MD ImageXpress Confocal HT.ai (Irchel) - 1: Start up

1. Instrument

Optional : if environmental control is needed, turn on sample heater

2. Laser (press button powerstatus)

3. Turn key to on

4. Turn on computer

Log in with your ZMB credentials.

Start the 20wi40pa10pa40wi profile

Login to MDCStore

username: use the username for the SQL database MDCStore.

password: use your SQL password

Enter acquisition setup

The Plate Acquisition Setup dialog provides three primary functions:

Configuration control - for setting up plate acquisition protocols.

Run - for doing a plate acquisition according to a selected protocol.

The Plate Map that displays the plate layout to control movable components of the system by simply right clicking.

The Eject Plate/Load Plate button switches between the following two options:

Eject Plate - Opens the top door and plate clamp to load or remove a plate.

Load Plate - Closes the top door and the plate clamp so that the instrument can acquire the plate.

Use the Plate Map to select the wells to acquire:

To exclude wells from the image acquisition, click on a well, click a row or column header, or drag the cursor over a section of wells. Excluded wells display in gray.

To include or exclude all wells, click the upper left corner triangle.

The Site Map represents the number of sites in each well and the position of the sites to acquire for each well. Sites you select to acquire display green and sites you choose to not acquire display gray.

Use the Active Wavelength drop-down to select an acquisition wavelength.

Use the Snap icon to acquire an image without performing (or re-performing) an autofocus.

Use the Start Live icon to start and stop Live mode

Use the Focus icon to perform the Autofocus routine and then acquire an image

Use the Test icon to perform the Autofocus routine and then acquire a Z series and present the corresponding 2D projection when you set up a z acquisition.

Use the Preview icon to perform the Autofocus routine and then acquire a Z series at each of the wavelengths you set up in the protocol and present the corresponding 2D projection when you set up a Z acquisition.

Use the Protocol drop-down to select from the eight most recently used plate acquisition protocols.

Use the Load Protocol button to select a protocol that you saved from a previous session for plate acquisition during the current session.

If you want to generate a new protocol, save a protocol first before modifying it. Save protocols to file in your private folder and not to database.

Click the Magnification drop-down and select the setting of the objective to use for the protocol.

For 20x and 40x water immersion: control the water immersion system and add water immersion by clicking on Make Ready.

Change camera binning if a different resolution is required or to increase the signal to noise signal-to-noise ratio.

Dual disk unit allowing selection between 3 modes:

Widefield imaging mode.

50 micron pinhole disk for your basic confocal requirements (better optical sectioning but less sensitive). Spacing between pinholes 500 um

51 micron slit for high throughput confocal requirements (faster acquisition but lower optical sectioning). Spacing between pinholes 250 um

When you select a water immersion objective the WI Status icon displays. Click on the icon to access the water immersion control.

Create a water bolus by clicking on "Make Ready".

As soon as water bolus is created and objective is ready to use the overall status will turn green "ready".

The ZMB provides you with common plate types that are already configured, from which you can choose.

If the plate type is not available in the Plate Name list, you can enter the manufacturer’s plate specifications including plate bottom settings (if available).

The manufacturer generally cannot provide values for the plate bottom settings, including optical thickness, which is not the same as physical thickness, and bottom variation. You must run the Laser Autofocus Wizard to measure the values to ensure proper focus.

Click Laser Autofocus Wizard and follow the instructions to calculate plate bottom measurements.

You configure the number of sites that are to be acquired for each well in a plate acquisition protocol on the Sites to Visit tab

Click to acquire a Single site.

Click to acquire a Fixed Number of Sites, configure the site layout, and then deselect the unwanted sites.

Click to acquire an Adaptive Acquisition is a computational algorithm that counts the number of cells on the fly during sample acquisition. This increases the chances of collecting valid data in every well. The number of sites the software acquires per well is based on the number of cells per well.

Click multi-well acquisition to acquire high density plates faster. This option uses the size of the camera field of view to acquire several wells simultaneously while taking a single image, thereby reducing plate acquisition time. The software automatically calculates the number of wells in which a site can be simultaneously acquired.

The Acquisition tab provides two autofocus options:

Laser Based Focusing is generally set to find the bottom of the well, and then moves the objective a distance up from the well bottom. This method is the fastest and does not cause photo damage to your wells. This method might not be sufficient if the distance above the bottom of the well varies in your sample.

Image Based Focusing uses a contrast-based algorithm to identify the best focus image. This option works best for experiments that use low-power objectives or when the sample distance above the bottom of the plate varies. Performance can be slower than Laser Based Focusing and focusing can fail if out-of-focus debris is in a sample.

Acquire Time Series acquires images at multiple time points. When you select this option, specify the set of images to acquire at each time point.

Continuous time series acquisition

With discontinuous time lapse acquisition, you can return the sample plate to the incubator in between time points, and you can read other plates in the interim.

Acquire Z Series acquires individual opticalsections (planes) in sequence through a sample that can produce a 3-D image of the sample. When you select this option, specify the number of steps and the step size to move through the sample.

Journals: macro based complex tasks that should be run while acquiring (normally not selected).

Analyze images after acquisition (normally not selected).

Allow appending to existing plate: data will be added to an already acquired plate. Can be used e.g. for multiplexing experiments.

Adjust focusing routine with laser settings and where the focus will be acquired.

Configure image based focusing (if selected in the previous step)

Specify the finding of sample and where to focus

Use the Wavelengths tab to specify the total number of acquisition wavelengths for an experiment.

When you specify more than one wavelength, subtabs display below the Wavelength tab where you configure exposure time, autofocus, and time lapse settings for each acquisition wavelength.

Enter the refinement level.

Click the Illumination drop-down and select the illumination setting; for example, FITC.

Specify the exposure and illumination power.

Calculate Offset to calculate the offset for all subsequent wavelengths.

Specify the z-series (3D, projection) and how the projection should be calculated

Digital confocal is a deconvolution computation. Normally this is not selected

Use the Timelapse tab to configure time series acquisitions (if activated previously).

Use the Z Series tab to configure Z series options (only if activated previously).

Specify the journals that should be run.

Specify how and if images should be displayed during data acquisition

Click Save Protocol to display the Save Acquisition Protocol dialog.

Select the Save to File Rather Than Database check box.

Navigate to the location to save the file in your private data folder (transfer server will be deleted wihtin 7 days).

Specify folder and plate names for organizing the data within the database.

Click to snap an image.

Click to test your settings.

Click Acquire Plate to acquire images from a plate based on the settings for the loaded protocol.