Cancer Institute Microscopy core Facility Standard ... · Zeiss LSM880 Airyscan Confocal Microscope Double click on the ZZEN [ Black software icon to open the software Select ^Start

Cancer Institute – Microscopy core Facility

Standard Operating Procedure

Zeiss LSM880 Airyscan Confocal Microscope

Double click on the ‘ZEN’ Black software icon to open the software Select “Start System”. The system will now initialise. Please do not use the microscope during initialisation.



Menu tabs:

Locate – Instrument is in Widefield mode

Load your sample

Put the sample in focus via the eyepiece

Check fluorescence

Find a good Field of View (FOV)

Acquisition – Instrument is in Confocal mode Control lasers

Set up confocal parameters

Acquire Z-Stacks and other multi-dimensional images

Processing – acquired images can be processed Maximum Intensity Projections (MIP)

Airyscan deconvolution processing

Other advanced features…



Locate

Remove the sample holder and place your slide coverslip facing down into the holder. Do not hold the screws to lift the holder. If you are planning on using an oil immersion objective:

Select an appropriate objective first (40x or 63x only). Please apply Oil to the slide where you have tissue/cells. Please use the correct oil for;

o Room temperature 23oC, warm chamber 30oC or heated chamber 37oC

There are already light path illumination settings set up for you to use to view your sample down the oculars of the microscope. The settings can also be selected from the microscope touch pad. The objectives on this system are very expensive. It is crucial for use with the airyscan that the objective provides a perfect PFS result. Care must be taken at all times when using the objectives on this microscope system. It is crucial that they do not get damaged as this will affect the PFS.

Or…

Objectives on the system: 5x Dry 10x Dry 20x Dry 40x Oil* 63x Oil* *All oils are compatible

Fluorescent cubes (for widefield): DAPI GFP PI Texas Red



Vessels that you can and cannot use on this microscope.

Slides: Coverslips mounted in the centre of the slide

Chambered Slides: Only the two centre wells can be imaged.

35mm glass bottom dishes: Only the central area can be imaged



Confocal Imaging setup – previous settings

Once you have focused on your sample and found an area of interest return to the Acquisition tab. If you are continuing with an experiment and want to re-use previous settings:

1. Click on:

File -> Open -> ‘locate a .lsm or .czi file’ -> select ‘Open’

2. Select: ‘Re-Use’



Confocal Imaging setup – New settings

Once you have focused on your sample and found an area of interest return to the Acquisition tab.

Select ‘Smart Setup’ From the drop down menu, select the fluorophores which you have used in your sample. It is important that these match your fluorophores exactly. Click ‘Apply’. Common combinations include:

DAPI ---- AlexaFluor 488 ---- AlexaFluor 555/568 ---- AlexaFluor 633/647

Once these have been inputted, the software will provide 3 different ways to image. If your sample

is fixed, you should always choose: ‘Best Signal’. Unless you have been trained otherwise.

Summary of different modes: Fastest: All lasers ON All detectors ON Time to scan: Fast Bleedthrough: High Gating flexibility: Medium Recommended only for samples with 2 or less fluorophores which are well enough separated spectrally. Ie. Green + Red fluorophore combination.

Best Signal: One Laser on at a time One detector on at a time Time to scan: Slow Bleedthrough: Very low Gating flexibility: Very high Recommended for all fixed samples with 2+ colours.

Smartest (line): Up to 2 Lasers on at a time Up to 2 detectors on at a time Time to scan: Medium Bleedthrough: Low/medium Gating flexibility: Very low Recommended only for live cell imaging.



Under the ‘Channels’ tab, you will see your newly created ‘Tracks’. A track contains the settings for one scan, it can contain multiple lasers/detectors depending how you setup your experiment. Highlight each track and then change the gating to what you want for each fluorophore.

In the acquisition mode window set up the following parameters: Select Scan Mode: Frame Resolution: 1024x1024 Speed: max Averaging Number: 4 Mode: line Method: mean Bit depth: 16 bit Direction: Bi-directional Scan Area Zoom: 0.6



The pinhole is the first parameter to setup as the Laser power and detector gain is dependent on this. The pinhole defines the resolution in Z with 1.0AU being optimal size (resolution vs intensity). The easiest way to setup the pinhole on a 3 colour experiment is to select the middle-track and select 1.0AU. You will then have a read-out of the size in Z. Typically, 1.0 AU on AF488 emission using the 40x oil objective will yield a section size of 0.9um. The section size then has to be translated to the other channels and this may not necessarily require the same pinhole diameter. You can use the same size pinhole to do this.



Setup Laser power and Detector Gain It is usually easier to work with one channel at a time to set the correct Laser power and Gain for the detector. To do this, un-check two tracks -> select ‘Live’ and the instrument will start scanning. Click on -> ‘Range indicator’ Blue = no signal/background Red = Oversaturated pixels

Firstly, set the gain between 600-900

Use the Laser power slider to set the level

You are aiming for no red pixels in the image as these cannot be accurately quantified. You should not oversaturate the detector at any one time. Increase the gain and reduce the laser power sequentially to prevent the detector becoming oversaturated if your sample has strong staining.



Once your imaging settings have been optimised, re-check all channels. Now you can click on ‘Snap’ to take an overlay image



Saving confocal images

Select file save as and save as a Zeiss .czi file. This is the raw data file that should be kept. This file can be used to reload and reuse settings. The .czi file holds all the image information.

Save as “Raw data” or “contents of image window” or full resolution image window



Once an image has been saved as a Zeiss .czi file the image file can be re-opened in the software and the settings reused for other images.



How to Zoom in on an area of your image Select the crop tool from bottom of the image window Move red square to the zoom and co ordinates that you require. Rescan the image and it will zoom into the area that you have selected.

To undo the zoom select reset all in the acquisition mode window.



How to take a Z stack Set up the sample and optimize the same way as you would do for a single plane. Select Z from the main acquisition window (tick box) Open up the Z control window. This should appear at the bottom of acquisition parameter windows. Select First/Last method (default) If you chose best signal when setting up your imaging, select only one channel which will determine the top and bottom of your cell (ideally some sort of membrane/cytoplasm marker) Now select ‘Live’ and scan you sample. With the fine focus knob, focus to one end of your sample and on the Z-stack menu click on ‘Set First’. Now focus the other way to find the bottom and select ‘Set Last’.



Stop the Live scan.

Select Start Experiment and the Z stack will be captured. Save as Zeiss .czi file format. The images can also be saved as a tiff series.

We have a Piezo stage fitted which allows for more accurate and faster movements in Z. If you wish to use this, please check the box. Click ‘Advanced’. You can choose to optimise sectioning automatically if you wish. Match Pinhole: Keeps pinhole optimal but may result in slight thicker z slices. Optimal: Images at 2 fold Nyquist settings X:Y:Z = 1: Matches the settings in Z to X and Y resulting in a cubical voxel.

Documents

Cancer Institute Microscopy core Facility Standard ... · Zeiss LSM880 Airyscan Confocal Microscope Double click on the ZZEN [ Black software icon to open the software Select ^Start