FLUOS

Imaging experiments were carried out using a multi-photon installation comprising a Radiance 2000 imaging system (Zeiss-BioRad) optically linked to a femtosecond laser MaiTai (SpectraPhysics) and integrated with a single-cell electrophysiology set-up (Rusakov and Fine, 2003 (link)). Granule cells were held in whole-cell mode and loaded with two fluorophores, a morphological tracer Alexa Fluor 594 (20 μM) and a Ca²⁺ indicator (Fluo-4, Fluo-5F or Oregon Green BAPTA-1, as specified). In granule cells with intact axons (see Results and Supplemental Fig. 1), 15-20 min were initially allowed for indicator equilibration before switching the system into fluorescence mode to trace the axon into stratum lucidum. Fluorophores were excited in two-photon mode at 810 nm, with the laser power optimized for emission detection at different depths in the slice.
The axon was followed from the soma into stratum lucidum using frame mode scanning (256 × 256 pixels, 500 Hz; the number of taken frames was kept small to minimize phototoxic damage) and the system was focused on a giant MFB identified by its distinct morphology, at the maximum optical resolution (~0.2 μm, digital capture 70 nm per pixel). Recording started when the baseline fluorescence in both channels was stable (approximately one hour later; see Results and Supplemental Fig. 2); the experiment lasted for up to 3-4 hours and dye equilibration was routinely controlled post-hoc, by comparing the resting fluorescence of Alexa Fluor 594 in the end of the experiment with that recorded 1-1.5 hours earlier. We conducted two additional tests to verify that Ca²⁺ indicators are equilibrated along the axon and are not extruded from the cells appreciably with time. In the first test, we carefully pulled out the patch pipette following initial dye equilibration. The seal was confirmed by the fact that we obtained outside-out patches as a result and that cell could respond to extracellular stimulation by generating action potential dependent Ca²⁺ transients. The subsequent recordings indicated no detectable loss of the resting fluorescence F over 100-200 min (Supplemental Fig. 3). In the second test, we measured the baseline fluorescence in the axonal regions at different distances from the soma and found no spatial gradient (Supplemental Fig. 4). The results of both tests argued against any significant loss of fluorescence indicators from the axon.
Fluorescence responses were recorded in line-scan mode at 500 Hz (500 or 1000 ms sweeps, inter-sweep interval 30 s or 1 min) and stored for off-line analysis. The Ca²⁺-dependent fluorescence response ΔF/F (integrated over the visible MFB width) was routinely calculated as (F_post-F_pre)/(F_pre-F₀). The values of F_pre and F_post stand for the line scan fluorescence averaged over, respectively, 100 ms prior to the first spike and either 50 ms in the case of single-response amplitude measurements or 250 ms in the case of five-response amplitude measurements (20 Hz train of five APs) after the first spike onset. F₀ denotes the background fluorescence measured outside any cell structures filled with the indicator. Because special care was taken to avoid escape of the indicator from the pipette and because the site of imaging was hundreds of microns away from the pipette tip, F₀ was likely to represent the photomultiplier tube dark current. Image analyses were performed on stacks of stored line-scan images using a set of custom NIH Image macros. False color tables and averaged images were used for illustration purposes but the original (gray level) pixel brightness values in each line-scan image were used for the quantitative analysis. In most experiments, we reconstructed the axon trajectory using a collage of high-resolution Kalman-filtered z-stacks 15-20 μm deep. In total, we obtained full reconstruction of 43 axons, with an average distance between the recorded MFB and the soma of 686 ± 38 μm. Throughout the experiments, we observed no failures of spike-driven Ca²⁺ signals propagating along the main axonal trunk including giant MFBs. This, however, does not rule the possibility that propagation could fail at higher spiking frequencies and/or in thin axon collaterals.
The two-photon excitation probability profile is proportional to the squared illumination light intensity $I_{2p}^2$ (Zipfel and Webb, 2001 ):
$$I_{2p}^2(u,v)={\mid 2{\int }_0^1{J}_0\left(v_{\rho }\right)\exp (-\frac{i}{2}u{\rho }^2)\rho d_{\rho }\mid }^4$$
where the canonical co-ordinates $u=4k{\sin }^2\left(\frac{\alpha }{2}\right)z$ and v = ksin(α)r represent the axial distance z, radial distance r, the objective’s numerical aperture NA = sin(α) = 0.9 and wave number $k=n\frac{2\pi }{\lambda }$ (n = 1.33 is the medium refraction index and λ = 810 nm is the wavelength); J₀ denotes zero-order Bessel’s function of the first kind. This theoretical function, however, represents the lower limit estimate of the excitation profile: in reality, optical aberrations and imperfect alignment of the experimental optical system are likely to increase the spread of excitation. Similar considerations apply to the emission path. We therefore obtained an estimate of the excitation-emission profile by recording the point-spread function (PSF) of the system using 0.17 μm fluorescent beads (PS-Speck Microscope Point Source Kit, Invitrogen) as illustrated below.