Single-Cell Spatial MIST for Versatile, Scalable Detection of Protein Markers.

High-multiplex detection of protein biomarkers across tissue regions has been an attractive spatial biology approach due to significant advantages over traditional immunohistochemistry (IHC) methods. Different from most methods, spatial multiplex in situ tagging (MIST) transfers the spatial protein expression information to an ultrahigh-density, large-scale MIST array. This technique has been optimized to reach single-cell resolution by adoption of smaller array units and 30% 8-arm PEG polymer as transfer medium. Tissue cell nuclei stained with lamin B have been clearly visualized on the MIST arrays and are colocalized with detection of nine mouse brain markers. Pseudocells defined at 10 µm in size have been used to fully profile tissue regions including cells and the intercellular space. We showcased the versatility of our technology by successfully detecting 20 marker proteins in kidney samples with the addition of five minutes atop the duration of standard immunohistochemistry protocols. Spatial MIST is amenable to iterative staining and detection on the same tissue samples. When 25 proteins were co-detected on 1 mouse brain section for each round and 5 rounds were executed, an ultrahigh multiplexity of 125 proteins was obtained for each pseudocell. With its unique abilities, this single-cell spatial MIST technology has the potential to become an important method in advanced diagnosis of complex diseases.

Axonal architecture of the mouse inner retina revealed by second harmonic generation.

We describe a novel method for visualizing the network of axons in the unlabeled fresh wholemount retina. The intrinsic radiation of second harmonic generation (SHG) was utilized to visualize single axons of all major retinal neurons, i.e., photoreceptors, horizontal cells, bipolar cells, amacrine cells, and the retinal ganglion cells. The cell types of SHG+ axons were determined using transgenic GFP/YFP mice. New findings were obtained with retinal SHG imaging: Müller cells do not maintain uniformly polarized microtubules in the processes; SHG+ axons of bipolar cells terminate in the inner plexiform layer (IPL) in a subtype-specific manner; a subset of amacrine cells, presumably the axon-bearing types, emits SHG; and the axon-like neurites of amacrine cells provide a cytoskeletal scaffolding for the IPL stratification. To demonstrate the utility, retinal SHG imaging was applied to testing whether the inner retina is preserved in glaucoma, using DBA/2 mice as a model of glaucoma and DBA/2-Gpnmb+ as the nonglaucomatous control. It was found that the morphology of the inner retina was largely intact in glaucoma and the presynaptic compartments to the retinal ganglion cells were uncompromised. It proves retinal SHG imaging as a promising technology for studying the physiological and diseased retinas in 3D.

High-pulse-energy multiphoton imaging of neurons and oligodendrocytes in deep murine brain with a fiber laser.

Here we demonstrate high-pulse-energy multiphoton microscopy (MPM) for intravital imaging of neurons and oligodendrocytes in the murine brain. Pulses with an order of magnitude higher energy (~ 10 nJ) were employed from a ytterbium doped fiber laser source at a 1-MHz repetition rate, as compared to the standard 80-MHz Ti:Sapphire laser. Intravital imaging was performed on mice expressing common fluorescent proteins, including green (GFP) and yellow fluorescent proteins (YFP), and TagRFPt. One fifth of the average power could be used for superior depths of MPM imaging, as compared to the Ti:Sapphire laser: A depth of ~ 860 µm was obtained by imaging the Thy1-YFP brain in vivo with 6.5 mW, and cortical myelin as deep as 400 µm ex vivo by intrinsic third-harmonic generation using 50 mW. The substantially higher pulse energy enables novel regimes of photophysics to be exploited for microscopic imaging. The limitation from higher order phototoxicity is also discussed.