This Article contains information on mulitacquisition from various versions of Nanoimager software. Most of the functionalities remained the same but new functionalities and some changes have been introduced in user interface. 

In Multi Acquisition setup every selected FOV can be imaged using three different ways of laser excitation.

1). Single                            2). Multiple acquisition                  3). Light program

Please note: Lasers selected in multiacquisition window will be activated only after starting the acquisition.

1). Single: Here you can choose laser excitation just like the way from the main acquisition panel and select any laser at desired laser power, exposure time and number of frames.

2). Multiple acquisitons: Here you can select laser excitations in multiple steps and each step can have its own independent exposure time, laser power and number of frames. Data from each acquisition is saved as separate file. New step can be added or removed using add or remove button.

3). Multistep advanced (Light program):

This will call the settings from the light program window. Make sure to set light program before using this option. This option will take number of frames as set in light program and light program for a selected FOV can be repeated using the number of repetitions.

Priority

In this option, you can change the way software is going to acquire a data set. You can choose to image sample with different laser at first and then change FOV or Z stack. alternatively, you can choose to image with one laser and change the FOV/Z stage and then follow the same process with second laser in the acquisition. 

Multiple acquisition setup – Multiple stage position acquisition

Overview scan set the first step towards automated imaging. The ability to acquire desired FOVs, image sample at set time intervals and at multiple optical sections provide actual platform to the Nanoimager user to make imaging automated. These features are included in Multi Acquisition setup tab positioned above the overview scan.

1). Acquire frame at multiple positions

2). Acquire Z stack and

3). Perform a Timelapse.

For acquiring multiple positions, tick the acquire frame at multiple stage position to activate the position setup tab.

Position setup operates in two manners:

1). Regular grid

2). List of positions

User can toggle between these options. The selected option will be executed by the software.

Regular grid

In an experimental setup where sample is evenly distributed, for example single molecule FRET experiment with DNA molecules bound to surface, user can acquire images in predefined pattern and choose the number of FOVs in both X and Y directions. The space or overlap between each FOVs within a pattern (highlighted in red below) can also be defined as per the requirement of the experiment.

Stages will move in the pattern selected by user (selected pattern will be highlighted as shown in the image above). Simple X and Y pattern will acquire total number of images from different positions calculated by simple formula.

Wait time after stage movement can also be entered.

IMPORTANT: It is crucial to use Z-lock for multiple position acquisition for best results.

List of Position

Sometime the distribution of sample on the coverslip is not uniform for example transfected cells in an ibidi chamber. This will prompt user to acquire selected stage positions. Once can add and remove list of positions required to imaging. List of positions registers four parameters to acquire image namely X, Y, Z and Z offset position.

• Bring sample in focus
  
• Run Overview scan with Z-lock on.
  
• Now open multi position acquisition setup
  
• Click on position setup to active position tab and select list of positions
  
• Put both overview scan and list of positions side by side as shown below or toggle between two windows
  
• In Overview scan double click on the FOV you want to image and add that position in the position setup using add current stage position. Adjust the focus, if require, using Z offset. Repeat this to add more positions
  

• Now choose the Z reference and saving file options as explained in next steps
  
• Set exposure time and laser powers/use light program
  
• Click Start

Autofocus and Z reference

Each stage position can have different Z offset values, it is therefore very important to choose the Z focus reference as values mentioned in position setup. Click on the Autofocus and Z reference tab and select Z reference set to values specified in position setup as highlighted in red box below.

Saving

Multi Acquisition setup has separate option to name the Dataset tag (folder name) and acquisition tag (file name) under the saving tab.

In Acquisition tab, for every position define the exposure time and number of frames needed to be acquired at each FOV. It also provides option to use light program.

Switch the lasers on if not using light program option and click start to begin the acquisition.

2). Z stack

For acquiring Z stack, open the Multiacquisition setup and select acquire Z stack to activate the Z stack tab.

Increasing the number of Z slices will activate the Z top and Z bottom tabs as shown in the image below.

Z stack tab assumes current stage position as ‘0’. Move the focus to the middle/center slice of the sample and then enter the Z top (positive values in microns) and Z bottom ( negative (-) values in micron) of the sample you want to image. For example, if you want to image 5 micron depth of sample using Z stack then focus on centre of the sample and enter 2.5 micron in the Z top and - 2.5 micron in the Z bottom tab.

Now enter the size of each optical section in the Z slice separation tab.

You can choose the motion of stage from bottom to top or top to bottom by simple selecting any of the option.

Set the acquisition parameters in the acquisition tab.

To save and open the each Z slice as hyperstack in ImageJ, select the combine dataset option.

To acquire the image, switch on the desired lasers or select light program. Click Start

Multiacquisition in version 1.0 and earlier

This is a more refined version that allows you to do Time-lapse/Multi-positions and Z-stacks. To access it, press the multidimension button which can be found just below the focus camera view.Th

You can also add specific list of positions. DZ is the offset in relationship to the focal plane set during autofocus calibration

On the Z-stack tab, you can select how many slices you want and what spacing between each Z-slice. Since the stage moves and not the objective, a positive movement of the Stage is equivalent to going down through the sample

For time lapse imaging where you want to acquire a preset combination of lasers and then have a period of no laser exposure and no acquisition..