High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

Abstract

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.

Figure 1

Preparation of serial thin sections. (a) Block face with parallel top and bottom edges. (b) Carbon coated coverslip submerged in Jumbo Histo knife. Use eyelash tool to detach ribbon (arrow) from knife-edge (1) and move it to attach to metal bar holding water in place (2) then slowly and carefully remove the coversip from boat so ribbon lays down flat. (c) FESEM image of ribbon of ultrathin serial sections mounted on carbon-coated coverslip. Scale bar 100 μm. Animal use in this experiment was conducted in strict accordance to our institutional animal care and use committee guidelines.

Figure 2

LM photomicrograph of unstained 0.5 micron semi-thin section of epoxy embedded mouse cortex shows crisp cellular details and good contrast. NCB Neuronal cell body, BV blood vessel. Scale bar 10 μm. Animal use in this experiment was conducted in strict accordance to our institutional animal care and use committee guidelines.

Figure 3

Set of 3 representative FESEM images of 70 nm sections from series of 25 on carbon coated glass coverslip. Arrows (a, b) and asterisks (c) mark two different synaptic contacts onto a dually innervated spine. Panel d shows a 3D reconstruction of the dual innervations and accompanying synaptic vesicles in red and blue. Scale bar, 200 nm. Animal use in this experiment was conducted in strict accordance to our institutional animal care and use committee guidelines.

Figure 4

FESEM images of ROTO (reduced osmium OTO)/en bloc lead aspartate staining of adult mouse brain tissue. Note the homogeneous staining of the tissue and the absence of contamination obtained after using this method. Representative regions (squares) near cell somas (a1) and neuropil (a2) are displayed at higher resolution. Observe the high contrast staining of all cellular membranes, myelin and organelles, including mitochondria, Golgi apparatus, endoplasmic reticulum, ribosomes, and synapses. Bars represent 10 (a), 1 (a1) and 1 (a2) μms respectively. Animal use in this experiment was conducted in strict accordance to our institutional animal care and use committee guidelines.

Figure 5

FESEM images of mouse cortex epoxy resin thin sections en bloc stained Osmid/OTO/copper sulfate lead citrate overnight at 20° C. a) Low magnification of 70 nm ultrathin section showing well contrasted specimen. NCB, neuronal cell body BV, blood vessel. Scale bar 2 μm. b) Higher magnification of boxed area reveals high contrast membranes and myelin. Arrow points to spine synapse with electron-dense post-synaptic density. Scale bar 200 nm. Animal use in this experiment was conducted in strict accordance to our institutional animal care and use committee guidelines.

Figure 6

3View Block Face FESEM of Zebrafish Optic Tectum. a) 3View Block Face FESEM pictures of larval 10 dpf zebrafish en bloc stained with Osmid, OTO, copper sulfate lead citrate overnight at 37°C. Scale bar 100 μm. b) Higher magnification 3View FESEM image of optic tectum showing good contrast and intact structure. En bloc staining as in panel (a). Scale bar 2 μm. Animal use in this experiment was conducted in strict accordance to our institutional animal care and use committee guidelines.

Figure 7

FESEM images of adult Drosophila brain ultrathin section mounted on Kapton tape. (a) overview of entire brain (b) Higher magnification of boxed area in a shows neuropil and (c) axon fascicles. Higher magnification of boxed area in (c) shows detail of neuropil in d and illustrates excellent contrast of membranes and synaptic vesicles. Osmid/OTO en bloc staining. Scale bars: a is 40 μm, b is 20 μm, c is 5 μm and d is 1 μm. Animal use in this experiment was conducted in strict accordance to our institutional animal care and use committee guidelines.