Oct 10, 2025

Public workspacePre-MALDI Autofluorescence Microscopy of Lung Tissues

DOI
https://dx.doi.org/10.17504/protocols.io.q26g75z23lwz/v1
  • Brittney Gorman1,
  • Christopher Anderton1
  • 1Pacific Northwest National Laboratory
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: [email protected]
Brittney Gorman
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g75z23lwz/v1
Protocol Citation: Brittney Gorman, Christopher Anderton 2025. Pre-MALDI Autofluorescence Microscopy of Lung Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g75z23lwz/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: Working
We use this protocol and it's working
Created: March 24, 2025
Last Modified: October 10, 2025
Protocol Integer ID: 124931
Keywords: Autofluorescence, Zeiss, MALDI, optical imaging, HuBMAP, Lung, PNNL, autofluorescence microscopy images of lung tissue section, autofluorescence microscopy image, brightfield image of each tissue section, lung tissue, lung tissue section, visualization of tissue morphology, imaging, tissue morphology, additional imaging modality, brightfield image, tissue section, tissue, image
Funders Acknowledgements:
Hubmap
Grant ID: U54HL165443
Abstract
Obtain autofluorescence microscopy images of lung tissue sections. This will produce an RGB plus brightfield image of each tissue section that enables visualization of tissue morphology, and these images can be co-registered with additional imaging modalities.
Guidelines
Carefully handle all slides with gloves
Materials
Zeiss Axio Zoom Microscope
Troubleshooting
Autofluorescence Microscopy Imaging
If tissue-mounted slides are frozen, allow them to equilibrate to room temperature (~20 ℃) before opening the vacuum-sealed bag (~30 min); otherwise, proceed directly to step 2.
Place the microscope slide(s) onto the stage of the Zeiss Axio Zoom Microscope
Open Zeiss Zen software
Set up the method for acquiring autofluorescence (RGB-channels) and transmitted light images

Note
Magnification: 100 x
Objective: PalnNeoFluar Z 2.3x

Blue: Chroma DAPI (ex. 353 nm, em. 465 nm)
Light Source Intensity: 100%
Exposure time: 350 ms
- Captures NAD(P)H excitation present in cells and elastin in the extracellular matrix

Green: Chroma GFP (ex. 488 nm, em. 509 nm)
Light Source Intensity: 100%
Exposure time: 500 ms
- Captures Flavins in metabolically active zones (increased in the vasculature)

Red: Chroma DSRED (ex. 545 nm, em. 572 nm)
Light Source Intensity: 100%
Exposure time: 1300 ms
- Captures lipopigments often found in muscle

Transmitted Light:
Light Source Intensity: 100%
Exposure time: 1450 ms
- General morphological assessment of the tissue

Define imaging regions to include each individual tissue section within a single region
Perform manual focus adjustment at ~6 'support points' per tissue section
Switch to the green channel, press 'Live Scan', and perform focus adjustments at each support point
Press 'Start Scan' to begin image acquisition. The microscope will automatically scan each tissue section and interpolate the focus between support points
The method will acquire a tiled brightfield and (auto)fluorescence image.

Export each image region as a Tiff and PNG using Zen
(The .czi files can be visualized using Zen or QuPath software)
Under the processing tab use the 'Split Scenes' method to create individual .czi files for each region
Under the processing tab, use the 'Image export' method to export as a Tiff and PNG
Resize: 50%