Expansion & structured illumination microscopy: the perfect combo to raise resolution

Nowadays, Thy1-GFP transgenic mice are widely used in neurobiological studies where fluorescent labeling of neural tissues is required (for further details on Thy1-GFP mouse line see Feng et al., Neuron, 2000). In particular, mature neurons are easily detectable thanks to GFP fluorescence at the cell surface and are suitable for super‐resolution imaging.

Here we show a whole Thy1‐GFP mouse brain section processed by an expansion microscopy protocol (ProExM protocol for tissues, visit website for details). By measuring the gel size before (100 um vibratome section) and after expansion protocol, it has been possible to appreciate an increase of 4.5 times the starting sample dimensions. Afterwards, the sample (about 450 µm thickness) has been acquired with X-Light V3 Confocal Spinning Disk using a 4x objective to obtain a large view of the entire expanded section (Figure A).

Working with a fully automated system it is easy to use this whole brain overview to identify specific regions of interest to be analyzed via super resolution. In this particular case, a more detailed analysis has been conducted on region containing neural spines (Figures B-D). These super-resolved images have been acquired with CrestOptics Structured Illumination (SIM) system by using a 60x objective (1.40 NA). After expansion protocol, the spine size is clearly increased (Figure C) and this allow a better resolution not only of the single spine itself, but also of synaptic puncta functional analysis (e.g. Bassoon and Homer proteins).

In conclusion, the combo of sample expansion methods with SIM microscopy push the resolution gain to smaller nanometer scale compared to each approach alone.

Figure A:
large view by confocal microscopy

Figure B:
region of interest by structured illumination microscopy

Single plane shown

Maximum Intensity Projection (12.3 um Z section)

Figure C:
spines before and after expansion

Normal sample
Expanded sample

Figure D:
3D volume movie and view in depth code

The application note has been prepared in collaboration with Emerald Perlas (Head of Histology, EMBL Rome) and Alvaro Crevenna (Head of Microscopy Service, EMBL Rome)


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