Cellular & Molecular Imaging

Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in fungiform, circumvallate and foliate taste papillae in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue.

Fluorescence imaging across the near-infrared (NIR, 700–1000 nm) and shortwave infrared (SWIR, 1000–2000 nm) regions offers significant advantages for biomedical applications, yet photophysical enhancements achieved with NIR and SWIR chromophores observed in solution often fail to translate to complex biological environments. Fluorous-soluble fluorophores, fluorofluorophores, face additional challenges, exhibiting poor brightness and photostability when dissolved in perfluorocarbons (PFCs) due to unfavorable interactions with the fluorous phase. Here, we report counterion exchange as a strategy to enhance the photophysical properties of two heptamethine cyanine fluorofluorophore for NIR and SWIR imaging. Exchanging the small chloride counterion with a large, fluorinated aryl borate counterions significantly improved the brightness (10-fold) and photostability (57-fold) in PFCs. These enhancements were successfully translated across multiple biological systems from macrophage cells to NIR imaging zebrafish retinal tissue and finally to SWIR imaging in mice. These results demonstrate that strategic counterion modification provides a straightforward approach to optimize fluorofluorophores, with solution-phase improvements that translate to in vivo NIR and SWIR imaging.

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

All symptoms of malaria disease are associated with the asexual blood stages of development, involving cycles of red blood cell (RBC) invasion and egress by the Plasmodium spp. merozoite. Merozoite invasion is rapid and is actively powered by a parasite actomyosin motor. The current accepted model for actomyosin force generation envisages arrays of parasite myosins, pushing against short actin filaments connected to the external milieu that drive the merozoite forwards into the RBC. In Plasmodium falciparum, the most virulent human malaria species, Myosin A (PfMyoA) is critical for parasite replication. However, the precise function of PfMyoA in invasion, its regulation, the role of other myosins and overall energetics of invasion remain unclear. Here, we developed a conditional mutagenesis strategy combined with live video microscopy to probe PfMyoA function and that of the auxiliary motor PfMyoB in invasion. By imaging conditional mutants with increasing defects in force production, based on disruption to a key PfMyoA phospho-regulation site, the absence of the PfMyoA essential light chain, or complete motor absence, we define three distinct stages of incomplete RBC invasion. These three defects reveal three energetic barriers to successful entry: RBC deformation (pre-entry), mid-invasion initiation, and completion of internalisation, each requiring an active parasite motor. In defining distinct energetic barriers to invasion, these data illuminate the mechanical challenges faced in this remarkable process of protozoan parasitism, highlighting distinct myosin functions and identifying potential targets for preventing malaria pathogenesis.

Sagittal section images showing cross-section of the stomodeal valve at point of greatest opening of 15 sand flies (Lutzomyia longipalpis) at various stages of infection with Leishmania mexicana. The images show the effects of an infection and of a second blood meal on the morphology of the foregut and midgut (especially thoracic midgut) compared to uninfected, control flies. Three flies were imaged by micro-CT scan in each of five groups: T7CON flies were 7 days after a non-infected blood meal. T9INF and T12INF flies were 9 and 12 days, respectively, after an infected blood meal. T6INF+ and T9INF+ flies were 6 and 9 days, respectively after an infected bloodmeal and 1 and 4 days, respectively, after a second, non-infected blood meal. The data are discussed in a paper published on 27th August 2021 in PLoS NTDs, which is accessible through the following link: https://doi.org/10.1371/journal.pntd.0009682

Mono-ADP-ribosylation, a regulatory modification of both protein and nucleic acids, has been implicated in innate immunity. In cells, this modification is catalyzed by PARP enzymes, some of which are induced in response to type I interferons. Mono-ADP-ribosylation is fully reversible by hydrolases that include proteins with macrodomains, highly conserved protein domains found in all kingdoms of life. Macrodomains encoded by certain positive sense single-stranded RNA viruses, such as Chikungunya virus and SARS-CoV2, antagonize host MARylation to enhance viral replication and suppress the immune response. While macrodomain hydrolase activity is essential for CHIKV replication, in SARS-CoV2, it predominantly contributes to immune evasion, underscoring viral macrodomains as attractive antiviral drug targets. Efforts to develop macrodomain inhibitors have included computational modeling, crystallography-based methods, and in vitro assays. However, tools to study macrodomain activity directly in cells remain rare. In this study, we established a cell-based assay using PARP15 isoform 1, which we identified to form nuclear foci dependent on its ADP-ribosyltransferase activity. Active macrodomains dissolve these foci, providing a means to monitor hydrolase activity in living cells. Using stable cell lines, this system enables the screening of macrodomain inhibitors while simultaneously addressing cell permeability, toxicity, and physiological relevance. Adaptable to various macrodomains, this platform offers a versatile approach to study macrodomain function, analyzing mutants, and advancing drug discovery efforts.

Imaging dataset for PMID: 39314434

raw image files analyzed for publication; source data files

All images used to generate the figures of this manuscript

The deformability of red blood cells (RBCs) is a critical factor in understanding cardiovascular diseases and serves as a key determinant of RBC lifespan. Red blood cells with different deformities were observed to exhibit significant differences in their morphological characteristics. To further investigate this relationship, we propose a deep learning-based approach to explore the correlation between RBC deformability and its morphological features. We utilize a dielectrophoretic microfluidic approach to assess deformability and capture microscopic images of erythrocytes. These images are then classified according to the deformability of the cells. Based on the above research, this study contains three datasets. We characterize the deformability of RBCs using the strain of RBCs under the same dielectrophoretic force with the strain formula ε = (a−a0)/a0, where a is the length of the long axis of the RBCs stretched by the dielectrophoretic force and a0 is the original length of the RBCs. the strain between 0~0.3 is called hard, between 0.3~0.6 is called deformable, and the rest is called soft.