Image Processing and Luminescent Probes for Bioimaging Techniques with High Spatial Resolution and High Sensitivity

The balance of microenvironmental factors (including temperature, pH, ROS species, etc.) plays a crucial role in maintaining normal living organisms’ normal physiological activities and physiological functions. Therefore, armed with the unique superiorities of high spatial resolution, non-invasion, high sensitivity, real-time monitoring, and simple operation, luminescent imaging technology has been widely used in real-time and accurate monitoring of microenvironmental factors in these organisms to prevent, diagnose and treat related diseases in time. However, due to its optical imaging characteristics, it is also faced with such interference factors as relatively shallow imaging penetration depth, background fluorescence (biological autofluorescence) interference in a complex environment, uncertain probe concentration, and unstable laser power in the imaging process, which are not related to the analyte. As for the problems in imaging, such as the uncertainty of probe concentration and the fluctuation of instrument laser power, the ratio detection, and imaging technology with self-calibration function can effectively avoid these problems. As for background fluorescence interference in imaging, probes with long-life emission can be used in imaging. The long-life luminescence of probes from background fluorescence can be recognized by time-resolved luminescence imaging technology to reduce its impact. This paper briefly introduces and summarizes the relative research of ratio detection and imaging technology and time-resolved luminescence imaging technology.

Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network

Surveillance and detection of polioviruses (PV) remain crucial to monitoring eradication progress. Intratypic differentiation (ITD) using the real-time RT-PCR kit is key to the surveillance workflow, where viruses are screened after cell culture isolation before a subset are verified by sequencing. The ITD kit is a series of real-time RT-PCR assays that screens cytopathic effect (CPE)-positive cell cultures using the standard WHO method for virus isolation. Because ITD screening is a critical procedure in the poliovirus identification workflow, validation of performance of real-time PCR platforms is a core requirement for the detection of poliovirus using the ITD kit. In addition, the continual update and improvement of the ITD assays to simplify interpretation in all platforms is necessary to ensure that all real-time machines are capable of detecting positive real-time signals. Four platforms (ABI7500 real-time systems, Bio-Rad CFX96, Stratagene MX3000P, and the Qiagen Rotor-Gene Q) were validated with the ITD kit and a redesigned poliovirus probe. The poliovirus probe in the real-time RT-PCR pan-poliovirus (PanPV) assay was re-designed with a double-quencher (Zen™) to reduce background fluorescence and potential false negatives. The updated PanPV probe was evaluated with a panel consisting of 184 polioviruses and non-polio enteroviruses. To further validate the updated PanPV probe, the new assay was pilot tested in five Global Polio Laboratory Network (GPLN) laboratories (Madagascar, India, Philippines, Pakistan, and Democratic Republic of Congo). The updated PanPV probe performance was shown to reduce background fluorescence and decrease the number of false positives compared to the standard PanPV probe.

PEAR: a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells

ABSTRACTPrime editing is a recently developed gene engineering tool that allows the introduction of short insertions, deletions or substitutions into the genome. However, the efficiency of prime editing, generally reaching around 10-30% editing, has not resembled its versatility. Here, Prime Editor Activity Reporter (PEAR), a sensitive fluorescent tool is introduced for the identification of single cells with prime editing activity. Possessing no background fluorescence, PEAR specifically reports on prime editing events in individual cells. By design, it ensures unrestricted flexibility for sequence variations in the full length of the target sequence. The application of PEAR as an enrichment marker of prime editing can increase the edited population by up to 70% and alleviate the burden of the otherwise time and labour consuming process of cloning of the correctly edited cells, therefore considerably improving the applicability of prime editing in fundamental research and biotechnological uses.

Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans

ABSTRACT Fluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence. IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.

Photochromic fluorophores enable imaging of lowly-expressed proteins in the autofluorescent fungus Candida albicans

Fluorescence microscopy is a standard research tool in many fields, though collecting reliable images can be difficult in systems characterized by low expressions levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly- or endogenously-expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungals agents in the fungal pathogen C. albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by a high background fluorescence.ImportanceUnderstanding the spatial and temporal organization of proteins-of-interest is key to unravel cellular processes and identify novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans and resistance mechanisms against these therapeutic agents is rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable imaging of lowly-expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems allowing the visualization of intricate processes.

Harnessing Long-lived Visible Phosphorescence to Eliminate Background Interference from Fingermark Images

Minimising background fluorescence can enhance the visible details of treated fingerprints. Here, a 4-tpt fingerprint powder exhibiting long-lived phosphorescence is applied to this end. The powder was found to suppress...