Journal: Biophysical Reports

Article Title: Multimodal single-molecule microscopy with continuously controlled spectral resolution

doi: 10.1016/j.bpr.2021.100013

Figure Lengend Snippet: Multicolor single-particle tracking of axonal transport in live neurons. ( A ) Schematic illustration descripting distal axonal uptake of the different neurotrophic factors and their transportation to the proximal axon and cell body through endosomes. The three neurotrophic factors used in this experiment, proBDNF, BDNF, and NGF, were labeled with Qdot565 ( green ), Qdot625 ( red ), and Qdot800 ( magenta ), respectively. ( B ) False-colored overlay of time-averaged localization (RPA = 180°) full FOV time lapse, processed with background removal. In each frame, four consecutive acquisitions with emission filter switching were taken ( white , no filter; cyan , 575/15; yellow , 605/15; and magenta , 809/81) and 405 nm excitation. The FOV shows the signaling activity in the proximal axons near neuronal cell bodies. Orange rectangle marks the axon presented in ( E ). Scale bars, 30 μ m. ( C ) PSF examples of an endosome transporting all three neurotrophic factors through the axon marked in ( B ). Left: three sequential localization (RPA = 180°) acquisitions with emission filters switching (same color code as in ( B )). Middle: single localization (RPA = 180°) acquisition with no emission filter. Right: CoCoS color-detection mode with no emission filter and optimal dispersion (RPA = 172°); color bar on the right illustrates the spectral-dispersion map (nonaccurate). ( D ) Theoretical spectra of the three Qdots with PSF visual representation for illustration purposes (nonaccurate. For a theoretically calculated dispersed PSF, see Fig. S13 ). ( E ) Example time-lapse frames followed by a kymograph of the marked axon in ( B ), showing the retrograde transport of the endosome presented in ( C ). Left: three consecutive acquisitions per frame are shown with RPA = 180°, 405 nm excitation, and 575/15 ( cyan ), 605/15 ( yellow ), and 809/81 ( magenta ) emission filters. A spatiotemporal mismatch between the acquisitions is clearly visible when the transport velocity is high (0, 3.2, and 6.4 s frames). Right: a single acquisition per frame is shown with no emission filter and with RPA = 172°. At the bottom of each time lapse, a kymograph of the full time lapse is presented, showing a halt in transportation at cell death after 30 s of imaging. Horizontal scale bars, 3 μ m; vertical scale bars, 30 s. ( F ) Example of CoCoS PSFs for each of the protein combinations inside the transporting endosomes as detected in the FOV of ( B ). Left: single acquisition with RPA = 172°. Right: false-color overlay of three consecutive acquisitions with RPA = 172° and emission filters switching.

Article Snippet: Meanwhile, human BDNF-biotin (B-250-B; Alomone Labs), human pro-BDNF-biotin (B-256-B; Alomone Labs), and mouse NGF-biotin (N-240-B; Alomone Labs) were mixed separately in a molar ratio of 3:1 with Qdot-625 streptavidin, Qdot-565 streptavidin, and Qdot-800 streptavidin, respectively (Q10131MP; Q10143MP; and Q10173MP; Molecular Probes, Eugene, OR).

Techniques: Single-particle Tracking, Labeling, Activity Assay, Imaging