Perkin Elmer Operetta (Lengg) -2: Acquire an Image in xy and z

Go to Setup

Choose the Optical Mode

Choose Binning

Activate Live Preview

Symbols for Temperature and CO2:

Start-up: the stated value has not yet reached the target value

Regulating: the target value is almost reached, but it is not yet stable (< 4 ° C below the target value). Check the messages panel for specifics

Ready: the target values have been reached (+/- 1°C around target temperature, and +/- 0.5 % around CO2 concetration)

In the Global Control, choose the objective that gives you the needed magnification for your imaging objective

Note that the collar of the objective sometimes needs to be adjusted to match the refractive index of the bottom of the plate

If the message "The correction collar needs to be adjusted..." appears, click "Yes". The selected objective is then moved into service position and the lid unlocks

The lid will open automatically and you can remove the objective from its position (12 o’clock position in the objective turret) by just lifting it up

If necessary, adjust the correction collar to the given value [mm]

Only touch the objective as shown in the picture, do not touch the lens. Make sure not to screw the objective out of its mount when adjusting the correction collar. The objective must remain screwed in firmly

Ensure that the red dot on the objective holder is placed correctly on top of an identical dot inside the microscope

When choosing one of the two water immersion objectives, you must first flush the objective

Go to "Settings"

Then "Operetta CLS"

Then "Flush Water Objective" twice consecutively

Open the front lid and watch that water falls inside the left bottle. This means that the flushing is working

In the "Define Layout" menu, choose a well and

as position inside the well for testing

In the Navigation Panel on the right side of the screen, select a well or a slide with a left click. The selection will become orange

If you clikc "Select", the well will become gray

By default the center of the well/slide is selected

To change the test position inside the well/slide, left click on the position of interest, then "Select", and "Deselect" the previous position, as only one position is needed for testing

Add the fluorescent channels you are aiming to image in the "Channel Selection" panel and click "+"

First choose your nuclear staining to test your imaging settings, or whichever staining is the most ubiquitous in your sample. DAPI for example

Adjust the exposure "Time"

Not that for confocal mode, you can only enter exposure times which are a multiple of 20 ms (e.g. 20, 40, 60 ms)

"Power" of the light source

and "Height" of focusing in case that you would like the focus to be slightly above or below to where the autofocus has been found

Note that for confocal imaging mode your will need significantly less power and exposure time. Ensure to adjust those accordingly before testing and possibly bleaching the well

Click on "Snapshot" for checking just one channel's settings

Check the channel's histogram to avoid saturation. If the image is satisfactory, add more channels

if needed adjust the height of focus, exposure and power pf the illumination to improve image quality

You can also modify the excitation wavelength and detection windows, when you need to customize the channel independently

Once you check each channel independently, you can check the full image with clicking "Test"

Change the look-up-table bydouble clicking the color bar

Set up Z-stack settings in the "Layout Selection" pannel

Go to "Stack" to define which planes of the well are measured (Z-stack). It is defined (and measured) 'bottom-up'

The "First Plane" defines the bottom of the stack

Give the "Number of Planes"

And the "Distance" between planes

Ensure that "Use in test" is checked to see it online when testing

If you define a stack, the height defined in each individual channel will be ignored

The "Stack" panel on the right will display the Z-stack you define

On the right "Navigation" panel, go to "Test Images"

You can click on the well and the areas where you have made a test stack

Click on the stack and navigate it with the arrow keys, or clicking on each plane with the mouse and the images will show on the Image panel

If you would like to update the start and end of the stack you can go to the previous step and adjust accordingly

Go to the "Time Series" panel

Select the number of sequences (different sequences for different time points)

Provide the amount of "Timepoints" where the imaging should be done

Give the interval between time points on "Fixed Interval"

Uncheck "As Fast as Possible"

In the Navigation panel go to Define Layout

Select the wells you want to image by clicking on each well (they become orange), and then click on select (they become gray)

On the Well window also select the areas you would like to image

Go to Run Experiment

Give a name to the plate

Click start

After the imaging is done, you can check the images in the Navigation panel

Click each well and location