Jun 19, 2023
Autofluorescence Microscopy QC for Multimodal Molecular Imaging
- Katerina Djambazova1,
- Nathan Heath Patterson1,
- Lukasz Migas2,
- Angela R.S. Kruse1,
- Melissa Farrow1,
- Raf Van De Plas2,
- Jeff Spraggins1
- 1Vanderbilt University;
- 2Delft University of Technology
- VU Biomolecular Multimodal Imaging Center / Spraggins Research Group
- KPMP
Protocol Citation: Katerina Djambazova, Nathan Heath Patterson, Lukasz Migas, Angela R.S. Kruse, Melissa Farrow, Raf Van De Plas, Jeff Spraggins 2023. Autofluorescence Microscopy QC for Multimodal Molecular Imaging . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjm5dlmk/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: February 08, 2023
Last Modified: October 18, 2023
Protocol Integer ID: 76686
Keywords: autofluorescence microscopy qc for multimodal molecular imaging, autofluorescence microscopy qc, autofluorescence qc, fluorescence, fluorescence intensity, fluorescence channel, multimodal molecular imaging, microscope performance, imaging, tissue af, egfp
Abstract
Autofluorescence QC is performed prior to collecting on-tissue AF. Fluorescence intensity can be tracked for each fluorescence channel (DAPI, EGFP, and DsRed), allowing the microscope performance and the LED light source to be benchmarked and tracked over time.
Materials
StarLight Calibration Slides (Bang Laboratories) - Glacial Blue (SL1GB), Dragon Green (SL1DG), Envy Green (SL1EG)
Zeiss AxioScan Z1 Microscope
Autofluorescence Microscopy QC
Fluorescence QC is collected prior to any on-tissue AF collection.
Use fluorescence calibration slides - StarLight Calibration Slides - Bang Laboratories
DAPI (excitation-360nm, emission-450nm), Dragon green (excitation-480nm, emission-520nm), and Envy green (excitation-525nm, emission-565nm)
Load calibration slides into Zeiss AxioScan.Z1 slide scanner
Select the same ~ 25 x 25 mm square region in each calibration slide
Note
To reproducibly sample the exact region each time, the square region can be saved as a "region of interest", which can be imported for each slide.
Acquire fluorescence microscopy data for each calibration slide.
Note
Example set-up and relative ranges:
DAPI QC: DAPI (ex. 360 nm, em. 450 nm) Calibration Slide
LED Light: 385 nm
Light Source Intensity: 90%
Exposure time: 20 ms
eGFP QC: Dragon Green (ex. 480 nm, em. 520 nm) Calibration Slide
LED Light: 475 nm
Light Source Intensity: 90%
Exposure time: 60 ms
DsRed QC: Envy Green (ex. 525 nm, em. 565 nm) Calibration Slide
LED Light: 567 nm
Light Source Intensity: 90%
Exposure time: 250 ms
Data Analysis:
Once data are collected, background pixels are subtracted, and the average intensity of each channel is calculated and monitored over time.