TITLE: Sample preparation and imaging for large scale 3D spectral confocal imaging of tissues
AUTHORS: Tarek Ashkar, Michael Ferkowicz

[DESCRIPTION]
Frozen participant samples are first sized and preserved for high-resolution 3D imaging. Preserved biopsy sections are imaged, without staining, to understand the sample’s structure and condition. Next, the biopsy sections are stained with antibodies and fluorescent dyes to identify specific structures and cells with pathological significance. This stained sample is imaged in its entirety giving a 3D image. Finally, the 3D image is analyzed by experts to assess known and novel pathologies.

This protocol summarizes both label-free imaging and labeling for fluorescence imaging including the labeling, mounting and imaging approaches. The fluorescent labels used herein include conjugated or fluorescent small molecules and conjugated antibodies. Protocol 2 uses both directly conjugated primary antibodies for identifying specific antigens and indirect labeling with a fluorophore conjugated secondary antibody that recognizes the primary antibody.

Multiple rounds of imaging are enabled by the use of a non-hardening mounting media, low charge slides and removable rubber cement as a sealant. Details are described below.

Two imaging approaches are outlined: protocol 1 and 2. Protocol 1 involves label free imaging of auto-fluorescent species and second harmonic generation (SHG) with multiphoton excitation. These modalities are followed by protocol 2, which involves staining with fluorescent small molecules and immuno-fluorescence and subsequent large scale 3D confocal fluorescence imaging. There are three different staining strategies used in protocols, summarized in Table 1.

These protocols start with fixed 50 µm tissue sections prepared as described in the Cryopreservation protocol.

  
  
  


 Version 3.0
 
     Structure/cell type      Target      Species      Vendor (cat #)       Dilution [stock]      Direct labeling      Indirect labeling        Nuclei      DAPI      -      ThermoFisher (D1306)      1:200 [1 mg/mL]      -      -        vasculature      Phalloidin      -      ThermoFisher O7466      1:200 [2 U/mL]      Oregon Green      -        glomeruli       "      -      "      "      "      -        brush borders      "      -      "      "      "      -        TAL      THP      Sheep      R&D Systems
  (AF5144)      1:1000  [0.2 mg/mL]      Alexa 546      -        Proximal tubules      AQP1      Goat      Santa Cruz      (sc-9878)      1:50 [0.2 mg/mL]      -      donkey anti-goat Alexa
  568 (1:200)        Neutrophils      MPO      Rabbit      Abcam (ab9535)      1:50      -      donkey anti-rabbit DyLight 594 (1:200)        macrophages      CD68      Mouse      Dako (M0876)      1:50 [0.3 mg/mL]      -      donkey anti-mouse
  Alexa 633 (1:200)        T-cells      CD3      Mouse      BD Pharmingen (557706)      1:50      Alexa 647      -        vasculature      CD31      Mouse      NovusBio (NB600-562ss)      1:50  [1 mg/mL]      Alexa 660      -   

Version 4.0
 
     Structure/cell type      Target      Species      Vendor (cat #)       Dilution [stock]      Direct labeling      Indirect labeling        Nuclei      DAPI      -      ThermoFisher (D1306)      1:200 [1 mg/mL]      -      -        vasculature      Phalloidin      -      ThermoFisher O7466      1:200 [2 U/mL]      Oregon Green      -        glomeruli       "      -      "      "      "      -        brush borders      "      -      "      "      "      -        TAL      THP      Sheep      R&D Systems
  (AF5144)      1:1000  [0.2 mg/mL]      Alexa 546      -        proximal tubules      AQP1      Goat      Santa Cruz  (sc-9878)      1:50 [0.2 mg/mL]      -      donkey anti-goat Alexa
  568 (1:200)        neutrophils      MPO      Rabbit      Abcam (ab9535)      1:50      -      donkey anti-rabbit DyLight 594 (1:200)        macrophages      CD68      Mouse      Dako (M0876)      1:50 [0.3 mg/mL]      -      donkey anti-mouse
  Alexa 633 (1:200)        most hematopoietic
  cells      CD45      Mouse      Biolegend (304056)      1:50 [0.5 mg/mL]      Alexa 647      -        vasculature      CD31      Mouse      NovusBio (NB600-562ss)      1:50 [1 mg/mL]      Alexa 660      -   



 
 





 
 




 
 


 
 




 
 


[GUIDELINES]
Excitation and filter settings
Label-free imaging was performed with femtosecond pulsed 910 nm excitation and de-scanned emissions were collected with custom band-passes of 435-465 nm for collecting SHG and 500- 550 for collecting auto-fluorescence. Spectral confocal imaging was performed with sequential scanning at each z frame for 4 excitations with 4 band-passes each giving 16 total channels. The configuration and scan settings are given in Table 2.

Protocol Quality Control metrics

1. Antibody validation: All primary and secondary antibodies are validated either by CLIA or GUDMAP citation, or by internal validation. 
2. Negative controls: When tissue is available, secondary only controls will be included.
3. Tissue qualification:
            a.A low resolution image of a 12 µm section of the entire biopsy labeled with PAS is collected (dissecting microscope, 6 µm resolution) and evaluated for tissue morphology and sample integrity. 
            b.The entire biopsy is imaged in 3D prior to labeling using multiphoton excitation to collect second harmonic and autofluorescence images (910 nm excitation, 430-460 nm and 460-660 nm emissions, respectively). Second harmonic images are used to identify/quantify the degree of fibrosis and along with tissue autofluorescence, to characterize tissue structure.
            c.The entire biopsy is imaged in 2D using confocal microscopy prior to labeling.(A mosaic of confocal images of a single focal plane collected from the middle of the tissue section, using the 16 channel protocol used to image labeled tissue).These images are used to characterize levels of background tissue autofluorescence in each channel to identify potentially problematic regions and to inform the process of spectral deconvolution of the labeled tissue.
4. Microscope image collection: Prior to collection of labeled tissues, the performance of the microscope system is verified by quantitative analysis of images collected from a slide containing with 0.1 µm and 4 µm TetraSpeck fluorescent microspheres (Blue – 360 nm excitation, 430 nm emission, Green – 505 nm excitation, 515 nm emission, Orange – 560 nm excitation, 580 nm emission and Dark Red – 660 nm excitation, 680 nm emission).By exciting the fluorescence of the microspheres with each of the four laser lines, and collecting the fluorescence in the four detectors, this procedure provides a sensitive test of the integrated performance of the microscope. Performance is quantified as signal-to-noise in the images of each of the four fluorophores in the 4 µm beads. Departures in signal-to-noise or resolution exceeding 1 standard deviation of the previously established values would be grounds to delay image collection until the microscope deficiency has been remediated.

Table 4. QC metrics scan settings

   Metric 

5. Digital image analysis:Digital image analysis is used to (1) distinguish 8 fluorescent probes in the 16-channel confocal image volumes (spectral deconvolution), (2) segment individual nuclei in the resulting image volumes and (3) quantify the amount of fluorescence associated with each nucleus in each of the 8 channels.
        a. Spectral deconvolution is validated by analysis of 16 channel images of beads labeled each of the fluorescent probes in specific proportions. The number of each type of bead is determined using spectral deconvolution, which is judged valid if the relative proportions of the different beads fall within 10% of the known proportions.
        b.The accuracy of nuclear segmentation is quantified as an F1 score (composite of precision and recall) obtained from 5 random fields (quantified relative to manually annotated).Automated nuclear segmentation is judged valid if F1 scores are greater or equal than 0.8
        c.Linearity in image collection is ensured by automatic elimination of regions of signal saturation (voxel values exceeding 4095) and detector dropout (voxel values of 0).
 
 
 
 
 
 
 
 

[STEPS]
SECTION: Label Free Imaging of Tissue
1. Mount sample on uncharged slide in PBS using prepositioned piece of spacer tape to support coverslip.
SECTION: Label Free Imaging of Tissue
2. Gently push coverslip to the sample and seal with rubber cement dispensed from a syringe and 16 G needle.
SECTION: Label Free Imaging of Tissue
3. Configure the microscope for multiphoton excitation at 910 nm for detecting SHG and auto-fluorescence (AF) (Table 2).
 

SECTION: Label Free Imaging of Tissue
4. Configure emission path by opening confocal pinhole and adjust the bandpasses on 2 to 4 detectors to collect SHG (~455 nm) and AF as desired (refer to “Excitation and filter settings”). Use PMTs to avoid detector “overloading” and data loss.
SECTION: Label Free Imaging of Tissue
5. Configure x, y and z limits to cover the entire tissue.  Scan in x and y at 0.5-1 µm/pixel.  Sample in z with 1-2 um steps.
SECTION: Label Free Imaging of Tissue
6. Perform scans with standard galvanometers set to 400 Hz, and averaging by line 2x.
SECTION: Label Free Imaging of Tissue
7. Fluorescence lifetime imaging is under development and may be included at this step.
SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
8. Recover specimen by removing rubber cement and gently lifting coverslip. 
 

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
9. Washing: Sample is washed 1x in PBS >10 min

 

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
10. Blocking: Remove wash and block non-specific binding sites with PBSTS 4–8 hours at Room temperature  on rocking platform.

 

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
11. Indirect primary antibody staining (Table 1): Incubate tissue in 200 µL staining solution for all the indirect detection primary antibodies 8-16 hours at Room temperature  on rocking platform.

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
12. Washing: Wash sample with 2 mL  PBST 2 times for a total of 360 min.

 

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
13. Blocking: Remove wash and block non-specific binding sites with PBSTS 240 min to 480 min  at Room temperature  on rocking platform.
 




SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
14. Indirect secondary antibody staining (Table 1): Incubate tissue in 200 µL staining solution for all the indirect detection secondary antibodies 480 min  to 960 min  at Room temperature  on rocking platform.

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
15. Washing: Wash sample with 2 mLPBST twice for a total of 360 min.

 

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
16. Blocking: Remove wash and block non-specific binding sites with PBSTS 240 min to 480 min at Room temperature  on rocking platform.

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
17. Directly conjugated antibody staining (Table 1): Incubate tissue in 200 µL staining solution for all the direct detection primary antibodies 480 min to 960 min  at Room temperature  on rocking platform.

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
18. Washing: Wash sample with 2 mLPBST twice for a total of 360 min.

 

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
19. Mount tissue with Prolong Glass or Fluoromount on pre-cleaned sides with silicone grease as needed to support coverslip.
 

 

 

SECTION: Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence
20. Seal cover slip to slide with rubber cement applied with a syringe.
SECTION: Large-scale 3D Spectral Confocal Imaging
21. Configure the microscope for spectral confocal imaging as described in Table 2 below.

  

 

SECTION: Large-scale 3D Spectral Confocal Imaging
22. Configure x, y and z limits to cover the entire tissue.  Scan in x and y at 0.5-1 µm/pixel.  Sample in z with    steps.

SECTION: Large-scale 3D Spectral Confocal Imaging
23. Perform scans with standard galvanometers set to 400 Hz.  The multispectral 16 channels are configured as indicated in Table 2 with 4 sequences of 4 detectors.
SECTION: Preparation of Bead Slide for Reference Spectra
24. Incubate 20 µL of CML latex beads (ThermoFisher, cat# C37253) with 100 µg  of fluorophore conjugated secondary overnight.
 




SECTION: Preparation of Bead Slide for Reference Spectra
25. Wash the beads after centrifuging at 1000 x g, 30 s .  Repeat twice. Dilute and mix individually labeled beads in MM (Prolong Glass or Fluoromount) such that 5-10 beads of each individually labeled beads is visible in a field size of 500 x 500 µm.

SECTION: Preparation of Bead Slide for Reference Spectra
26. Beads are mounted under #1.5 coverglass and cured for 2880 min to 5760 min  
 


SECTION: Genearating Matrix for Linear Unmixing
27. Scan slide-mounted beads labeled with known fluorophores with settings given in the table below for channels 1-16.

  

 

SECTION: Genearating Matrix for Linear Unmixing
28. Select “Process” -> “Dye Separation” -> “Channel Dye Separation”
SECTION: Genearating Matrix for Linear Unmixing
29. Select regions of interest including beads intensity and select “Add” to add to spectra.
SECTION: Genearating Matrix for Linear Unmixing
30. Select Rescale to “Per Channel.”
SECTION: Genearating Matrix for Linear Unmixing
31. Select “Load” to load a saved matrix.  The saved matrix will be displayed near the bottom of the window.
SECTION: Genearating Matrix for Linear Unmixing
32. Select “Apply”.
SECTION: Applying Unmixing Matrix for Linear Unmixing
33. In the LASX software select image to unmix.
 

SECTION: Applying Unmixing Matrix for Linear Unmixing
34. Select “Process” -> “Dye Separation” -> “Automatic Dye Separation”
SECTION: Applying Unmixing Matrix for Linear Unmixing
35. Set fluorescent dyes to “8.”
SECTION: Applying Unmixing Matrix for Linear Unmixing
36. Set Rescale to “Per Channel.”
SECTION: Applying Unmixing Matrix for Linear Unmixing
37. Select “Load” to load a saved matrix.  The saved matrix will be displayed near the bottom of the window.
SECTION: Applying Unmixing Matrix for Linear Unmixing
38. Select “Apply”.
SECTION: Stitiching/Merging of Unmixed Confocal Volumes
39. Select images to merge.
 

SECTION: Stitiching/Merging of Unmixed Confocal Volumes
40. Select “Process” -> “Mosaic Merge”
SECTION: Stitiching/Merging of Unmixed Confocal Volumes
41. Check “Autostitching”, “Smooth” overlap and “Linear Blending”.
SECTION: Stitiching/Merging of Unmixed Confocal Volumes
42. Select “Apply”.
SECTION: Cytometric analysis of stitched and merged unmixed confocal volumes
43. All 3D cytometry is performed with the ImageJ plugin Volumetric Tissue Exploration and Analysis (VTEA). The settings for processing, segmentation and gating are given in analysis files. As software improves, new versions of VTEA may be used.