axio observer z1 inverted widefield microscope  (Carl Zeiss)

Journal: bioRxiv

Article Title: MMV687794 blocks Plasmodium falciparum invasion of red blood cells by targeting a Surface-associated Lipid-Interacting Rhoptry Protein, Pf SLIRP

doi: 10.64898/2026.02.24.707847

Figure Lengend Snippet: A 2D widefield immunofluorescence assay (IFA) images of C-WT parasites showing HA-tagged Pf SLIRP in schizonts at the periphery of rhoptry bulbs. EXP2 marks the parasitophorous vacuole membrane, RhopH3 marks rhoptry bulbs, and DNA is stained with DAPI. Super-Resolution Radial Fluctuations (SRRF) processing was applied to enhance spatial resolution. The white box in the merged schizont image indicate zoomed area to the right. B Confocal maximum intensity projections of expanded C-WT schizonts illustrating wild-type Pf SLIRP–HA localisation at the rhoptry membrane. RAP1 marks rhoptry bulbs, GAP45 labels the merozoite periphery, and SYTOX™ blue stains DNA. The white boxes in the merged image indicate zoomed areas at the bottom. B = rhoptry bulb, N = rhoptry neck. C Confocal maximum intensity projections of rhoptries from expanded C-C36W schizonts (Figure S11) illustrating C36W Pf SLIRP-HA localisation at the rhoptry membrane. D Western blot of parasite proteins from ring (R), early trophozoite (ET), late trophozoite (LT), and schizont stages, probed with anti-HA to detect Pf SLIRP. HSP70-1 serves as a loading control. Densitometry values were normalised to HSP70-1, a constitutively-expressed housekeeping protein. Pf SLIRP expression peaks at the schizont stage for C-WT, and the ring stage for C-C36W. Western blot is representative of three biological replicates (refer to Figure S12 for all replicates). Error bars represent the standard deviation of the mean of three biological replicates, each with two technical replicates. Statistical analyses were performed with GraphPad Prism 10, with Welch’s t -test between the late trophozoite and schizont stages within both parasite lines, and between the same stages across both parasite lines. ** p < 0.01, **** p < 0.0001. No bar indicates not significant. E Sequential lysis of C-WT and C-C36W schizont saponin pellets by freeze–thaw and sodium carbonate extraction. Western blot shows Pf SLIRP is found in the carbonate soluble (Na2CO3 Sup.) and insoluble (Na2CO3 Pel.) fractions but not in the PBS soluble fraction (PBS Sup.). Control proteins included soluble protein Pf HSP70-1, peripheral membrane protein Pf HSP101, and integral membrane protein Pf EXP2. Mouse anti-HA was used for Pf SLIRP detection; rabbit antibodies were used to detect the control proteins. * denotes cross-reactive bands produced by rabbit HSP70-1 antibody (refer to Figure S14 for all cross-reactive bands produced by the antibody).

Article Snippet: Images were captured using a Zeiss Axio Observer Z1 inverted widefield microscope with a Plan-Apochromat 100×/1.40 Oil DIC objective and the super-resolution radial fluctuations (SRRF) algorithm was used to capture super-resolution images.

Techniques: Immunofluorescence, Membrane, Staining, Western Blot, Control, Expressing, Standard Deviation, Lysis, Extraction, Produced

Journal: Nucleic Acids Research

Article Title: RecN and RecA orchestrate an ordered DNA supercompaction response following ciprofloxacin-induced DNA damage in Escherichia coli

doi: 10.1093/nar/gkaf437

Figure Lengend Snippet: DNA supercompaction in wild-type E. coli cells (KV21) after CIP exposure. All cells were grown in LB at 37°C and imaged at 2-min intervals using live-cell imaging. ( A and B ) Cells were immobilized in microfluidic channel slides and imaged using widefield microscopy. (A) Representative images of wild-type cells with HU-mCherry fluorescence (green) at 0, 8, and 16 min after CIP exposure, showing DNA distribution; scale bar: 5 μm. (B) Analysis of DNA distribution along the cells’ long axis before and after CIP, quantified by measuring the distance between the outer bounds of symmetrical fluorescence peaks at 80% of maximum averaged intensity for each time point. Results are averaged from 34 to 141 cells from a single representative biological replicate (see “Materials and methods” section for detailed explanation). ( C and D ) Cells were immobilized on agar pads and imaged using spinning disk microscopy. Results shown are from images captured 12, 14, 16, and 18 min after CIP exposure, averaged from 40 tracked cells from a single representative biological replicate. (C) Average HU-mCherry fluorescence intensity along the cells’ long axis. (D) Kymograph heat map of relative HU-mCherry intensity distribution over time. ( E ) DNA compaction phenotype distribution for baseline unchallenged cells (dots) and cells after CIP exposure (lines). Dots and lines represent means from three biological replicates, while error bars and shaded regions indicate standard deviation. A.u., arbitrary unit.

Article Snippet: To examine the cells’ immediate response to CIP exposure, we employed a microfluidic setup to image cells using a Zeiss Axio Observer Z1 widefield inverted microscope with a 63x oil objective (Zeiss Plan Apochromat 1.4 NA, DIC), a Colibri 7 LED light source, a Hamamatsu ORCA-Flash4.0 V3 digital CMOS camera, as well as a heated incubation chamber and mounting frame, both maintained at 37°C.

Techniques: Live Cell Imaging, Microscopy, Fluorescence, Standard Deviation

Journal: Nucleic Acids Research

Article Title: RecN and RecA orchestrate an ordered DNA supercompaction response following ciprofloxacin-induced DNA damage in Escherichia coli

doi: 10.1093/nar/gkaf437

Figure Lengend Snippet: ΔrecN cells exhibit limited nucleoid reorganization after CIP exposure. All cells were grown in LB at 37°C and imaged at 2-minute intervals using live-cell imaging. ( A and B ) Cells (MR16) were immobilized in microfluidic channel slides and imaged using widefield microscopy. (A) Representative images of ΔrecN cells at 0, 8, and 16 min after CIP exposure, showing HU-mCherry fluorescence (green) to represent DNA distribution. Scale bar: 5 μm. ( B ) Analysis of DNA distribution along the cells’ long axis before and after CIP, quantified by measuring the distance between the outer bounds of symmetrical fluorescence peaks at 80% of maximum averaged intensity for each time point. Results are averaged from 17 to 61 cells from a single representative biological replicate (see “Materials and methods” section for detailed explanation). ( C and D ) Cells (KV68) immobilized on agar pads and imaged using spinning disk microscopy. Results are shown from images captured 12, 14, 16, and 18 min after CIP exposure, averaged from 40 tracked cells from a single representative biological replicate. (C) Average HU-mCherry fluorescence intensity along the cells’ long axis. (D) Kymograph heat map of relative HU-mCherry intensity distribution over time. ( E ) DNA compaction phenotype distribution for ΔrecN cells (KV68) at unchallenged baseline (dots) and after CIP exposure (lines). Dots and lines represent means from three biological replicates; error bars and shaded regions indicate standard deviation. A.u., arbitrary unit.

Article Snippet: To examine the cells’ immediate response to CIP exposure, we employed a microfluidic setup to image cells using a Zeiss Axio Observer Z1 widefield inverted microscope with a 63x oil objective (Zeiss Plan Apochromat 1.4 NA, DIC), a Colibri 7 LED light source, a Hamamatsu ORCA-Flash4.0 V3 digital CMOS camera, as well as a heated incubation chamber and mounting frame, both maintained at 37°C.

Techniques: Live Cell Imaging, Microscopy, Fluorescence, Standard Deviation