A Novel Morphological Marker for the Analysis of Molecular Activities at the Single-cell Level

Abstract

Abbreviations: H&E, hematoxylin and eosin; BAC, bacterial artificial chromosome; FRET, Förster resonance energy transfer; TPEM, two-photon excitation microscopy; FP, fluorescent protein; WPRE, woodchuck hepatitis virus (WHP) posttranscriptional regulatory element; DCGAN, deep convolutional generative adversarial networks. AbstractFor more than a century, hematoxylin and eosin (H&E) staining has been the de-facto standard for histological studies. Consequently, the legacy of histological knowledge is largely based on H&E staining. Due to the recent advent of multi-photon excitation microscopy, the observation of live tissue is increasingly being used in many research fields. Adoption of this technique has been further accelerated by the development of genetically encoded biosensors for ions and signaling molecules. However, H&E-based histology has not yet begun to fully utilize in-vivo imaging due to the lack of proper morphological markers. Here, we report a genetically encoded fluorescent marker, NuCyM (Nucleus, Cytosol, and Membrane), which is designed to recapitulate H&E staining patterns in vivo. We generated a transgenic mouse line ubiquitously expressing NuCyM by using a ROSA26 bacterial artificial chromosome (BAC) clone. NuCyM evenly marked the plasma membrane, cytoplasm and nucleus in most tissues, yielding H&E staining-like images. In the NuCyM-expressing cells, cell division of a single cell was clearly observed as five basic phases during M phase by three-dimensional imaging. We next crossed NuCyM mice with transgenic mice expressing an ERK biosensor based on the principle of Förster resonance energy transfer (FRET). Using NuCyM, ERK activity in each cell could be extracted from the FRET images. To further accelerate the image analysis, we employed machine learning-based segmentation methods, and thereby automatically quantitated ERK activity in each cell. In conclusion, NuCyM is a versatile cell morphological marker that enables us to grasp histological information as with H&E staining.

DeTerm: Software for automatic detection of neuronal dendritic branch terminals via an artificial neural network

“…It is evident that artificial neural network‐based workflows are increasingly being incorporated in multiple levels of automatic or semi‐automatic biomedical image analysis, such as histopathological diagnosis (Capper et al, ), segmenting neuronal dendrite arborizations to identify dendritic spines (Smirnov, Garrett, & Yasuda, ), and segregating subcellular structures in solid tissues (e.g., plasma membrane vs. nucleus in Imanishi et al, ). These methods, including DeTerm, are expected to dramatically improve the efficiency of image analysis such as phenotypic screening of a large number of genotype‐by‐environment conditions contributing to dendrite development.…”

Biliverdin Reductase-A Deficiency Brighten and Sensitize Biliverdin-binding Chromoproteins

“…NuCyM mice have been reported previously (Imanishi et al, 2018). In these mice, the morphological marker NuCyM is expressed throughout the body, on a B6 albino background.…”

“…pose, we generated NuCyM +/-Blvra -/-mice by crossing Blvra -/-mice with NuCyM mice, which ubiquitously express a set of morphological marker proteins throughout the body (Imanishi et al, 2018). NuCyM consists of iRFP713 fused with a nuclear export signal (NES), mCherry fused with histone H1, and mCherry fused with the CAAX domain of K-Ras, all of which are cleaved off from a single peptide by means of the P2A self-cleavage peptides (Fig.…”

“…Therefore, the extracellular transport machinery of BV may also affect the availability of BV. The different subcellular localization of iRFP713 among organs is probably due to incomplete cleavage of the P2A peptide (Imanishi et al, 2018). To explore the application of Blvra -/-mice to neuroimaging, we observed the cerebral cortex of NuCyM +/-Blvra -/-mice through an imaging window (Yang et al, 2010).…”

Experimental pathology by intravital microscopy and genetically encoded fluorescent biosensors

“…In addition, we recently established mouse lines expressing a morphology marker named NuCyM, which enables us to distinguish the nucleus, plasma membrane and cytoplasm by 2PEM. 15 With NuCyM, images acquired by 2PEM can be converted to H&E-like images.…”