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. 2014 Jan 16;5:3024. doi: 10.1038/ncomms4024

Figure 1. SPT in organotypic brain slices using QDs coupled to a monoclonal antibody against GFP.

Figure 1

(a) Z-projection of anti-GFP QDs inside an organotypic hippocampal slice acquired with our CSU set-up: anti-GFP QDs at dendrites of a GPI–GFP-transfected neuron were recorded at different depths inside the slice with an acquisition frequency of 10 Hz and an axial sampling rate of 0.1 μm. The colour encodes the z-depth of localized molecules within the stack projection, starting from 5 μm (blue) to 60 μm (deep red). Scale bar, 10 μm. (b) Magnified view of the positions of detected molecules at four different depths/locations, as indicated in a. Extracted trajectories are plotted with different colours in the right panel. Note that the labelling specificity remains similar between the four given examples. (c) Example of freely diffusing unspecific QDs. Note that the large cloud of detected QDs only results in very few trajectories because of the quick escape of QDs from the focal plane. Scale bar, 1 μm (b,c). (d) Projections of z-scans along a GPI–GFP-transfected neuron within an organotypic hippocampal slice using a two-photon microscope. Scale bar, 10 μm. Enlarged subregions at different depths show specific labelling with anti-GFP QDs without a loss of labelling density. Scale bar, 5 μm. (e) Quantification of relative labelling density for three experimental settings (red, single-photon excitation, oil immersion objective × 100/NA 1.4, blue, single-photon excitation, water immersion objective × 60/NA 1.1, black, two photon excitation, water immersion objective × 60/NA 1.1, error bars represent±s.e.m.). The labelling density was calculated by normalizing the number of QDs detected within optical slices to the density at 3 μm penetration depth. The inset demonstrates that there is only minor change in the specificity of labelling within the first 40 μm (error bars represent±s.e.m.). Data obtained with × 60/NA 1.1 water immersion objective are from 11 neurons in 7 organotypic slice cultures and data acquired with a × 100/NA 1.4 oil immersion objective are from 7 neurons in three slice cultures. Two-photon data are from six neurons in four slice cultures.