Fig 1.
Workflow for the preparation of cryo-FIB milled glutamatergic synaptosomes for cryo-ET studies.
Fig 2.
FSEC analysis of isolated glutamatergic synaptosomes.
Detection of (A) vGLUT1-mVenus and (B) AMPAR bound to 15F1 Fab-mCherry in isolated synaptosomes using Venus (λex: 510 nm, λem: 530 nm) and mCherry (λex: 580 nm, λem: 610 nm) channels, respectively, via FSEC.
Fig 3.
Cryo-LSM images of glutamatergic synaptosomes on cryo-EM grids isolated using (A-C) sucrose, (D-F) Ficoll and (G-I) Percoll density gradient centrifugation. Fluorescence signals of vGLUT1-mVenus and AMPAR-15F1 Fab-mCherry at the pre and postsynaptic compartments of synaptosomes are in the green and red channel, respectively. Zoomed-in images of glutamatergic synaptosomes prepared using (J) Ficoll and (K) Percoll density gradient centrifugation corresponding to areas enclosed in cyan dashed box in (F) and (I). Glutamatergic synaptosomes are highlighted with white arrows. Scale bar in (A-I): 5 μm; scale bar in (J,K): 2 μm.
Fig 4.
Cryo-LSM images of glutamatergic synaptosomes on cryo-EM grids passed through 1 μm filter using a thermobarrel extruder.
Fluorescence signals from green, red and both green & red channels corresponding to (A) vGLUT1-mVenus, (B) AMPAR-15F1 Fab-mCherry and (C) both vGLUT1-mVenus & AMPAR-15F1 Fab-mCherry. (D) Zoomed-in area enclosed in cyan dashed box in (C) with glutamatergic synaptosomes highlighted with white arrows. Scale bar in (A-C): 5 μm and scale bar in (D): 2 μm.
Fig 5.
Cryo-SEM images of synaptosomes at different stages of FIB-milling.
(A) Cryo-SEM images of the whole cryo-EM grid with the selected squares for FIB-milling highlighted with cyan sphere. (B) Representative cryo-SEM image of a lamella preparation during rough milling. The lamella lies between the two milling patterns shown as yellow bars. Lateral micro-expansion joints [58], marked with yellow arrow, are created on both side of the lamella (only shown for the right-hand side) (C) Representative image of a polished synaptosome lamella with the sample area that can be imaged using cryo-ET enclosed in cyan dashed box. The platinum (Pt) gas injection system (GIS) layer is marked with yellow double arrow. Scale bar: 5 μm.
Fig 6.
Cryo-LSM images of glutamatergic synaptosomes in two cryo-FIB-milled lamellae, (A-C) lamella 1 and (D-F) lamella 2. Fluorescence signal from green, red and both green & red channels corresponding to (A) vGLUT1-mVenus, (B) AMPAR-15F1 Fab-mCherry and (C) overlay of vGLUT1-mVenus and AMPAR-15F1 Fab-mCherry, respectively. Glutamatergic synaptosomes are highlighted with white arrows. Scale bar: 5 μm.