Improving the detection sensitivity of calcium transients in densely labeled neuronal tissue with pinhole illumination – A low-cost approach

Optical recording from large numbers of neurons is an indispensable technique for studying neuronal ensembles. We use optical sectioning through pinhole illumination to reduce the background fluorescence (F ₀ ) and increase the optical signal (ΔF/F ₀ ) in ex vivo brain slices densely labeled with GCaMP6f, allowing an ordinary fluorescence microscope to capture calcium transients from over 300 individual CA1 neurons – a marked increase compared to ordinary wide field fluorescence illumination.

Multiple layers of overlapping neurons can be identified by their locations and the shape in space of their ΔF/F ₀ images. A single pinhole mask was placed at the field stop of a wide field illuminator, and the image of the pinhole was projected onto the tissue by a 20X NA 0.95 water immersion objective (Olympus). This created an illuminated disk with a diameter of ∼200 μm and optical sections of hippocampal CA1 pyramidal layer tissue ∼100 μm thick. This illumination blocked a large fraction of the F ₀ , which in turn increased the ΔF/F ₀ 5-10-fold compared to that of wide field illumination. When putative pyramidal neurons fire sparsely in the brain slice, up to 300 partially superimposed neurons can be identified by their shape and spatial location in the thick (∼480 μm) ex vivo slice in the CA1 area surrounding the pinhole image. The signal-to-noise ratio was adequate even at a low excitation light level of ∼20k photoelectrons per pixel well on the camera, allowing for 3,000 seconds of total recording time without significant bleaching. This pinhole “half confocal” method has created a useful way to sample calcium transient signals in thick tissue with a large population of neurons densely labeled with GCaMP-6f.