Manual for Olympus BX microscope with Hamamatsu camera

https://sils.fnwi.uva.nl/bcb/BX_2016/MD/bx_macros.html

N.Vischer, 08-Feb-2017

see also: First-time installation and download

Basically, the macro set provides a two-step dialog (settings and exposures), and then provides shortcut keys such as "Grab next channel [F1]" to fill a hyperstack of the desired structure.

• Open Micro-Manager (shortcut on desktop, Fig 1). You will be asked to confirm the config file, click OK.

Fig. 1: Start-up window shows used config file

• This will automatically install the "StartUpMacros.txt" and the Micro-manager window will become visible (Fig 2).

Fig. 2: Micro-Manager window

• You can optionally play with buttons "Snap" and "Live", which will open the "Snap" window and take pictures with the selected Exposure [ms]. But during normal operation, it is enough that the "Snap" window exists and "Live" is switched off.

• Now let us use the installed macros to capture image sequences in an organized way. Via key strokes, images will be grabbed and copied from the 'Snap' window to a hyperstack with the desired structure, using individual shutter behaviour and exposure times per channel.

Fig. 3: Two-step dialog, acquisition structure (left) and exposures (right)

• Make the ImageJ window active, and choose menu Plugins> Macros> Dialog [F5] or press the key F5. First time the dialog BX-Settings step 1 will appear (Fig 3, left).
• Enter a file name and numerical suffix (or supply this info later when you are asked to save the stack).

• choose a Channel Pattern consisting of letters b (=brightfield) and f (=fluorescence). For example, the pattern 'bff' will create a 3-channel stack, where the UV shutter will be opened only for the second and third snapshot.

• choose Field Size (currently 2048*2048 or 1300*1300). The latter option will only store the central part of the Snap window, useful if the peripheral part of the sensor chip is poorly illuminated in case of fluorescence images.

• choose the Number of Frames. For example, if 3 channels and 8 frames are selected, an acqueisition stack of 3*8 = 24 images will be created to receive the individual snaps. With a larger number of frames per stack we have to handle less files.

• Click OK to leave the first and enter the second step of the dialog (Fig 3, right), and select the desired exposure times.

• Subsequent calls of "Dialog [F5]" will lead directly to "Dialog step 2" with the exposure times, but you can go back to step 1 by checking "Change Settings Above". In this case, you may be asked to save any (half full) acquired stack.

• Now you can collect snapshots in the desired sequence. Choose menu Plugins> Macros> Grab Next Channel [F1] or press key F1. An image will be captured and and populate the current acquisition stack. The 'Acquisition Stack" shows at the top the exposure time and channel name, which will be saved as metadata (see Fig 5).

• Zooming: when the first channel is acquired, the zoom factor is optimized to show the entire image content on a maximal visible area at the left of the screen. Pressing the "+" key will zoom in at the vicinity of the cursor. You can choose go back to show the entire image via Plugins> Macros> Zoom to Fit [0] or press key 0 (zero).

Fig.5: Channel, frame and exposure are shown at the top of the image

• When the acquisistion stack is full, you are asked to save it. You still can change the name and the suffix index (suffix is number after underscore such as "4" in "MyStack_04.tif"). In this case, the new name and suffix number will be used for the next acquisition stacks.
• When the exposure times needs to be changed, choose Plugins> Macros> Dialog [F5] and change the values.

• You can redo the most recent acquisition by choosing Plugins> Macros> Redo Channel [F2] or press key F2.

• There are commands to operate the shutter: Plugins> Macros> Open Shutter [F6] and Plugins> Macros> Close Shutter [F7]. The shutter can also be operated via the two hardware buttons on the black shutter box.

Customizing the BX macros

Rather than using 'StartupMacros.txt' described above, you can use your own macros with different defaults for channel names and exposure times.
Create your own macro file as follows:
- Choose "Plugin> Macros> Startup Macros..."
- Choose menu File> Save As...>" and save it in your personal folder e.g. as "Modified-BX-macros.txt".
- change the text by entering different settings - note that declarations end with comma, not semicolon - also change the version text
- Save the file again with the changes. - For installing your personal macro file, just choose menu "Plugins> Macros> Install..." and thus replace the current settings with your own ones.
Alternatively, you could simply drag your personal macro file from the Finder/Explorer onto the ImageJ main window, which then shows something like "8 macros installed".

Below is a copy of the current default values:

// begin user-changeable values
    mVersion = "Version N.V. 22-aug-2016",
    defaultField = 2048,  //2048 or 1300
    expArray = split("22 150 330 100 100 100 100 100 100 100"),
    gChnPattern = "bff", 
    gFrames = 4,
    gChannelNames = "",//example: "phc gfp dapi"
    gName = "Collection",
// end user-changeable values

Download adresses: https://sils.fnwi.uva.nl/bcb/BX_2016/download/