• Startup
• Loading a sample
• Image and navigation tabs
• First imaging settings (Acquisition mode)
• Fix incorrect sample carrier in navigation tab
• Preview imaging
  • First focus and an image
  • Preview scan
    • • Objectives
• Channel settings
  • Wide-field
  • Confocal
  • Airy scan
• Select areas to image (Navigation tab)
  • Tiles for a well-plate
  • Positions
  • Copy areas to selected wells
  • Add images to navigation
    • Show recorded image in navigation
• Focus strategy
• Stacks
• Time series
• Incubator settings
• Shutting Down

Startup

Computer must not be on when any hardware is put on. If computer was on already then it must be shut down before you put any hardware on!

1. Rocker switch bottom right of the instrument box.
2. Button switch on the instrument display panel.
3. (Optional) Lasers on by a key, box under the instrument.
   • The two switches above laser power should be always on.
4. Wait for display panel to be ready.
   • If you use lasers, it's enough to wait until light next to key goes out
5. Start computer
   • 5b If there is a nosepiece error on instrument display, choose retry. Do this before starting a software. If you started software with this error, you should turn computer and instrument off (not lasers) and starting again.
6. Start Zen Blue, select "Full ZEN Functionality".
7. Select option "Acquire New Image" on project selection.

Loading a sample

There are sample carriers for slides and dishes. Well-plates can be loaded as they are. Other carriers require different sample carrier settings for start up. Ask help if you don't know what to do.

1. Click "Eject Tray".
2. Insert well-plate or other sample carrier with correct insert. Check that plate or insert is not tilted.
3. Check options:
   • Measure Bottom Thickness
   • Determine Bottom Material
   • Prescan Options:
     • Sample Carrier detection
     • Create overview
     • Automatic Sample Carrier Calibration (for well-plates with 96 wells or more)
4. Click "Load Sample"

Image and navigation tabs

The main window has buttons for different oparating modes (Sample, Acquisition, Processing).

Sample mode In sample mode you can adjust the properties of your sample if automatic detection did not got them correct. You can also remeasure bottom thickness when you change sample (dish, chamber or slide). Correct bottom thickness  and material are important if you want highest resolution.  

Images are shown as tabs on main part of the window. 

• If you selected "Create Carrier Overview", the first image will be an overview of the sample carrier (CarrierOverview.czi). This is taken with a dedicated camera. The idea is to document the experiment, for example markings made on a plate lid with a black marker will be visible. You can also move to different dishes or slides when looking this tab.
• Second tab is Navigation View where you can move around the carrier and take preview images to help locating the i9nterestion position of the sample
• rest of the tabs are images

First imaging settings (Acquisition mode)

1. Smart Setup
   1. Select WF
   2. Dyes & Contrast Methods / TL Oblique
   3. OK
2. Channels tool
   • With 5x magnification, drop illumination power to 5% and exposure time to 5ms.

Fix incorrect sample carrier in navigation tab

Sometimes the sample carrier in the navigation doesn't initially correspond with carrier that was loaded. In this case, it is necessary to update it. This is how you can make that happen:

1. Create imaging settings as above
2. Select navigation tab
3. Select overview tab
4. Select navigation tab

At this point the software should notice there is a mismatch and proposes to update the experiment. Say yes.

Preview imaging

First focus and an image

1. Select navigation tab.
2. Double-click to navigate to an area where there should be a sample.
3. On the right, click Definite focus / Find surface. This will find the bottom of the sample.
4. Click live to see sample with oblique illumination.
5. To focus, press Ctrl and use mouse roll to move up/down. You can change the speed of the manual focus by selecting the speed by mouse when you are imaging live and you have ctrl pressed down.
6. Click "Snap" to record an image.

Preview scan

It is usually good to do a preview scan of your sample so you will get a low resolution image on Navigation View which shows where your sample is. Then you can use navigation view to move to different parts or wells of your sample. Quite often an Oblique illumination with 5x objective is enough but it is certainly possible to use fluorescence channels and/or higher magnification if oblique does not give you enough information. The image you took after focusing should give you an idea what you can expect.

1. Choose one of the tile imaging options from the left of the window and choose one or multiple tiles. Go to the middle of the area or middle well and refocus.
2. Choose preview button
3. Start button at Navigation tab

This will create a preview image in the navigation view covering area or wells you marked. The preview imaging does not use autofocus so if your focus varies a lot and you have a big area, you might need to use proper imaging and autofocus settings instead of preview imaging. The image is not saved but it is only on navigation view. Do not worry if it is not a perfect one because it is meant to be a quick preview to help you to locate the interesting positions of your sample.

Objectives

Change objectives and tubelens magnification. 20x yellow, is a long working distance 20x, 20x green is high resolution 20x,  and 50x is water immersion. When you change an objective, wait until it is active (blue background) before changing tubelens. 50x objective takes longer because the system also add or remove water immersion automatically.

Next check your fluorophores by Wide Field imaging. Try to get the highest signal 25-75% of maximum by adjusting light intensity and exposure time. Avoid overexposure. Refocus by one fluorescent channel using 20x or 50x objective. You should do this first even if you are going to use confocal or Airyscan for imaging.

Channel settings

In most cases you can get a good base for imaging settings using Smart Setup. Search exact fluorophore you have in your sample if possible. Use 'a similar' color only if your fluorophore is not found from the list of fluorophores.

Wide-field

Wide field channels can be combined with either confocal or AiryScan settings but it is usually not recommended

Confocal

Airy scan

Select areas to image (Navigation tab)

Tiles for a well-plate

1. Select wells you want to image.
2. Select "Set up new tile regions from an underlying sample carrier".
3. Select and set "Fill Factor" or "Columns/Rows".
4. Click "+".

At this point you can still edit the number of rows and columns.

To delete the tiles:

1. Select the wells in the small Carrier tab at the bottom.
2. Press Delete key.

Positions

Copy areas to selected wells

Add images to navigation

You can have images with multiple resolution in Navigation View and they can overlap each other. Clicking on image so that it has a white border selects it so you can adjust display range for that image or you can remove it by delete-button from keyboard. If the image was only for preview then you can't get it back. Recorded images are on disk and they are not removed totally when you remove them from navigation view.

Show recorded image in navigation

• Open Navigation & Tiles tab.
• Select an image in the "Images and Documents" list on the right.
• Drag the image on to the navigation view

Focus strategy

Stacks

Time series

Incubator settings

Shutting Down

1. Check from OpenIRIS,
   • If there is next booking within 24h at 37C or 30C, put the warming on at ZEN - Incubation 
   • If there is next booking within 2h you should leave system on. If next user is using Widefield, you should turn off lasers at step 7. Check configuration at  booking.
2. Change objective to 5x
3. Close ZEN
   • If you are leaving warming on, software will ask you if you want to shut it off. Answer no.
   • Save the last images. Software will ask to save all images you have not save yet, Unselect images you don't want to save.
4. Wait 20s so ZEN Service is also shut down.
5. Choose 'End session Now' from OpenIRIS Agent clock and immediately choose Shut Down Windows
6. Press Eject Sample from instrument screen if you still have sample inside
7. If there was no next booking within 2h at step 1, then shut down lasers and instrument. If you are unsure, don't leave the system on. If next user is using widefield, turn off the lasers. 
8. Wait until the small screen has shut down and switch off the main power switch. If you left the warming on at Step 1, leave the main power switch on.