Imaging Morphological Changes in Bacteria Using Live-Cell Fluorescence Microscopy

Abstract

Source: Brzozowski, R. S. et al. Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria. J. Vis. Exp. (2019)

This video demonstrates live-cell fluorescence microscopy to visualize real-time morphological changes in bacteria. It outlines the steps involved in imaging, time-lapse acquisition, and deconvolution to obtain high-resolution images in real time.

Protocol

1. Imaging

1. On the day of the experiment, turn on the microscope system. Start the imaging software (see Table of Materials) by clicking the appropriate icon on the desktop. Initialize the microscope by clicking the Initialize Microscope option (button depicted with a microscope on it) on the software’s start-up dialog box. Ensure that the objective is fully lowered using the microscope coarse adjustment prior to initialization.
   NOTE: Following initialization, three additional dialog boxes should appear in addition to the start menu: resolve3D, data collection, and filter monitor.
2. Place a drop....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	Fisher BioReagents	BP160-100	Molecular Biology Grade - Low EEO
FM4-64	Invitrogen	T3166	Microscopy
Glass bottom dish	MatTek	P35G-1.5-14-C	Microscopy
Microscope	GE	DeltaVision Elite	Customized Olympus IX-71 Inverted Microscope Stand; Custom Illumination Tower and Transmitted Light Illuminator Module. Objectives: PLAPON 60X (N.A. 1.42, WD 0.15 mm); OLY 100X OIL (N.A. 1.4, WD 0.12 mm); DIC Prism Nomarski for 100X Objective; CoolSnap HQ2 camera; SSI Assembly 7-color; Environmental control chamber - opaque.
PC190723	MilliporeSigma	3445805MG	FtsZ inhibitor
SoftWorx	GE		Manufacturer-supplied imaging software