How to use Extended Depth of Focus (EDF image) on a manual microscope

Using this method you can combine the sharp regions from the individual sections of a Zstack image to form an extended depth of focus (EDF) single image. The focus planes of all the z-positions will be calculated to one EDF image. This enables you to display a considerably larger depth of field than is possible with a single focus

By activating the Extended Focus Module in the software (Zen Lite/Pro/Desk), you can manually acquire an EDF image using a simple microscope without motorized z focus. (EDF image can be also acquired automaticaly through the Processing Module using. microscopes with motorized stage)

The procedure for Manual EDF

To activate manual EDF Module go to Tools-Modules Manager

Select Manual EDF

Now the Manual EDF Module is activated
Go to Locate-Open Manual Extended Depth of Focus tab

In the Manual EDF Module

Z-Stack - If activated, the image with extended depth of focus will be calculated out of already

existing Z-Stack image and will be opened in a new image container.

Mode:

Acquisition with F12 key mode: Snap, Move, Snap, Move, Get the final result

In this mode you can achieve different time intervals between the individual focus steps. By pressing

the F12 key on your keyboard, you can manually record a new image. Between the acquisitions, you

can change the focus position

 Select the F12 Key mode

 Click on the Start button. The Start button will only be active when the live image is closed

Press the F12 key on your keyboard to initiate manually, each acquisition with a different time

step

 Select manually a new focus position. Repeat the last two steps until you have sharp images from

all desired areas of your sample

 To finish the acquisition, click on the Stop button

Acquisition with Timer mode:
In this mode, after a freely selectable interval, a new picture will be automatically added. In the
period between the acquisitions you set a different focus positions. After each recording a new
image with an extended depth of focus is immediately calculated. When you finish the acquisition
you will get the image which was calculated last

Quality - Here you can select the quality level that you want the function to work with (Low/
Medium/ High/ Highest)

Registration Method- Here you can select the method (or a combination of these) to be used to align
the images

Translation - The neighboring sections of the Z-stack image are shifted in relation to
each other in the X and Y direction

Rotation - The neighboring sections of the Z-stack image are rotated in relation to
each other

Iso Scaling - The magnification is adjusted from section to section

Skew Scaling - The neighboring sections of the Z-stack image are corrected for
skewness / shearing

Affine - The neighboring sections of the Z-stack image are shifted in the X and Y
direction, rotated and the magnification is adjusted from section to section

Interpolation - Here you can select how you want interpolation to be performed if a pixel in the EDF
image is calculated from several individual pixels

Nearest Neighbor -The output pixel is given the gray value of the input pixel that is closest to
it

Linear- The output pixel is given the grey value resulting from the linear combination of the
input pixels closest to it

Cubic- The output pixel is given the gray value resulting from a polynomial function of the
input pixels closest to it