Na water immersionobjective

In Supplementary Fig. 2, mCherry-tagged “seeds” of MyD88^FL and MAL^FL were purified using N-terminally tagged MyD88^FL and C-terminally tagged MAL^FL. Proteins were expressed in LTE by addition of the respective template DNAs in 10 µL lysate at a final concentration of 40 nM. The expression was allowed to be carried out for 3.5 h at 27°C. To produce the “seeds”, the samples were then spun down at 13,000 x g for 5 min. 80% of the supernatant was discarded and the solution was sonicated for 1 min in a water bath. In the meantime, GFP-tagged MyD88^FL or MAL^FL were expressed for 2.5 h, from 2 nM DNA template, and diluted 10 times before being placed under the microscope. Two lasers (488 nm and 561 nm) were focused in solution using a 40x/1.2 NA water immersion objective (Zeiss). Fluorescence was collected and separated using a 565 nm dichroic mirror; signal from GFP was passed through a 525/20 nm band-pass filter, while fluorescence from mCherry was filtered by a 580 nm long-pass filter. The fluorescence of the two channels was recorded simultaneously in 1 ms time bins. Fluorescence time traces were recorded for 75 s before 1 µL of “seeds” was introduced into the mixture and the measurements were carried out for an additional 300 s.

Cortical neurons transduced with relevant Lentivirus as described above were imaged at DIV 15 (7 days after iCre transfection) in imaging solution (HBSS with 2 mM GlutaMAX, 1 mM sodium pyruvate, and 10 mM HEPES pH 7.4). As a membrane-localized reference channel, Crimson RFP^{26 (link)} with a C-terminal farnesylation motif (CAAX) for membrane targeting was co-expressed with an ASAP variant via IRES. The epifluorescence from the cells were imaged on an Axiovert 200M inverted microscope with a 40x 1.2-NA water-immersion objective (Zeiss) and an X-Cite 120 metal-halide lamp (Exfo) as the excitation light source. For ASAP, excitation and emission filters were BP450-490 and BP515-565 (Zeiss). For Crimson, excitation and emission filters were HQ535/50m and HQ625/60m (Chroma). For each cell, 15 focal planes spaced 1 μm apart were captured by a Flash4.0LT+ camera using μManager. To quantify membrane localization, a custom-written MATLAB (MathWorks) code was used. Membrane masks of the neurons were generated by applying the graythresh function on Sobel-filtered red channel images. The soma masks were manually selected using the roipoly function on red channel images. Then the two masks were applied to background-subtracted green channel images to obtain the membrane fluorescence and the soma fluorescence of the ASAP reporter. The total fluorescence is the sum of the two.

The fluorescence lifetime images were acquired using a LSM 7 MP 2-photon microscope (Carl Zeiss, Weimar, Germany) coupled to the Becker and Hickl (BH) simple-Tau-152 system. Chameleon Ti:sapphire laser system with an 80 MHz repetition rate was used to excite the sample at a wavelength of 1000 nm. Images were acquired through a Zeiss 20 × 1 NA water-immersion objective. A Zeiss dichroic mirror (LP 760) was used to separate the excitation and the emission light. Emission light between 625 and 760 nm was collected via a hybrid GaAsP detector (HPM-100-40, BH, Berlin, Germany). The image acquisition time was 150−180 s to collect a sufficient number of photons.
For the photostability measurements, 50 images were collected for 23 min every 25 s. Images were analyzed using the SPCImage software (BH).