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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><div class = "text-block">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.</div><div class = "text-block">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.</div><div class = "text-block">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.</div><div class = "text-block">These protocols start with fixed 50 µm tissue sections prepared as described in the Cryopreservation protocol.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/2f06ffaf97a411d8b024ac07fbf0d125b1ba7e72ab795411cfbcef42ae0901dc/bzj9bbgep.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/2f06ffaf97a411d8b024ac07fbf0d125b1ba7e72ab795411cfbcef42ae0901dc/bzkabbgep.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/2f06ffaf97a411d8b024ac07fbf0d125b1ba7e72ab795411cfbcef42ae0901dc/bzkbbbgep.png" /></div></div>

[STEPS]
?. [Label Free Imaging of Tissue]
Mount sample on uncharged slide in PBS using prepositioned piece of spacer tape to support coverslip.
?. [Label Free Imaging of Tissue]
Gently push coverslip to the sample and seal with rubber cement dispensed from a syringe and 16 G needle.
?. [Label Free Imaging of Tissue]
Configure the microscope for multiphoton excitation at 910 nm for detecting SHG and auto-fluorescence (AF) (Table 2).
?. [Label Free Imaging of Tissue]
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.
?. [Label Free Imaging of Tissue]
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.
?. [Label Free Imaging of Tissue]
Perform scans with standard galvanometers set to 400 Hz, and averaging by line 2x.
?. [Label Free Imaging of Tissue]
Fluorescence lifetime imaging is under development and may be included at this step.
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Recover specimen by removing rubber cement and gently lifting coverslip.
Lifting the coverslip may be easier if done after or while soaking the slide in PBS.
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Washing: Sample is washed 1x in PBS >
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Blocking: Remove wash and block non-specific binding sites with PBSTS 4–8 hours at   on rocking platform.
0 Room temperature
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Indirect primary antibody staining (Table 1): Incubate tissue in  staining solution for all the indirect detection primary antibodies 8-16 hours at   on rocking platform.
200 µl
0 Room temperature
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Washing: Wash sample with   PBST 2 times for a total of .
2 ml
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Blocking: Remove wash and block non-specific binding sites with PBSTS  to   at   on rocking platform.
0 Room temperature
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Indirect secondary antibody staining (Table 1): Incubate tissue in  staining solution for all the indirect detection secondary antibodies   to   at   on rocking platform.
200 µl
0 Room temperature
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Washing: Wash sample with PBST twice for a total of .
2 ml
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Blocking: Remove wash and block non-specific binding sites with PBSTS  to  at   on rocking platform.
0 Room temperature
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Directly conjugated antibody staining (Table 1): Incubate tissue in  staining solution for all the direct detection primary antibodies  to   at   on rocking platform.
200 µl
0 Room temperature
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Washing: Wash sample with PBST twice for a total of .
2 ml
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Mount tissue with Prolong Glass or Fluoromount on pre-cleaned sides with silicone grease as needed to support coverslip.
?. [Staining of Tissue with Fluorescent Small Modules and by Immunofluoresence]
Seal cover slip to slide with rubber cement applied with a syringe.
?. [Large-scale 3D Spectral Confocal Imaging]
Configure the microscope for spectral confocal imaging as described in Table 2 below.
The settings for laser power, detector, voltage, gain and offset may vary based on the microscope configuration.
?. [Large-scale 3D Spectral Confocal Imaging]
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.
?. [Large-scale 3D Spectral Confocal Imaging]
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.
?. [Preparation of Bead Slide for Reference Spectra]
Incubate  of CML latex beads (ThermoFisher, cat# C37253) with of fluorophore conjugated secondary overnight.
20 µl
100 µg
Reference spectra for DAPI and Oregon Green are collected from tissue labeled only with DAPI or Oregon Green. The remaining reference spectra from fluorophores outlined in Table 1 are collected from single-fluorophore labeled latex beads.
?. [Preparation of Bead Slide for Reference Spectra]
Wash the beads after centrifuging at  .  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.
Centrifuge: 1000 34, 30 s
?. [Preparation of Bead Slide for Reference Spectra]
Beads are mounted under #1.5 coverglass and cured for  to
?. [Genearating Matrix for Linear Unmixing]
Scan slide-mounted beads labeled with known fluorophores with settings given in the table below for channels 1-16.
Data collected is subjected to linear unmixing with Leica LASX software
?. [Genearating Matrix for Linear Unmixing]
Select “Process” -> “Dye Separation” -> “Channel Dye Separation”
?. [Genearating Matrix for Linear Unmixing]
Select regions of interest including beads intensity and select “Add” to add to spectra.
?. [Genearating Matrix for Linear Unmixing]
Select Rescale to “Per Channel.”
?. [Genearating Matrix for Linear Unmixing]
Select “Load” to load a saved matrix.  The saved matrix will be displayed near the bottom of the window.
?. [Genearating Matrix for Linear Unmixing]
Select “Apply”.
?. [Applying Unmixing Matrix for Linear Unmixing]
In the LASX software select image to unmix.
Once a matrix file is generated (.sdm) it may be used again to apply collected images.
?. [Applying Unmixing Matrix for Linear Unmixing]
Select “Process” -> “Dye Separation” -> “Automatic Dye Separation”
?. [Applying Unmixing Matrix for Linear Unmixing]
Set fluorescent dyes to “8.”
?. [Applying Unmixing Matrix for Linear Unmixing]
Set Rescale to “Per Channel.”
?. [Applying Unmixing Matrix for Linear Unmixing]
Select “Load” to load a saved matrix.  The saved matrix will be displayed near the bottom of the window.
?. [Applying Unmixing Matrix for Linear Unmixing]
Select “Apply”.
?. [Stitiching/Merging of Unmixed Confocal Volumes]
Select images to merge.
Following unmixing, the confocal dataset is merged with LEICA LASX software.
?. [Stitiching/Merging of Unmixed Confocal Volumes]
Select “Process” -> “Mosaic Merge”
?. [Stitiching/Merging of Unmixed Confocal Volumes]
Check “Autostitching”, “Smooth” overlap and “Linear Blending”.
?. [Stitiching/Merging of Unmixed Confocal Volumes]
Select “Apply”.
?. [Cytometric analysis of stitched and merged unmixed confocal volumes]
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.