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Although it is well known that the morphology of Gram-negative rods changes on exposure to antibiotics, the morphology of antibiotic-resistant bacteria in the absence of antibiotics has not been widely investigated. Here, we studied the morphologies of 10 antibiotic-resistant strains of Escherichia coli and used bioinformatics tools to classify the resistant cells under light microscopy in the absence of antibiotics. The antibiotic-resistant strains showed differences in morphology from the sensitive parental strain, and the differences were most prominent in the quinolone-and β-lactam-resistant bacteria. A cluster analysis revealed increased proportions of fatter or shorter cells in the antibiotic-resistant strains. A correlation analysis of morphological features and gene expression suggested that genes related to energy metabolism and antibiotic resistance were highly correlated with the morphological characteristics of the resistant strains. Our newly proposed deep learning method for single-cell classification achieved a high level of performance in classifying quinolone-and β-lactam-resistant strains.

Imaging Methods: recorded image

Organisms: Escherichia coli

Collective migration of epithelial cells is a fundamental process in multicellular pattern formation. As they expand their territory, cells are exposed to various physical forces generated by cell–cell interactions and the surrounding microenvironment. While the physical stress applied by neighbouring cells has been well studied, little is known about how the niches that surround cells are spatio-temporally remodelled to regulate collective cell migration and pattern formation. Here, the authors analysed how the spatio-temporally remodelled extracellular matrix (ECM) alters the resistance force exerted on cells so that the cells can expand their territory. Multiple microfabrication techniques, optical tweezers, as well as mathematical models were employed to prove the simultaneous construction and breakage of ECM during cellular movement, and to show that this modification of the surrounding environment can guide cellular movement. Furthermore, by artificially remodelling the microenvironment, they showed that the directionality of collective cell migration, as well as the three-dimensional branch pattern formation of lung epithelial cells, can be controlled. Their results thus confirm that active remodelling of cellular microenvironment modulates the physical forces exerted on cells by the ECM, which contributes to the directionality of collective cell migration and consequently, pattern formation.

Imaging Methods: recorded image phase contrast microscopy time lapse microscopy fluorescence microscopy confocal microscopy

Organisms: Homo sapiens Canis lupus familiaris

Meiotic errors of relatively small chromosomes in oocytes result in egg aneuploidies that cause miscarriages and congenital diseases. Unlike somatic cells, which preferentially mis-segregate larger chromosomes, aged oocytes preferentially mis-segregate smaller chromosomes through unclear processes. Here, the authors provide a comprehensive three-dimensional chromosome identifying-and-tracking dataset throughout meiosis I in live mouse oocytes. This analysis reveals a prometaphase pathway that actively moves smaller chromosomes to the inner region of the metaphase plate. In the inner region, chromosomes are pulled by stronger bipolar microtubule forces, which facilitates premature chromosome separation, a major cause of segregation errors in aged oocytes. This study reveals a spatial pathway that facilitates aneuploidy of small chromosomes preferentially in aged eggs and implicates the role of the M phase in creating a chromosome size–based spatial arrangement.

Imaging Methods: recorded image confocal microscopy time lapse microscopy

Organisms: Mus musculus

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) was applied to measure EGFR receptor in resting and EGF-stimulated fixed HeLa cells. Pre-incubated antibody-nanobody construct was used to label EGFR. R3 docking strand was conjugated to the nanobody and complimentary imager strand was coupled to Cy3b fluorophore.

Organisms: Homo sapiens

Immunofluorescent images of male adolescent rat brain. Rats were exposed to 3,3'-dichloro-biphenyl-4-ol (sham, low, medium, and high groups) in a 28-day polymeric implant study. Four brain regions were analyzed.

Imaging Methods: confocal microscopy

Organisms: Rattus norvegicus

During ovariogenesis, more than two-thirds of germ cells are sacrificed to improve the quality of the remaining oocytes. However, the detailed mechanisms behind this selection process are not fully understood in mammals. Here, we developed a high-resolution, four-dimensional ovariogenesis imaging system to track the progression of oocyte fate determination in live mouse ovaries. Through this, we identified a cyst-independent oocyte phagocytosis mechanism that plays a key role in determining oocyte survival. We found that oocytes act as individual cells, rather than connected cyst structures, during ovarian reserve construction. In this process, dominant oocytes capture and absorb cell debris from sacrificed oocytes to enrich their cytoplasm and support their survival. Single-cell sequencing indicated that the sacrificed oocytes are regulated by autophagy. When oocyte sacrifice was inhibited using autophagy inhibitors, the pool of surviving oocytes expanded, but they failed to fully develop and contribute to fertility. Our study suggests that mammals have evolved a cyst-independent selection system to improve oocyte quality, which is essential for sustaining a long reproductive lifespan.

Imaging Methods: spinning disk confocal microscopy

Organisms: Mus musculus

Biomolecular condensates mediate dynamic compartmentalization of cellular processes. The multivalent interactions that underlie biomolecular condensation are often promoted by intrinsically disordered regions (IDRs) within proteins. While the role of IDRs in biomolecular condensates is well appreciated, predicting whether an IDR forms condensates in cells remains challenging. Here, we developed a machine learning model to accurately predict the condensation behavior of IDRs, analyzing 215 IDRs from fusion oncoproteins in HEK293T cells. We identified distinct sequence-derived physicochemical features associated with condensation. Leveraging these data, our model predicts that ~12% of the ~13,000 IDRs in the human proteome are likely to form cellular condensates, establishing a robust framework for proteome-wide analysis of IDR-mediated biomolecular condensation. Notably, proteins with condensate-forming IDRs are significantly enriched in RNA processing and splicing functions and are predominantly localized to membrane-less organelles (MLOs), highlighting a central role of IDR-mediated biomolecular condensation in cellular organization and RNA biology.

Imaging Methods: confocal microscopy

Organisms: Homo sapiens

Phase separation (PS) of biomolecular condensates is often assumed to be driven by interactions involving nucleic acids and intrinsically disordered regions (IDRs) of proteins. PGL-3 is a component of P granules, biomolecular condensates in C. elegans, that contains two structured domains in tandem (D1-D2), an internal IDR, and a C-terminal IDR rich with RGG motifs. Theoretical and in vitro studies have implicated the internal IDR and RGG motifs in driving PGL-3 PS via self-interactions and binding to RNA. Studies in cells, however, have implicated the D1 and D2 domains. Here, we investigate the molecular basis of PGL-3 PS in vitro using microscopy, crosslinking mass spectrometry and biophysical measurements. We find that D1-D2 oligomerizes and is necessary and sufficient for PS. The terminal RGG domain interacts with D1-D2 in a manner that enhances PS even in the absence of RNA.. In contrast, the internal IDR is neither necessary nor sufficient for PS. These findings support a new model for PGL-3 PS that is driven by oligomerization of structured domains and enhanced by RGG repeats.

Imaging Methods: confocal microscopy

Organisms: Caenorhabditis elegans

Oblique plane microscopy (OPM) is a form of light-sheet fluorescence microscopy (LSFM) employing a single microscope objective at the sample for both fluorescence excitation and detection. Dual-view OPM (dOPM) is an optically folded form of OPM. We present an improved dOPM system employing a 60×/1.2NA water immersion primary objective and measure the spatial resolution and fluorescence collection efficiency for illumination angles of 35° and 45° with respect to the coverslip. Illumination at 35° provides slightly better lateral resolution and collection efficiency. Collection efficiency measurements are compared to a full vectorial raytracing simulation of the system. Using a light-sheet angle of 35°, the median bead FWHM for 100 nm diameter fluorescent beads in x, y and z and the optical sectioning strength were measured over a volume of 100×100×100 μm³, to be 0.29, 0.31, 0.83 and 2.45-3.00 μm respectively when the two dOPM views are fused. We demonstrate less photobleaching in time-lapse dOPM of live mEmerald-expressing organoids compared to widefield epi-fluorescence z-stack imaging under the condition of equal detected fluorescence signal from a point object in focus. We demonstrate dOPM for multi-field-of-view 3D imaging of biological samples in 96-well plates and apply it to imaging cells in collagen gel and quantifying the FUCCI cell-cycle reporter to provide drug dose-response curves in spheroids. We also use it to perform time-lapse multi-field-of-view imaging and demonstrate the detection of organoid lumen closure and reopening, organoid migration within a collagen gel and observing dynamic events in arrays of ex vivo tissue slices.

Organisms: Homo sapiens Mus musculus

Human triple negative breast cancer tissue samples including cores and resections from Neoadjuvant Chemotherapy (NACT) responders and non-responders. Samples were acquired as part of the Wellcome Leap DeltaTissue project and were stained with the nuclear dye SiR-DNA followed by imaging using FLIM to detect chromatin compaction

Imaging Methods: fluorescence lifetime imaging microscopy

Organisms: Homo sapiens

See details in Sato Y, et. al. (2023) Development. Jun 1;150(11):dev201275.

Imaging Methods: recorded image time lapse microscopy confocal microscopy

Organisms: Coturnix japonica

Biological systems are inherently noisy; however, they produce highly stereotyped tissue morphology. Drosophila pupal wings show a highly stereotypic folding through uniform expansion and subsequent buckling of wing epithelium within a surrounding cuticle sac. The folding pattern produced by buckling is generally stochastic; it is thus unclear how buckling leads to stereotypic tissue folding of the wings. The authors found that the extracellular matrix (ECM) protein, Dumpy, guides the position and direction of buckling-induced folds. Dumpy anchors the wing epithelium to the overlying cuticle at specific tissue positions. Tissue-wide alterations of Dumpy deposition and degradation yielded different buckling patterns. In summary, they propose that spatiotemporal ECM remodeling shapes stereotyped tissue folding through dynamic interactions between the epithelium and its external structures.

Imaging Methods: recorded image confocal microscopy time lapse microscopy multi-photon microscopy

Organisms: Drosophila