160_100_stack.tif’ (AO).
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig1-data1-v1.zip
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Figure 1—source data 2
Source image stacks of retinal neuronal processes (Figure 1C).
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig1-data2-v1.zip
Figure 1—figure supplement 1
Characterization of aberrations introduced by ETL and alignment.
(A) 3D rendering of the eye imaging module. (B) Additional corrective wavefronts for system aberrations measured with 0, 20, 40, 60, and 80 mA ETL currents, relative to the corrective wavefront …
Figure 1—figure supplement 2
Contact lens and eye gel application improve image and wavefront sensing quality.
(A) Design of the customized contact lens (CL). (B) 2PFM single-plane images of (left) retinal vasculature and (right) retinal cells acquired (i) with CL and eye gel and (ii) without CL or eye gel. …
Figure 1—figure supplement 3
Zernike decompositions and corrective wavefronts for all experiments.
All corrective wavefronts and Zernike decompositions were calculated excluding piston, tip, tilt, and defocus.
Figure 2 with 1 supplement
In vivo imaging of mouse retinal vasculature with AO-2PFM.
(A,B) MIPs of image stacks (580×580×128 µm3) of vasculature measured (A) without and (B) with AO, respectively, normalized to AO image. Red asterisk: center of 19×19 µm2 wavefront sensing (WS) area. …
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Figure 2—source data 1
Source image stacks of retinal vasculature (Figure 2A, B and D).
‘1-NoAO_8fAvg_stack.tif’ (No AO) and ‘2-AO_8fAvg_stack.tif’.
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig2-data1-v1.zip
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Figure 2—source data 2
Source image stacks with AO measured at different locations (Figure 2F).
‘AO [1]’ stack: ‘1-AO[1]_Location_0_0_stack-9fAVG.tif’, ‘AO [2]’ stack: ‘2-AO[2]_Location_N15_N18_stack-9fAVG.tif’ ‘AO [3]’ stack: ‘3-AO[3]_Location_P4_P25_stack-9fAVG.tif’.
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig2-data2-v1.zip
Figure 2—video 1
In vivo 2-photon image stacks of retinal vasculature in a wildtype mouse measured without and with AO performed at different locations in the field of view (red asterisks).
Same data as shown in Figure 2F. Image volume: 580×580×110 µm3; Z step: 3.26 µm.
Figure 3 with 2 supplements
In vivo imaging of mouse retinal neurons with AO-2PFM.
(A,B) MIPs of image stacks (580×580×80 µm3) of a Thy1-YFP-16 retina, measured (A) without and (B) with AO, respectively, normalized to AO images. Red asterisk: center of a 19×19 µm2 WS area. Top: …
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Figure 3—source data 1
Source image stacks of retinal neurons (Figure 3A and B).
‘1-NoAO_60_84_8fAvg.tif’ (No AO) and ‘2-AO_60_84_8fAvg.tif’ (AO).
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig3-data1-v1.zip
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Figure 3—source data 2
Source image stacks of retinal neurons (Figure 3E).
‘1-NoAO_8fAvg_stack.tif’ (No AO) and ‘2-AO_8fAvg_stack.tif’ (AO).
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig3-data2-v1.zip
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Figure 3—source data 3
Source image stacks of retinal neurons (Figure 3F).
‘1-central_AO_20_28_8fAvg.tif’ (Central AO) and ‘2-local_AO_20_28_8fAvg.tif’ (Local AO).
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig3-data3-v1.zip
Figure 3—figure supplement 1
AO improves the visualization of subcellular features in vivo.
(A) Lateral images of neurons in a Thy1-YFP-16 retina, measured (top) without and (bottom) with AO, respectively. Insets with white borders highlight the subcellular features within the white dashed …
Figure 3—video 1
In vivo 2-photon image stacks of retinal neurons in a Thy1-YFP-16 mouse measured without and with AO.
Same data as shown in Figure 3A, B and E. Image volume: 580×580×80 µm3/193×193×50 µm3; Z step: 1.63/0.98 µm.
Figure 4
Larger WS areas enlarges the effective region of AO correction for 3D cellular resolution imaging.
(A) Top: AO/NoAO pixel ratio maps for corrections with differently sized WS areas (yellow dashed boxes; i, 19×19 µm2; ii, 95×95 µm2; iii, 190×190 µm2; iv, 380×380 µm2). Bottom: (for [i]) corrective …
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Figure 4—source data 1
Source image stacks with AO by measuring aberrations with different wavefront sensing areas.
‘No AO’ stack: ‘1_NoAO_9fAvg_stack.tif’‘AO [i]’ stack: ‘2_AO[i]_2_2.8_9fAvg_stack.tif’ ‘AO [ii]’ stack: ‘3_AO[ii]_10_14_9fAvg_stack.tif’ ‘AO [iii]’ stack: ‘4_AO[iii]_20_28_9fAvg_stack.tif’ ‘AO [iv]’ stack: ‘5_AO[iv]_40_56_9fAvg_stack.tif’.
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig4-data1-v1.zip
Figure 5 with 5 supplements
In vivo vasculature imaging in pathological and healthy retinas.
(A,B) Left: MIPs of image stacks of (A) VLDLR-KO/Sca1-GFP (580×580×94 µm3) and (B) WT/Sca1-GFP (520×520×120 µm3) mouse retinas, measured (arrow start) without and (arrow end) with AO. Asterisks: …
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Figure 5—source data 1
Source image stacks of VLDLR-KO/Sca1-GFP mouse retina (Figure 5A, full FOV).
‘1-NoAO_8fAvg_60_84_stack.tif’ (No AO) and ‘2-AO_8fAvg_60_84_stack.tif’ (AO). Source image stacks of VLDLR-KO/Sca1-GFP mouse retina (Figure 5A, zoomed-in view). ‘3-NoAO_20_28_8fAvg_stack.tif’ (No AO) and ‘4-AO_20_28_8fAvg_stack.tif’ (AO).
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig5-data1-v1.zip
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Figure 5—source data 2
Source image stacks of WT/Sca1-GFP mouse retina (Figure 5B, full FOV).
‘1-NoAO_60_84_Stack.tif’ (No AO) and ‘2-AO_60_84_Stack.tif’ (AO). Source image stacks of WT/Sca1-GFP mouse retina (Figure 5B, zoomed-in view). ‘3-NoAO_20_28_stack.tif’ (No AO) and ‘4-AO_reged_8fAvg_stack.tif’ (AO).
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig5-data2-v1.zip
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Figure 5—source data 3
Source image stacks of VLDLR-KO/Sca1-GFP mouse retina (Figure 5C, full FOV).
‘1-AO_8fAvg_stack.tif’. Source image stacks of VLDLR-KO/Sca1-GFP mouse retina (Figure 5C, zoomed-in view). ‘2-AO_zoomin_8fAvg_stack.tif’.
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig5-data3-v1.zip
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Figure 5—source data 4
Source image stack of VLDLR-KO/Sca1-GFP mouse retina after FITC injection (Figure 5D).
‘1-NoAO-8fAvg_stack.tif’.
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig5-data4-v1.zip
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Figure 5—source data 5
Source image stack of WT/Sca1-GFP mouse retina after FITC injection (Figure 5E).
‘1-NoAO-8fAvg_stack.tif’.
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig5-data5-v1.zip
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Figure 5—source data 6
Source image stacks of VLDLR-KO/Sca1-GFP mouse retina after EB injection (Figure 5F).
Day 1: ‘1A-Day1-NIR_EB_8fAvg_stack.tif’ and ‘1B-Day1-GFP_60_84_8fAvg_stack.tif’ Day 2: ‘2A-Day2-NIR_EB_8fAvg_stack.tif’ and ‘2B-Day2-GFP_60_84_8fAvg_stack.tif’ Day 3: ‘3A-Day3-NIR_EB_8fAvg_stack.tif’ and ‘3B-Day3-GFP_60_84_8fAvg_stack.tif’ Source image stacks of microglia (Figure 5G). ‘4A-Day3-microglia_t=0.tif’, ‘4B-Day3-microglia_t=20.tif’, and ‘4C-Day3-microglia_t=40.tif’.
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig5-data6-v1.zip
Figure 5—figure supplement 1
Ex vivo 2PFM imaging of dissected VLDLR-KO/Sca1-GFP and WT/Sca1-GFP mouse retinas.
(A,B) Ex vivo MIPs of image stacks from (A) VLDLR-KO/Sca1-GFP (1380×1380×82 µm3) and (B) WT/Sca1-GFP (1380×1380×71 µm3) mouse retinas. (C) Top: 3D projected view of an example capillary lesion (red …
Figure 5—figure supplement 2
In vivo AO-2PFM imaging of Evans Blue (EB) leakage in healthy retina.
(A–C) MIPs of image stacks of (580×580×130 µm3) WT/Sca1-GFP retina measured in the (A) near-infrared EB and (B) green GFP channels, and (C) merged images. (D–F) Single-plane images from (A–C) with …
Figure 5—video 1
In vivo two-photon image stacks of abnormal retinal capillaries in a VLDLR-KO/Sca1-GFP mouse measured without and with AO.
Same data as shown in Figure 5A. Image volume: 48×48×65 µm3; Z step: 3.26 µm.
Figure 5—video 2
Ex vivo two-photon image stacks of abnormal capillaries in dissected VLDLR-KO/Sca1-GFP mouse retinas.
Same data as shown in Figure 5—figure supplement 1A and C. Image volume: 1380×1380×82 µm3 / 78×78×82 µm3; Z step: 0.5 µm.
Figure 5—video 3
Ex vivo two-photon image stacks of normal capillaries in dissected WT/Sca1-GFP mouse retinas.
Same data as shown in Figure 5—figure supplement 1B and D. Image volume: 1380×1380×71 µm3/78×78×71 µm3; Z step: 0.5 µm.
Figure 6 with 1 supplement
In vivo calcium imaging of Lidocaine-suppressed RGC hyperactivity in rd1-Thy1-GCaMP6s mouse retina.
(A) Simultaneous cell-attached and 2PFM calcium recordings of two RGCs before, during, and 45 min after Lidocaine treatment. Representative data from >3 cells. (B) Top: average intensity projections …
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Figure 6—source data 1
Source data of ex vivo cell-attached and 2PFM recordings (Figure 6A): RGC #1.
‘1A-RGC1_before-and-during_lidocaine.tiff’ and ‘1A-RGC1_before-and-during_lidocaine.mat’ ‘1B-RGC1_lidocaine-washout.tiff’ and ‘1B-RGC1_lidocaine-washout.mat’ RGC #2. ‘2A-RGC2_before-and-during_lidocaine.tiff’ and ‘2A-RGC2_lidocaine-washout.mat’ ‘2B-RGC2_lidocaine-washout.tiff’ and ‘2B-RGC2_lidocaine-washout.mat’.
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig6-data1-v1.zip
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Figure 6—source data 2
Source image sequences of ex vivo 2PFM calcium imaging (Figure 6B).
‘1-Sequence-i.tif’, ‘2-Sequence-ii.tif’, and ‘3-Sequence-iii.tif’.
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig6-data2-v1.zip
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Figure 6—source data 3
Source images of rd1-Thy1-GCaMP6s mouse retina (Figure 6C, full FOV).
‘1-NoAO-large_FOV.tif’ (No AO) and ‘2-AO-large_FOV.tif’ (AO). Source images of rd1-Thy1-GCaMP6s mouse retina (Figure 6C, zoomed-in view). ‘3-NoAO-inset.tif’ (No AO) and ‘4-AO-inset.tif’ (AO).
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig6-data3-v1.zip
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Figure 6—source data 4
Source data for in vivo 2PFM calcium imaging (Figure 6D).
‘1-pre_Lido’, ‘2-after_Lido_1 min.tif’, ‘3-after_Lido_30 mins.tif’, and ‘4-after_Lido_60 mins.tif’.
- https://cdn.elifesciences.org/articles/84853/elife-84853-fig6-data4-v1.zip
Figure 6—figure supplement 1
Ex vivo multielectrode array (MEA) recordings of Lidocaine-modified RGC hyperactivity in rd1-Thy1-GCaMP6s mouse retina.
(A) MEA setup for RGC spontaneous spike activity recording. Inset: illustration of retina placement relative to MEA. (B) Raster and average firing frequency plots of RGCs in dissected rd1 mouse …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | C57BL/6 J | Jackson Laboratory | Stock #000664 | |
| Genetic reagent (M. musculus) | B6.Cg-Tg(Thy1-YFP)16Jrs/J | Jackson Laboratory | Stock #003709 | |
| Genetic reagent (M. musculus) | B6.Cg-Tg(Ly6a-EGFP)G5Dzk/J | Jackson Laboratory | Stock #012643 | |
| Genetic reagent (M. musculus) | B6;129S7-Vldlrtm1Her/J | Jackson Laboratory | Stock #002529 | |
| Genetic reagent (M. musculus) | C3H/HeJ | Jackson Laboratory | Stock #000659 | |
| Genetic reagent (M. musculus) | C57BL/6J-Tg(Thy1-GCaMP6s) GP4.3Dkim/J | Jackson Laboratory | Stock #024275 | |
| Software, algorithm | ImageJ software | http://imagej.nih.gov/ij/ | RRID:SCR_003070 | |
| Software, algorithm | GraphPad Prism software | https://graphpad.com | RRID:SCR_015807 | |
| Software, algorithm | MATLAB | https://www.mathworks.com/products/matlab.html | RRID:SCR_001622 | |
| Chemical compound, drug | Lidocaine | Phoenix | NDC: 57319-533-05 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/84853/elife-84853-mdarchecklist1-v1.pdf
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Supplementary file 1
Imaging parameters for all experiments.
- https://cdn.elifesciences.org/articles/84853/elife-84853-supp1-v1.xlsx
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Source code 1
Zemax file of the eye imaging module.
- https://cdn.elifesciences.org/articles/84853/elife-84853-code1-v1.zip
