405 nm diode laser

The detection system was set up on an epi-fluorescence microscope (Olympus IX81). Diode lasers (Toptica Photonics, Munich, Germany) were used for selective fluorescence excitation of GFP, YFP and Cy5/DiD at 488 nm, 514 nm and 640 nm, respectively. A 405 nm diode laser (Toptica Photonics, Munich, Germany) was used for bleaching of GFP/YFP fluorescence. Samples were illuminated in total internal reflection (TIR) configuration (CellTIRF, Olympus) using a 60 x oil immersion objective (NA  =  1.49, APON 60XO TIRF, Olympus, Munich, Germany). After appropriate filtering using standard filter sets, fluorescence was imaged onto a CCD camera (Orca-R2, Hamamatsu, Japan). Samples were mounted on an x-y-stage (CMR-STG-MHIX2-motorized table; Märzhäuser, Germany) and scanning of larger areas was supported by a laser-guided automated focus-hold system (ZDC-2; Olympus). For FRAP experiments single patterns were photobleached with a laser pulse (405 nm) applied for 100 ms. Recovery images were recorded at indicated time intervals. FRAP images were analyzed using the Multimeasure plugin of ImageJ [27] (link). Data were normalized by the pre-bleach image and curve fitting was done using Graphpad Prism. Resulting FRAP curves were plotted based on the standard error of the mean (SEM) and fitted using a bi-exponential equation. Kinetic FRAP parameters were directly obtained from curve fitting.

Diffusion analysis
was performed by a custom written MATLAB platform (based on @msdanalyzer).^{52 (link)} The spreadsheets with linked coordinates (output
from Trackpy tool) were used as input for @msdanalyzer. For the colocalization
analysis of internalized EVs and transferrin from human serum conjugated
with AlexaFluor647 (Invitrogen, Carlsbad, CA, USA), HeLa cells were
cultured for 48 h on glass slides coated with 1 μg/cm² fibronectin. eGFP-CD63 EVs were diluted 1:100 in FluoroBrite medium
supplemented with 1% EV-depleted FBS and incubated with HeLa cells
for 30 min at 37 °C in 5% CO₂ atmosphere. After EVs
were internalized by the cells, transferrin was added to the cells
and incubated for 10 min of incubation at 37 °C, 5% CO₂. Cells were washed and fixed with 4% paraformaldehyde (PFA) in PBS.
Imaging was performed in OxEA buffer (50 mM β-mercaptoethylamine,
3% oxyrase, 100 μM DL-lactate, 30v/v% glycerine) in PBS adjusted
to pH 8.0–8.5 with NaOH. Images were acquired as previously
described. Samples were illuminated for 20 ms, and a sequence of 10 000
frames was recorded with a delay between individual images of 33 ms.
During the camera chip read-out, UV light (405 nm diode laser, Toptica
Photonics) was additionally used to activate the probes, and the laser
power was adjusted during the measurements accordingly.

RR and SERR spectra were acquired with a Raman spectrometer (Jobin Yvon U1000, Edison, NJ, USA), equipped with a 1200 lines/mm grating and a liquid-nitrogen-cooled CCD detector, which was coupled to a confocal microscope. An Olympus 20× objective was used for laser focusing onto the sample and light collection in the backscattering geometry. Spectra were measured using a 405 nm diode laser (Toptica Photonics AG, Munich, Germany).
The RR spectra of CboDyP and TfuDyP were measured as previously described [26 (link)]. ScoDyP spectra were acquired at low temperature using ca. 2 μL of the enzyme sample placed in a microscope stage (Linkham THMS 600, Tadworth, UK) cooled to the desired temperature with liquid N₂.
RR experiments were performed with a 1.8 mW laser power and a 120 s accumulation time. SERR experiments were performed with a 1.3 mW laser power and 30–40 s accumulation time. Up to 16 spectra were co-added in each measurement to improve the signal-to-noise ratio (S/N). All spectra were subjected to polynomial baseline subtraction; the positions and widths of Raman bands were determined by component analysis as described previously [43 (link)].