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 sample is fully blurred above and below.
   1. Use the software associated with the microscope (see Table of Materials) to control the microscope.
   2. Focus on the middle of the cell using the microscope focus wheels. Under ND acquisition, check the Z box to take a z-stack. Click the Home button to set the middle of the cell as the starting point. Set the Step size to 0.1 µm and set the Range to 4 µm. Make sure that the Z device is set to the piezo stage.
   3. Set the fluorescent channels under the Lambda Window to the settings for the fluorescent molecules being imaged. In this experiment, GFP and mCherry were used.
      NOTE: Take an additional z-stack with the same Step Size and Range in the second color channel if the 3D distribution of an additional fluorescent channel is desired. In this experiment, cytoplasmic mCherry was used to determine cell shape and MreB-GFP was used as a second color channel.
   4. Ensure that the Order of Experiment is set to lambda (z series) so that it will take a complete z-stack in each color channel before switching.
   5. Click Run Now to start the image acquisition and save the file once one or both z-stacks are complete.
   6. Move to a new area on the pad and repeat steps 1.2.2-1.2.5.

2. Cell Reconstruction

1. Crop individual cells and save the images as a stacked tiff file so that there is only one cell per file. Ensure that this cell is well isolated from any other cells (i.e., roughly 5x the full-width half-maximum of the blurring function in xy.
   NOTE: This can be done using freely available image analysis software (see Table of Materials).
   1. Draw a box around an individual cell and duplicate that cell 2x, once for each channel. Make sure the duplicate hyperstack box is checked and change the channel to either 1 or 2, making sure that the slices include the entire z-stack.
      NOTE: If imaging only the shape of the cells and not an additional fluorescent protein, only one channel will be present.
   2. Once both stacks are available, go to Images | Stacks | Tools | Concatenate to combine the images with the protein channel first and the shape channel second.

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