Journal: bioRxiv

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

doi: 10.1101/2024.11.15.623168

Figure Lengend Snippet: All cells were grown in LB at 37°C and imaged at 2-minute intervals using live-cell imaging. ( A and B ) Cells were immobilized in microfluidic channel slides and imaged using widefield microscopy. ( A ) Representative images of HU-mCherry labelled (green) wild-type cells at 0, 8, and 16 minutes 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 34-141 cells from a single representative biological replicate (see Materials and methods 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 minutes 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. 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

Journal: bioRxiv

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

doi: 10.1101/2024.11.15.623168

Figure Lengend Snippet: All cells were grown in LB at 37°C and imaged at 2-minute intervals using live-cell imaging. ( A and B ) Cells were immobilized in microfluidic channel slides and imaged using widefield microscopy. ( A ) Representative images of ΔrecN cells at 0, 8, and 16 minutes 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 17-61 cells from a single representative biological replicate (see Materials and methods for detailed explanation). ( C and D ) Cells immobilized on agar pads and imaged using spinning disk microscopy. Results are shown from images captured 12, 14, 16, and 18 minutes 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. 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