Visualizing Bacterial Protein Distribution in Three Dimensions by Fluorescence Imaging

Abstract

Source: Bratton, B. P., et al., Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization. J. Vis. Exp. (2019)

This video demonstrates the method for acquiring and analyzing three-dimensional fluorescence images of bacterial cells to study protein organization at the single-cell level.

Protocol

1. Imaging

1. Insert the sealed slide onto the microscope and allow it to sit for 5 minutes to equilibrate the temperature with the surroundings. The microscope room may be at a different temperature from the sample preparation room.
2. Take a fluorescent z-stack of the sample.
   NOTE: The z-stack should entirely cover the sample with a z-spacing less than the depth of the field. For a 1.45 NA 100x objective and ~1 μm-thick E. coli cells, 40 steps at 100 nm per step work well. For larger cells or cells that do not lie perfectly flat on the surface, 50 or more steps may be necessary. Include enough steps and ensure that the samp....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	sigma-Aldrich	A9539
Cotton swab	Puritan Medical Products Company LLC	S304659	Used to appy VaLaP
Cover slips	VWR	16004-302
Fiji	ImageJ	https://fiji.sc	Used to cro cells
Lanolin	Sigma-Aldrich	L7387	Combine with paraffin and petroleum jelly to make VaLaP
LB growth medium	BD Difco	DF0446173
M63 medium	US Biological	M1015
MATLAB	Mathworks		Needed to run forward convolution scripts
Microscope slides	Fisher	12-550-133
NIS elements	Nkon
Paraffin	Sigma-Aldrich	327212
Petroleum jelly	Equate	49035-038-27
Piezo z stage	Nikon	77011589
Pipet tips -p200	USA Scientific	1111-0730
Pipet tips- p10	USA Scientific	1111-3730