Extended field-of-view ultrathin microendoscopes for high-resolution two-photon imaging with minimal invasiveness
- Optical Approaches to Brain Function Laboratory, Istituto Italiano di Tecnologia, Italy
- Nanostructures Department, Istituto Italiano di Tecnologia, Italy
- University of Genova, Italy
- Neural Coding Laboratory, Istituto Italiano di Tecnologia, Italy
- Neural Computation Laboratory, Center for Neuroscience and Cognitive Systems @UniTn, Istituto Italiano di Tecnologia, Italy
- Center for Mind and Brain Sciences (CIMeC), University of Trento, Italy
- Biological and Environmental Science and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), Saudi Arabia
Figures
Figure 1
Optical design of eFOV-microendoscopes.
(a-d) Ray-trace simulations for the four different eFOV-microendoscopes (type I-IV). The insets show the profiles of corrective polymeric lenses used in the different eFOV-microendoscopes. For each e…
Figure 2 with 4 supplements
Corrective lenses improve the simulated optical performance of ultrathin microendoscopes.
(a) Simulated diffraction PSFs to assess the Strehl ratio of the designed microendoscope (type I microendoscopes) without the corrective lens (uncorrected, left) and with the corrective lens …
Figure 2—figure supplement 1
Aberration correction improves the simulated PSF in the peripheral portions of the FOV in eFOV-microendoscopes.
(a) Lateral and axial projection of a simulated PSF positioned at the border of the FOV with the uncorrected (left) and corrected (right) type I microendoscopes. Scale bars: 1 μm (lateral), 10 μm …
Figure 2—figure supplement 2
Simulated spatial resolution is improved in eFOV-microendoscopes.
(a) Simulated axial (left) and lateral (right) spatial resolution (see Materials and methods for definition) as a function of the radial distance from the center of the FOV for type I uncorrected …
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Figure 2—figure supplement 2—source data 1
Simulated values of the axial and lateral spatial resolution as a function of radial distance from the center of the FOV in uncorrected and corrected microendoscopes.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig2-figsupp2-data1-v3.xlsx
Figure 2—figure supplement 3
Corrective lenses enlarge the simulated volume that can be scanned during 3D imaging.
(a) Pseudocolor map showing the simulated Strehl ratio on a x,z section of the image space for type I uncorrected (left) and corrected (right) microendoscopes. The dashed white line represents a …
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Figure 2—figure supplement 3—source data 1
Simulated Strehl ratio as a function of the lateral and axial displacement for type I-IV uncorrected and corrected microendoscopes.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig2-figsupp3-data1-v3.xlsx
Figure 2—figure supplement 4
Chromatic aberrations in eFOV-microendoscopes.
(a) Left panel: Simulated Strehl ratio at the border of the FOV as a function of wavelength. The red dashed line represents the diffraction-limited condition, which was set at 80% (Maréchal …
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Figure 2—figure supplement 4—source data 1
Simulated Strehl ratio as a function of wavelength.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig2-figsupp4-data1-v3.xlsx
Figure 3 with 7 supplements
Optical characterization shows enlarged effective FOV in corrected ultrathin microendoscopes.
(a) Schematic of the eFOV-microendoscope mount for head implant. The GRIN rod is glued to one side of the glass coverslip, the microfabricated polymer lens to the other side of the coverslip. The …
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Figure 3—source data 1
Experimental values of the axial and lateral spatial resolution as a function of radial distance from the center of the FOV in uncorrected and corrected microendoscopes.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Polymer lens fabrication using 3D micro-printing based on two-photon lithography.
(a) Schematic of the optical set-up for two-photon lithography. AOM, Acousto Optical Modulator; S, laser source; CCD, camera. (b) Scanning electron microscope image of a corrective polymer lens. (c) …
Figure 3—figure supplement 2
Set-ups for the assembly and characterization of eFOV-microendoscopes.
(a) Optomechanical stage used for microendoscope assembly. Red arrows indicate key components. The blue line indicates the plane used for the cross-section view shown at an expanded scale in b. (b) …
Figure 3—figure supplement 3
Aberration correction improves the PSF in the peripheral portions of the FOV in eFOV-microendoscopes.
(a) Lateral and axial projection of a z-stack of a subresolved fluorescent bead positioned at the border of the FOV and imaged with the uncorrected (left) and corrected (right) type I …
Figure 3—figure supplement 4
Field curvature in eFOV-microendoscopes.
(a,b) Magnification correction factor in the horizontal direction as a function of the radial position in uncorrected (a) and corrected (b) type II eFOV-microendoscopes. Plots show values obtained …
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Figure 3—figure supplement 4—source data 1
Magnification correction factor as a function of the radial position in uncorrected and corrected type II microendoscopes.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig3-figsupp4-data1-v3.xlsx
Figure 3—figure supplement 5
Corrected microendoscopes have extended effective FOV.
(a-d) Representative images of fixed cortical tissue expressing eGFP in neuronal cells acquired with type I (a), type II (b), type III (c), and type IV (d) microendoscopes without (uncorrected, left …
Figure 3—figure supplement 6
Hippocampal imaging with type I and type III eFOV-microendoscopes.
(a-a2), Confocal images of hippocampal CA1 neurons expressing GCaMP6s (a). Nuclei were counterstained with Hoechst (a1). Images are merged in (a2). Scale bar in (a) applies to a1-a2. (b-b1) Confocal …
Figure 3—figure supplement 7
VPM imaging with type II eFOV-microendoscopes.
(a-a2) Confocal images of thalamic neurons expressing GCaMP6s (a). Nuclei were counterstained with Hoechst (a1). Images are merged in (a2). Scale bar in (a) applies to a1-a2. (b) Confocal image …
Figure 4 with 2 supplements
eFOV-microendoscopes allow higher SNR and more accurate evaluation of pairwise correlation.
(a) Schematic of the procedure for in silico simulation of imaging data. Neuronal activity was simulated within spheres located in a 3D volume, integrated over an elliptical PSF (blue) that was …
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Figure 4—source data 1
Results of manual segmentation: # of ROIs, SNR, and pairwise correlations for simulated and experimental data.
Comparison between data obtained with uncorrected and corrected microendoscopes in silico and in vivo.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Manual vs. automated segmentation in simulated and experimental t-series.
(a), Recall values for the segmentation of simulated data in t-series obtained in uncorrected (red) and corrected (blue) microendoscopes for the manual (continuous line) and automated CaImAn (Giovann…
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Figure 4—figure supplement 1—source data 1
Comparison of manual vs automated segmentation methods in simulated and experimental data.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig4-figsupp1-data1-v3.xlsx
Figure 4—figure supplement 2
Improved description of neuronal signals in eFOV-microendoscopes is observed in t-series automatically segmented with CaImAn.
(a) Number of segmented ROIs as a function of the SNR threshold in artificial data from n = 9 simulated experiments. A two-way ANOVA with interactions showed a significant effect of SNR threshold …
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Figure 4—figure supplement 2—source data 1
Results of automated segmentation: # of ROIs, SNR, and pairwise correlations for simulated and experimental data.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig4-figsupp2-data1-v3.xlsx
Figure 5
Ultrathin microendoscope implantation preserves thalamo-cortical and cortico-thalamic connectivity between S1bf and VPM.
(a) Local injection of red retrobeads and AAVs transducing floxed eGFP was performed in the VPM of Scnn1a-Cre mice. (b) Confocal image showing a coronal slice from an injected control animal. Scale …
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Figure 5—source data 1
Percentage of labeled S1bf area with eGFP and retrobeads in control and implanted mice.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig5-data1-v3.xlsx
Figure 6
High-resolution population dynamics in the VPM of awake mice during locomotion and free whisking.
(a) Schematic of the experimental set-up for the recording of locomotion, whisker mean angle, and pupil size in awake head-fixed mice during VPM imaging using type II eFOV-microendoscopes. (b) …
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Figure 6—source data 1
Percentage of frames, ΔF/F0, and distribution of pupil diameter across behavioral states.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig6-data1-v3.xlsx
Figure 7 with 1 supplement
Cell-specific encoding of behaviorally-dependent information in distributed VPM subnetworks.
(a) Spatial map of neurons encoding whisking information. The pseudocolor scale shows significantly informative neurons (see Materials and methods). Data are pooled from 24 t-series from four …
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Figure 7—source data 1
Information theoretical analysis and non-negative matrix factorization results.
Values of cell-specific and population information analysis.
- https://cdn.elifesciences.org/articles/58882/elife-58882-fig7-data1-v3.xlsx
Figure 7—figure supplement 1
Cell-specific encoding of behavioral state-dependent information in distributed VPM subnetworks.
Spatial map of neurons encoding whisking information in 12 out of the total 24 analyzed time series. The pseudocolor scale shows significantly informative neurons (see Materials and methods).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | C57BL/6J | Charles River | RRID:IMSR_JAX:000664 | |
| Genetic reagent (M. musculus) | B6;C3-Tg(Scnn1a- cre)3Aibs/J | The Jackson Laboratory | RRID:IMSR_JAX:009613 | |
| Recombinant DNA reagent | pAAV.Syn.Flex. GCaMP6s.WPRE.SV40 | Penn Vector Core | RRID:Addgene_100845; Addgene viral prep # 100845-AAV1 | Chen et al., 2013 |
| Recombinant DNA reagent | pGP-AAV-syn- FLEX-jGCaMP7f-WPRE | Addgene | RRID:Addgene_104492; Addgene viral prep # 104492-AAV1 | Dana et al., 2016 |
| Recombinant DNA reagent | AAV pCAG- FLEX-EGFP-WPRE | Penn Vector Core | RRID:Addgene_51502; Addgene viral prep # 51502-AAV1 | Oh et al., 2014 |
| Recombinant DNA reagent | AAV.CaMKII0.4. Cre.SV40 | Penn Vector Core | RRID:Addgene_105558; Addgene viral prep # 105558-AAV1 | |
| Commercial assay or kit | Kwik-Cast | World Precision Instruments | Cat# KWIK-CAST | |
| Commercial assay or kit | Sylgard Silicone Elastomer | Dow Inc | Cat# Sylgard 164 | |
| Commercial assay or kit | Norland Optical Adhesive 63 | Norland | Cat# NOA 63 | |
| Commercial assay or kit | GRIN lens | Grintech | Cat# NEM-050- 25-10-860-S | |
| Commercial assay or kit | GRIN lens | Grintech | Cat# NEM-050- 43-00-810-S-1.0p | |
| Commercial assay or kit | GRIN lens | Grintech | Cat# GT-IFRL-035- cus-50-NC | |
| Commercial assay or kit | GRIN lens | Grintech | Cat# NEM-035- 16air-10–810 S-1.0p | |
| Chemical compound, drug | bisBenzimide H 33342 trihydrochloride (Hoechst) | Sigma-Aldrich | Cat# B2261; CAS: 23491-52-3 | |
| Chemical compound, drug | Red Retrobeads | LumaFluor Inc | Red Retrobeads | |
| Software, algorithm | Zemax OpticStudio 15 | Zemax | https://www.zemax.com/products/opticstudio | |
| Software, algorithm | MATLAB R2017a | Mathworks | RRID:SCR_001622; https://it.mathworks.com/products/matlab.html | |
| Software, algorithm | GraphPad PRISM | GraphPad PRISM | RRID:SCR_002798; https://www.graphpad.com/ | |
| Software, algorithm | ImageJ/Fiji | Fiji | RRID:SCR_002285; http://fiji.sc/ | |
| Software, algorithm | NoRMCorre | Pnevmatikakis and Giovannucci, 2017 | https://github.com/flatironinstitute/NoRMCorre | |
| Software, algorithm | CaImAn | Giovannucci et al., 2019 | https://github.com/flatironinstitute/CaImAn-MATLAB | |
| Software, algorithm | Population Spike Train Factorization Toolbox for Matlab Version 1.0 | Onken et al., 2016 | https://stommac.eu/index.php/code | |
| Software, algorithm | LIBSVM | Chang and Lin, 2011 | https://www.csie.ntu.edu.tw/~cjlin/libsvm/ | |
| Software, algorithm | Information Breakdown ToolBox | Magri et al., 2009 | N/A | |
| Software, algorithm | Software used in this paper for generation of artificial time series | https://github.com/moni90/eFOV_microendoscopes_sim | Figure 4a–h, Figure 4—figure supplement 1a–c and Figure 4—figure supplement 2a–c | |
| Software, algorithm | Software to compute recall, precision, and F1 score | Soltanian-Zadeh et al., 2019 | https://github.com/soltanianzadeh/STNeuroNet | |
| Other | Basler ace camera | Basler AG | Cat# acA800-510um | |
| Other | Optical encoder | Broadcom | AEDB-9140-A13 | |
| Other | Zortrax M200 3D printer | Zortrax | M200 | |
| Other | Z-ULTRAT 3D printer filament | Zortrax | Z-ULTRAT | |
| Other | Arduino Uno | Arduino | Arduino Uno |
Additional files
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Supplementary file 1
Characteristics of eFOV-microendoscopes and their application in awake mice.
Supplementary Table 1: Parameters for the fabrication of corrective lenses. Coefficients used in Equation (1) (see Materials and methods) for the aspherical corrective lenses used in type I-IV eFOV-microendoscopes. Supplementary Table 2: Simulated focal length in uncorrected and corrected microendoscopes. Supplementary Table 3: Spatial resolution and effective FOV of eFOV-microendoscopic probes. Values are reported as average ± sem. For statistical comparison of uncorrected (uncor.) vs. corrected (cor.) microendoscopes, Student’s t-test was used. Supplementary Table 4: Statistical comparisons of behavior state distributions as a function of pupil diameter. For the statistical comparison of Q, W, and WL state distributions in each range of pupil diameter, a two-way ANOVA with Tukey-Kramer post-hoc correction was performed.
- https://cdn.elifesciences.org/articles/58882/elife-58882-supp1-v3.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/58882/elife-58882-transrepform-v3.docx
