$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
1. Microscope Set-up and Time-Lapse Acquisition
NOTE: The protocol described here was developed for an inverted widefield microscope with autofocus, a 100X/1.30 NA oil PH3 objective, an X, Y motorized stage, a sCMOS camera, a light source, filters for green-fluorescent, red-fluorescent, or yellow-fluorescent proteins, and a temperature-controlled incubation chamber. This chamber keeps cells protected from light and at a constant temperature.
- Preheat the incubation chamber and the microscope to 32 °C for ~1 - 2 h before starting microscopy.
NOTE: Depending on the microscope set-up, heating may take longer. Pre-heating is essential to reduce drift and stabilizes the autofocus control system. - Switch on the microscope and start the microscope control software. Select the correct objective and the correct mirrors and filters to acquire phase contrast images as well as images of green-fluorescent, red-fluorescent, or yellow-fluorescent proteins.
NOTE: A microscope is typically supplied with preferred software for microscope control and image acquisition. Here, a commercially available software (see the Table of Materials) was used to control the microscope and image acquisition. - Add a drop of high-quality immersion oil to the objective lens and to the bottom of the sample, pre-incubated at 32 °C. Place the objective at the lowest possible Z-position to avoid damaging it when the sample is placed on the microscope stage. Place the metal frame with the sample onto the microscope stage, with the "hole side" towards the objective. Fasten the sample securely in the stage holder.
- Focus on the cells by moving the stage in the Z-direction closer to the objective. Move the stage slowly when the oil drops on the sample's bottom side and the objective lens makes contact. Move the stage in the X/Y direction until multiple single cells are visible in the region of view, when cells are in the focal plane. Make sure that at least one fluorescent microsphere is in the region of view in order to later align the acquired images.
NOTE: Under optimal conditions, a cell density of 15 - 30 cells per region of view (2,048 x 2,048 pixels or 133.1 x 133.1 µm) should be reached. - Open the Multi-Dimensional Acquisition wizard of the microscope control software to set up a time lapse experiment that allows the microscope to acquire images at multiple wavelengths and stage positions if required.
- In the Main tab, activate Timelapse and Multiple Wavelengths. Additional tabs will appear on the left side of the window.
- Click on the Saving tab and Select Directory to select an empty folder on the computer hard drive to save the acquired images. Activate Increment base name if file exists to make sure that consecutive datasets do not overwrite earlier ones. Then give the experiment a name with date and the strain name or title of the experiment.
- Click on the Time-lapse tab to adjust the time-lapse parameters. Set Duration to 24 h and set Time Interval to 20 min. The Number of Time Points will change automatically.
NOTE: The optimal time interval depends on the experiment and the cellular function to be analyzed. Frequent image acquisitions may cause photobleaching. Thus, a trade-off between temporal resolution and photobleaching must be empirically found. At a doubling time of 4 - 6 h, images can be easily acquired at an interval of 5 min (or even smaller intervals if desired) for phase contrast microscopy. If fluorescence microscopy over a time-course of 24 h is desired, images should be recorded at an interval of approximately 15 - 30 min. - Click on the Wavelengths tab. Select the number of wavelengths to acquire for each image at each time point by changing the number.
NOTE: For each wavelength, a new tab will appear on the left side of the Multi-Dimensional Acquisition" wizard and wavelengths will be acquired in the order from top to bottom. For each wavelength, the acquisition settings can be modified separately. - Click the first wavelength tab from the top. Select Phase contrast in the Illumination drop-down list. Select 100 ms for Exposure and select Every Time Point in the Acquire drop-down list. Deactivate Auto Expose by selecting Never in the drop-down list.
- Repeat step 3.5.5 for each wavelength that needs to be acquired at each time point. For the experimental set-up and fluorescently labeled proteins described here, use the following parameters for Exposure: 250 ms for mCherry fusion proteins, 200 ms for YFP(Yellow fluorescent protein) fusion proteins, and 1,000 ms for GFP (Green fluorescent protein) fusion proteins.
NOTE: The optimal illumination settings for each strain and fluorescent protein should be determined in advance by changing the lamp intensity and the image acquisition time for each wavelength. Too long image acquisition times will increase the phototoxic effect and ultimately lead to growth arrest and cell death. Therefore, a trade-off between image quality and cell viability should be achieved. - Acquire images from multiple stage positions to increase the number of cells recorded in the same experiment.
- To acquire images from multiple stage positions, activate Multiple Stage Positions in the Main tab. Then click on the Stage tab and click the Live button to look at the field of view.
- Move the stage in the X/Y-direction until a region of interest (ROI) is in the field of view. Save the X- and Y-coordinates by clicking the "+" in the Stage tab. Move the stage again in the X/Y-direction until a new ROI is found and save the coordinates again by clicking the "+". Go on until the desired number of regions is saved.
NOTE: In case of fluorescence image acquisition, make sure that regions of interest (ROIs) are not too close to each other to minimize phototoxicity.
- Check once more that the cells are in focus by clicking on the different saved X- and Y-positions and start the hardware autofocus by clicking AFC hold to keep the saved Z-position constant over the course of the experiment.
- Start the time-lapse recordings in the microscope control software by clicking Acquire in the Multi-Dimensional Acquisition wizard.
NOTE: One window will appear for each wavelength that is acquired, and an additional window will appear that shows the number of acquired time points and the time until the next picture acquisition. - Check that the cells are still in focus after the first few time points in the time-lapse recordings to maximize the quality of the images and refocus if necessary.