Quantitative Fluorescence In Situ Hybridization (FISH) of Magnetically Confined Bacteria Enables Early Detection of Human Bacteremia.

The current diagnosis of bacteremia mainly relies on blood culture, which is inadequate for the rapid and quantitative determination of most bacteria in blood. Here, a quantitative, multiplex, microfluidic fluorescence in situ hybridization method (µFISH) is developed, which enables early and rapid (3-h) diagnosis of bacteremia without the need for prior blood culture. This novel technology employs mannose-binding lectin-coated magnetic nanoparticles, which effectively opsonize a broad range of pathogens, magnetically sequestering them in a microfluidic device. Therein, µFISH probes, based on unique 16S rRNA sequences, enable the identification and quantification of sequestered pathogens both in saline and whole blood, which is more sensitive than conventional polymerase chain reaction. Using µFISH, Escherichia coli (E. coli) is detected in whole blood collected from a porcine disease model and from E. coli-infected patients. Moreover, the proportion of E. coli, relative to other bacterial levels in the blood, is accurately and rapidly determined, which is not possible using conventional diagnostic methods. Blood from E. coli-infected patients is differentiated from healthy donors' blood using cutoff values with a 0.05 significance level. Thus, µFISH is a versatile method that can be used to rapidly identify pathogens and determine their levels relative to other culturable and nonculturable bacteria in biological samples.

Visualizing Live Chromatin Dynamics through CRISPR-Based Imaging Techniques.

The three-dimensional organization of chromatin and its time-dependent changes greatly affect virtually every cellular function, especially DNA replication, genome maintenance, transcription regulation, and cell differentiation. Sequencing-based techniques such as ChIP-seq, ATAC-seq, and Hi-C provide abundant information on how genomic elements are coupled with regulatory proteins and functionally organized into hierarchical domains through their interactions. However, visualizing the time-dependent changes of such organization in individual cells remains challenging. Recent developments of CRISPR systems for site-specific fluorescent labeling of genomic loci have provided promising strategies for visualizing chromatin dynamics in live cells. However, there are several limiting factors, including background signals, off-target binding of CRISPR, and rapid photobleaching of the fluorophores, requiring a large number of target-bound CRISPR complexes to reliably distinguish the target-specific foci from the background. Various modifications have been engineered into the CRISPR system to enhance the signal-to-background ratio and signal longevity to detect target foci more reliably and efficiently, and to reduce the required target size. In this review, we comprehensively compare the performances of recently developed CRISPR designs for improved visualization of genomic loci in terms of the reliability of target detection, the ability to detect small repeat loci, and the allowed time of live tracking. Longer observation of genomic loci allows the detailed identification of the dynamic characteristics of chromatin. The diffusion properties of chromatin found in recent studies are reviewed, which provide suggestions for the underlying biological processes.