Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression

Abstract

Acquisition of detailed anatomical and molecular knowledge from intact biological samples while preserving their native three-dimensional structure is still a challenging issue for imaging studies aiming to unravel a system’s functions. Three-dimensional micro-CT X-ray imaging with a high spatial resolution in minimally perturbed naive non-transparent samples has recently gained increased popularity and broad application in biomedical research. Here, we describe a novel X-ray-based methodology for analysis of β-galactosidase (lacZ) reporter-driven gene expression in an intact murine brain ex vivo by micro-CT. The method relies on detection of bromine molecules in the product of the enzymatic β-galactosidase reaction. Enhancement of the X-ray signal is observed specifically in the regions of the murine brain where expression of the lacZ reporter gene is also detected histologically. We performed quantitative analysis of the expression levels of lacZ reporter activity by relative radiodensity estimation of the β-galactosidase/X-gal precipitate in situ. To demonstrate the feasibility of the method, we performed expression analysis of the Tsen54-lacZ reporter gene in the murine brain in a semi-quantitative manner. Human mutations in the Tsen54 gene cause pontocerebellar hypoplasia (PCH), a group of severe neurodegenerative disorders with both mental and motor deficits. Comparing relative levels of Tsen54 gene expression, we demonstrate that the highest Tsen54 expression is observed in anatomical brain substructures important for the normal motor and memory functions in mice.

Experimental Study on the Characterization of Orientation of Polyester Short Fibers in Rubber Composites by an X-ray Three-Dimensional Microscope

“…As shown in Figure 1 , An X-ray three-dimensional microscope (micro-CT) [ 9 , 10 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ] is mainly composed of a micro-focus ray source system, a precision sample stage system, a high-resolution optocoupler detector, and a flat panel detector system. An X-ray three-dimensional microscope is an instrument developed based on the CT imaging principle.…”

Gold-Nanostar-Based Surface-Enhanced Raman Scattering Substrates for Gastric Carcinoma Detection

“…Surface-enhanced Raman scattering (SERS) is a useful analytical technique for detecting trace levels of biochemical analytes. Because of strong plasmonic enhancement by noble-metal nanostructures, single-molecule identification has been demonstrated with SERS. − The current SERS , and other methods ,, of measurement are basically based on the reaction of 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-gal) with SA-β-gal to produce an insoluble blue substance as a marker for detection; however, this method has the problems of high cost and long staining time. In the construction of substrates, most of the substrates of other SERS methods used simple colloidal particles that showed lower uniformity and stability.…”

“…Because of strong plasmonic enhancement by noblemetal nanostructures, single-molecule identification has been demonstrated with SERS. 19−21 The current SERS 22,23 and other methods 12,24,25 substance as a marker for detection; however, this method has the problems of high cost and long staining time. In the construction of substrates, most of the substrates of other SERS methods used simple colloidal particles that showed lower uniformity and stability.…”

Gene Expression Detection in Developing Mouse Tissue Using In Situ Hybridization and µCT Imaging

“…Enhanced DAB detection was also the basis of a method where use of peroxidase reporter gene enabled staining of specific cell populations in Drosophila legs and mouse brain (Kuan et al, 2020). Also, a LacZ reporter gene system was recently established for μCT imaging of gene expression, utilizing the radiodensity of bromine in the product of the β-galactosidase reaction (Ermakova et al, 2021).…”