Sep 01, 2024

Public workspaceIMARIS Analyses of LAMP_CATD in vitro

DOI
dx.doi.org/10.17504/protocols.io.81wgbzozygpk/v1
  • Chiara Pavan1,
  • Stefano Frausin2,
  • Clare Parish1,
  • Lachlan Thompson2
  • 1Florey Institute for Neuroscience and Mental Health;
  • 2University of Sydney
courtney.wright Wright
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DOI: dx.doi.org/10.17504/protocols.io.81wgbzozygpk/v1
Protocol Citation: Chiara Pavan, Stefano Frausin, Clare Parish, Lachlan Thompson 2024. IMARIS Analyses of LAMP_CATD in vitro. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzozygpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: August 30, 2024
Last Modified: September 01, 2024
Protocol Integer ID: 106733
Keywords: ASAPCRN, cathepsin D, LAMP, in-vitro, iPSC, microscopy analysis, MAP2
Funders Acknowledgements:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol details how to analyse in-vitro LAMP and cathepsin D staining using Imaris software.
Materials
Software
Imaris Analysis Software (Oxford instruments Andor)
Before start
Select your image files folders. Drag the .LIF files into the Imaris Arena.
Double click. Do not open \LNG files
Creation of MAP2 surface
Creation of MAP2 surface
  1. Add new surface.
  2. Leave Creation Parameters as default
  3. Selected segment only ROI.
  4. Click next. Drag the yellow box to select the cell. Whole cell of interest.
  5. Click Next. Select the channel (for your marker of interest). Smooth Surface detail. 0.0360. Machine learning segmentation.
  6. Click Next. Draw thresholds; foreground and background w Shift-Click > 3 planes. Ensure the “pointer selection mode” is selected.
  7. Click Next. twice.
  8. Threshold to have only one mask included.
  9. Click Finish.
  10. Go to Statistics, detailed tab, average values, save/export data.
o Save with suffix Cell #_MAP2
Create LAMP1 Surface
Create LAMP1 Surface
  1. Add new surface.
  2. Add LAMP1 parameters. Ensure Select only a ROI is ticked. Ensure object-object stats is checked.
  3. Click Next.
  4. Drag yellow box to the ROI. Click Next.
  5. Select green (LAMP1) channel. Smooth 0.0361. Machine learning selected. Click Next.
  6. Add Machine Learning Thresholds. Foreground (green) and background (purple) w Shift-Click > 3 planes. Click Next.
  7. Check enable. Seed Point Diameter 0.181.
  8. Click Next. Adjust slide bar to highlight LAMP1 included regions. ClickNext.
  9. Click Next. again and adjust.
  10. Click Finish.
  11. Remove LAMP1 regions by selecting the pencil (edit) and click the regions outside the cell and ‘delete’. Move through all the planes (z-axis).
  12. Click Statistics, detailed tab; Average Values and export data.
o Save with suffix Cell #_LAMP1
Create Cathepsin Spots
Create Cathepsin Spots
  1. Create new ‘Spots’ for Cathepsin. Check the ‘different spot size (region growing). Click next.
  2. Select ROI using the yellow box. Click Next.
  3. Select the correct channel (red). Model XYZ diameter= 0.291, Estimated z diameter = 0.830. Check background subtraction. Click Next..
  4. Select threshold slider of Cat+ spots. For simplicity, select slice rendering for 3D spots. Click Next.
  5. Check absolute intensity. Click Next.
  6. Click Finish.
  7. Remove Cathepsin dots outside the cell by selecting the pencil (edit) and click the regions outside the cell and ‘delete’. Move through all the planes (z-axis).
  8. Click Statistics, detailed tab; Average Values and export data.
o Save with suffix Cell #_CatD
Determining CatD-LAMP1 coalescence
Determining CatD-LAMP1 coalescence
  1. Select filer. Add Shortest distance to LAMP1 surface #.
  2. Slide scale bar to remove spots that aren’t touching.
  3. Click duplicate selection.
  4. Click Statistics, detailed tab; Average Values and export data.
o Save with suffix Cell #_CatD_co