Leica Application Suite Software

The program is divided into four main options, Setup, Acquire, Browse and Process.

Image Browser

All images are saved to disk under a default option, hence all Users must first setup Capture Folder in Browse.

Click on Directory Browser option, then highlight folder option or create new folder by right clicking on mouse, then click on Capture Folder option, see below.

Setup

This is to configure the microscope objectives and the connections to the software and should not be accessed by Users.

Acquire

The Acquire option has three parts, Mic1, Camera and λ .

λ is the image acquisition part of the software, which allows users to look at images and set the correct exposure of the required fluorophores.

The Leica DM5000B has filter cubes for DAPI (LP – Long Pass), DAPI (BP – Band Pass), BGR option, FITC, TRITC (BP – Band Pass), TRITC (LP – Long Pass), TL-BF is the Bright Field option which can be changed manually on the microscope to Dark Field or Phase Contrast, and Cy5. To take an image with a specific fluorophore the box on the left side of the channel setup dialogue box should be ticked. Boxes with the ticked option should be clicked off if not required in the final image. The software collects a separate RGB image for each fluorophores highlighted by a tick in the dialogue box above.

By clicking on a filter option the image from the slide will appear in the viewing window. The exposure for that fluorophores can be adjusted by altering the time on Exposure.

Gamma function adjusts the gray scales for each fluorophores resulting in a change of intensity, see below.

The Gain control should not be used if possible as this enhances noise in the image.

The shutter can closed by clicking on the icon below.

To acquire images click on the Acquire Overlays option highlighted below.

If a single image is to be taken the overlay image button will be non-functionally and the user has to click on the option highlighted below.

The software takes the fluorophores from top to bottom, it is possible if you have a feint staining to change the order in which the fluorophores are list by highlighting a fluorochrome with a click of the mouse and then click on either of the icons highlighted below.

Process

The Process final area of the software allows the User to alter the image, in terms of annotation and enhance. Calib is an option to re-calibrate the scale-bar pixel rating, this is only required when fitting a new objective and should not be used by the User.

The Annotate option shown below is divided into several areas in which the User can, under Information add descriptions or file name to an image, as well as time and date details.

The software program holds information on the objectives the microscope is fitted with and hence the software can add a Scale Bar displaying any size, the User can change this by typing numbers into the µm box, highlighted above.

Lines and Arrows can also be added to the image with text added to these via the Line option highlighted below.

The Font type and size can also be changed via the Font option highlighted above.

Once all the changes are done these have to be merged into the image, this is done from the Actions option highlighted above. This merging action is permanent and the changes cannot be removed from the image. Thus it is important to duplicate the image before adding any annotation.

Enhance

This option allows the User change the brightness, saturation, orientation and crop images, see figure below.

To confirm any changes the User has to press Apply under Confirm.