Kocheril, P. A. | bioRxiv Science

bioRxiv2024

Wide-field bond-selective fluorescence imaging: from single-molecule to cellular imaging beyond video-rate

Wide-field (WF) imaging is pivotal for observing dynamic biological events. While WF chemical microscopy offers high molecular specificity, it lacks the sensitivity for single-molecule detection. In contrast, WF fluorescence microscopy provides live-cell dynamic mapping but fails to leverage the rich chemical information necessary for functional interpretations. To address these limitations, we introduce Wide-Field Bond-selective Fluorescence-detected Infrared-Excited (WF-BonFIRE) spectro-microscopy. This technique combines rationally optimized imaging speed and field-of-view (FOV) to achieve single-molecule sensitivity with bond-selective contrast. WF-BonFIRE outperforms its point-scanning counterpart, enhancing frame acquisition up to 10,000 times. We demonstrate WF-BonFIREs capabilities in imaging cells, astrocytes, and live neurons, capturing single FOVs up to 50 {micro}m x 50 {micro}m, with further expansion via multi-FOV mosaicking. Additionally, we have implemented a temporal-delay modulation scheme that allows real-time kilohertz imaging speeds up to 1500 Hz. This enables millisecond temporal resolution while monitoring random motion of live Escherichia coli. Overall, WF-BonFIRE significantly broadens the possibilities for chemical imaging, enabling high-speed observations at unparalleled sensitivity levels. One-Sentence SummaryWide-field bond-selective fluorescence imaging pushes chemical-sensitive microscopy platform into a new regime, achieving single-molecule sensitivity and speeds up to kilohertz.

biophysics↗

bioRxiv2022

Serum lipoproteins and lipoarabinomannan suppress the inflammatory response induced by the mycolactone toxin

Mycobacterium ulcerans is the causative agent of the chronic and debilitating neglected tropical disease Buruli ulcer (BU) which mostly affects children. The early detection and treatment of M. ulcerans infections can significantly minimize life-long disability resulting from surgical intervention. However, the disease is characterized by relatively few systemic systems as a result of complex host-pathogen interactions that have yet to be fully characterized, which has limited the development of both diagnostic and therapeutic approaches to treat BU. In this work, we study the interactions of the host immune system with two principle M. ulcerans virulence factors: mycolactone, an amphiphilic macrolide toxin, and lipoarabinomannan (LAM), a cell wall component of most mycobacterial pathogens. We observe that human lipoproteins have a profound effect on the interaction of both mycolactone and LAM with the immune system. Individually, both molecules are pro-inflammatory in the absence of serum and immunosuppressive in the presence of serum. When combined, mycolactone and LAM are immunosuppressive regardless of serum conditions. We also show that Toll-like receptor 2 (TLR2), a macrophage pathogen pattern recognition receptor, is critical for LAM immune stimulation but aids in mycolactone immunosuppression. These findings are a first step towards unraveling mycolactone-mediated immunosuppression during BU disease and may facilitate the development of effective diagnostics and therapeutics in the future. Author SummaryBuruli ulcer (BU) is a neglected tropical disease caused by the pathogen Mycobacterium ulcerans. The principal virulence factors associated with it are the macrolide toxin mycolactone and the major cell wall component lipoarabinomannan (LAM). Here, we examine the impact of the amphiphilic biochemistry of mycolactone and LAM on their interaction with the human immune system. We show that both mycolactone and LAM associate with serum lipoproteins, and that this association is critical for the immune evasion seen in early-stage M. ulcerans infections. In the absence of serum, mycolactone is pro-inflammatory. Immunosuppression occurs only in the presence of human serum lipoproteins. In the presence of LAM, mycolactone is immunosuppressive, regardless of serum conditions. Immunosuppression is a hallmark of BU disease, and understanding the mechanisms of this immunosuppression can support the development of effective diagnostic and therapeutic strategies.

microbiology↗