Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems

Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.Simons Foundation. Postdoctoral FellowshipLife Sciences Research FoundationBurroughs Wellcome Fund (Career Award at the Scientific Interface)Searle Scholars ProgramMichael J. Fox Foundation for Parkinson's ResearchUnited States. Defense Advanced Research Projects AgencyNational Institutes of Health (U.S.) (1-U01-NS090473-01

Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues

The biology of multicellular organisms is coordinated across multiple size scales, from the subnanoscale of molecules to the macroscale, tissue-wide interconnectivity of cell populations. Here we introduce a method for super-resolution imaging of the multiscale organization of intact tissues. The method, called magnified analysis of the proteome (MAP), linearly expands entire organs fourfold while preserving their overall architecture and three-dimensional proteome organization. MAP is based on the observation that preventing crosslinking within and between endogenous proteins during hydrogel-tissue hybridization allows for natural expansion upon protein denaturation and dissociation. The expanded tissue preserves its protein content, its fine subcellular details, and its organ-scale intercellular connectivity. We use off-the-shelf antibodies for multiple rounds of immunolabeling and imaging of a tissue's magnified proteome, and our experiments demonstrate a success rate of 82% (100/122 antibodies tested). We show that specimen size can be reversibly modulated to image both inter-regional connections and fine synaptic architectures in the mouse brain.United States. National Institutes of Health (1-U01-NS090473-01

Noninvasive Optical Measurement of Cerebral Blood Flow in Mice Using Molecular Dynamics Analysis of Indocyanine Green

<div><p>In preclinical studies of ischemic brain disorders, it is crucial to measure cerebral blood flow (CBF); however, this requires radiological techniques with heavy instrumentation or invasive procedures. Here, we propose a noninvasive and easy-to-use optical imaging technique for measuring CBF in experimental small animals. Mice were injected with indocyanine green (ICG) via tail-vein catheterization. Time-series near-infrared fluorescence signals excited by 760 nm light-emitting diodes were imaged overhead by a charge-coupled device coupled with an 830 nm bandpass-filter. We calculated four CBF parameters including arrival time, rising time and mean transit time of a bolus and blood flow index based on time and intensity information of ICG fluorescence dynamics. CBF maps were generated using the parameters to estimate the status of CBF, and they dominantly represented intracerebral blood flows in mice even in the presence of an intact skull and scalp. We demonstrated that this noninvasive optical imaging technique successfully detected reduced local CBF during middle cerebral artery occlusion. We further showed that the proposed method is sufficiently sensitive to detect the differences between CBF status in mice anesthetized with either isoflurane or ketamine–xylazine, and monitor the dynamic changes in CBF after reperfusion during transient middle cerebral artery occlusion. The near-infrared optical imaging of ICG fluorescence combined with a time-series analysis of the molecular dynamics can be a useful noninvasive tool for preclinical studies of brain ischemia.</p> </div

Definitions and interpretations of cerebral blood flow (CBF) map parameters.

*<p>Other reports may use T_max and I_max instead of T_peak and I_peak, respectively.</p><p>t, observation time point; I(t), intensity function of time; I_peak, intensity of the first peak; T_peak, time of I_peak; I_arrival, intensity at T_arrival; I_max, maximum intensity; T_max, time of I_max.</p

Types of CBF maps and a comparison of CBF parameters in normal and ischemic hemispheres.

<p>(A) Representative CBF maps using the four parameters described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048383#pone-0048383-t001" target="_blank">Table 1</a> were generated from a normal condition (left panels) and an ischemic condition in which the left MCA was occluded (right panels). (B) Average blood flow parameters of regions over left somatosensory cortices of nine mice before and after MCA occlusion (MCAO) surgery. T_rising and blood flow index (BFI) parameters showed significant differences between the normal and ischemic conditions (<i>t</i>-test, ***<i>p</i><0.001). (C) Average blood flow parameters of regions over ipsilateral ischemic somatosensory cortices were compared to those regions over contralateral normal cortices in nine mice during MCAO. All four parameters showed significant differences (paired <i>t</i>-test, ***<i>p</i><0.001). MTT, mean transit time; Contra, contralateral region; Ipsi, Ipsilateral region.</p

Detection of cerebral hemodynamic changes using CBF maps.

<p>(A) Representative T_rising maps for mice anesthetized with either 1.5% isoflurane or 0.1 mg/g ketamine and 0.01 mg/g xylazine. (B) Averaged CBF parameters of regions over left somatosensory cortices of six mice anesthetized with isoflurane and six mice anesthetized with ketamine and xylazine. T_arrival, T_rising, and MTT parameters increased significantly, and BFI parameter was decreased significantly, in ketamine and xylazine group (<i>t</i>-test, **<i>p</i><0.01; ***<i>p</i><0.001). (C) Timeline of the transient MCAO protocol (upper diagram) and representative T_rising maps (lower panels). Reperfusion (RP) was induced at 45 min after MCA occlusion. The six time points for imaging acquisition are indicated under the timeline.</p