Imaging Morphological Changes in Bacteria Using Live-Cell Fluorescence Microscopy

Samenvatting

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 of 1.517 (refractive index) oil on the 100x oil immersion objective supplied by the manufacturer (Numerical Aperture = 1.4, Working Distance = 0.12 mm; see Table of Materials).
   NOTE: It is important to choose the appropriate immersion oil for the temperature at which imaging is conducted.
3. Load the glass bottom dish containing the sample into the metal housing (coffin) and gently slide it into the stage clamp.
4. Use the coarse adjustment knob to raise the objective until the oil makes contact with the glass bottom of the dish. Use the eyepiece and fine adjustment knob to bring the sample into focus. Once the cells are in focus, turn the knob from the eyepiece to camera mode by moving the selector switch located on the front of the microscope body to the left.
5. Begin the experiment using the imaging software. On the resolve3D window, select the Design/run experiment icon depicted by a flask. A new dialog box should appear entitled design/run experiment.
   1. Set the number of Z-stacks and sample thickness using the design and then sectioning tab on the design/run experiment dialog box.
      NOTE: For the experiments in the representative results section, 17 Z-stacks at a 200 nm interval for still images and four Z-stacks at 200 nm interval for time-lapse microscopy were used.
   2. To measure the thickness of the cells in the sample, manually adjust the Z-plane incrementally by using the up and down arrows on the resolve3D dialog box. Mark where the cells go out-of-focus as the upper and lower limits for image acquisition. Import this information prior to running the experiment.
   3. To help minimize phototoxicity and photobleaching during time-lapse imaging, reduce the number of Z-stacks and choose the mid-plane of the cells for image acquisition.
6. Select the appropriate filter set for the experiment using the design and then channels tab on the design/run experiment dialog box (TRITC: EX 542/27; EM 597/45; FITC/GFP: EX 475/28; EM 525/48; mCherry: EX 575/25; EM 632/60; Cy5: EX 632/22; EM 676/34).
   1. Also, select a reference for the collection of POL/DIC information. Adjust percentage transmission (light intensity) and duration of exposure for individual channels selected prior to imaging by selecting the appropriate options on the resolve3D dialog box.
      NOTE: In a test field of view that is not considered for the experiment, test these settings to identify if the selected parameters obtain meaningful fluorescence data without missing weak signal or oversaturating it. Then import these parameters for the experimental set up.
7. Open the points list by selecting the Points list button on the resolve3D dialog box. A new dialog box should appear entitled points list. Mark several fields of view to be used in the experiment by finding appropriate fields of view using the microscope stage controls and selecting the mark point option on the points list dialog box.
   NOTE: A replace point will have to be selected each time the microscope is refocused on a point in the points list. This can be done by focusing the microscope on the appropriate point in the list and then selecting the replace point button. It is important not to touch the analog coarse/fine adjustment knob when refocusing; use only the software to adjust the focus.
8. Set time-lapse parameters by first selecting the design and then time-lapse tab on the design/run experiment dialog box. Select the time-lapse check box. Enter the appropriate time-lapse parameters in terms of time-lapse images/total time.
9. Set points to be imaged from the points list by first selecting the design and then points tab on the design/run experiment dialog box. Select the visit points list option and enter points to be imaged in the text box separated by commas or hyphens if it is a complete sequence.
10. Prior to beginning an experiment, edit file names and file locations using the run tab on the design/run dialog box. File location can be changed by selecting the settings button, selecting the data folder, and then selecting the appropriate folder or creating a new one. Change file names by entering the new file name in the image file name text box.
11. Begin the experiment by selecting the play button on the begin experiment dialog box.
    NOTE: For time-lapse, continually check focus at each field of view throughout the experiment using DIC setting (to avoid unnecessary photobleaching) and refocus and update the information in the points list as cells tend to go out of focus over time.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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