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Abstract

Source: Nyström, S., et al., Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging. J. Vis. Exp. (2017).

This video demonstrates analyzing amyloid structures with fluorescence lifetime imaging microscopy or FLIM, using hFTAA dye to reveal compact cores as blue/green and unstable peripheries as red/yellow in color-coded images, reflecting amyloid organization.

Protocol

1. Preparation of Tissue Samples

NOTE: Many tissue types can be imaged using heptamer-formyl thiophene acetic acid (hFTAA) as an amyloid marker. hFTAA is sensitive to aggregate conformation. The staining should, hence preferably, be performed on undisrupted aggregates with no epitope exposure. Optimal spectral quality is achieved if the tissue fixation is kept to a minimum. Hence, fresh frozen material, gently fixed in ethanol at the time of staining, is preferred. However, it is possible to detect amyloid deposits also in tissue that has been fixed with e.g., formalin. hFTAA generally penetrates the tissue well. Select a specimen thickness that is compatible with the intended imaging technique.

1. If formalin-fixed, paraffin-embedded sections are used, they should be deparaffinized in xylene overnight. Then, dip the sections in consecutive baths of 99% ethanol, 70% ethanol, dH₂O, and phosphate-buffered saline (PBS), 10 min in each. Allow the tissue sections to dry under ambient conditions.
   Caution: Xylene is always handled in a chemical fume hood. Xylene and other organic solvents are harmful.
   1. Thaw cryosections at room temperature. Fix the tissue sections in 10% formalin overnight and rehydrate by dipping them in consecutive baths of 99% ethanol, 70% ethanol, dH₂O, and PBS, 10 min in each. Allow the tissue sections to dry under ambient conditions.
2. Add droplets of the hFTAA working solution (approximately 200 µL) to the tissue sections to cover it. The droplet should stay in place by surface tension. Incubate for 30 min at room temperature for staining.
3. Rinse off the staining solution with 500 µL PBS using a pipette and then immerse the slide in the PBS bath for 10 min. Allow the section to dry under ambient conditions.
4. Mount using the fluorescence mounting medium. Allow mounting medium to settle overnight.

2. FLIM
NOTE: The confocal microscope is equipped with a fluorescence lifetime imaging microscopy (FLIM) unit.

1. Set up the following parameters for the confocal microscope: Pinhole, 20; excitation wavelength, 490 nm; laser intensity, 0.5% (corresponding to an average power of 7.5 µW). Use pulsed lasers at 40 MHz.

2.     In the FLIM software, set up photon counting over 550 nm. In the Display parameters window, follow the photon counting until the Max count is around 4,000 photon counts.

3.     Save the file and export it as a single-photon counting (SPC) image.

4.     Fit the data to a 2-component exponential decay in the FLIM software. A fit that gives a χ² < 2 is good. The value on the y-axis is the number of counts for the given lifetime.

5.     Select a threshold for counts to include, e.g., 100. Color code by T1 and select lifetime range. The decay is dependent on the amyloid structure where the hFTAA is bound. Fluorescence lifetimes between 300 and 1,000 ps have been observed. Save the file.

6.     Export the raw data by using the export options and save the desired data in a new folder.

Materials

hFTAA/Amytracker545	Ebba Biotech
Dako fluorescene mounting medium	Agilent technologies	GM304
LeicaDM6000	Leica
Lumen 200	Prior
Spectraview system	ASI spectral imaging
Spectraview software	ASI spectral imaging
LSM780	Zeiss
Zen 2010b v6.0 software	Zeiss
FLIM system	Becker & Hickl
Ar/ML 458/488/514	Zeiss
Tunable Laser In Tune	Zeiss