Getting Started

After you have finished constructing your macroscope, connect the camera and stage’s USBs to your computer.

1. Opening up the GUI
2. Connect to the stage
3. Connect to the camera
4. Calibrate the camera and stage relationship
5. Dual-color calibration (optional)
6. Set recording file path

Opening up the GUI

Connect to the stage

If you have never connected to the Zaber stage before, install and open Zaber Launcher. In there, you can find the connection port name to your stage, usually COM3 for Windows or /dev/ttyUSB0 for Linux, and you can also update your stage firmware to the latest version. Afterward, close the Zaber Launcher and open up the GlowTracker. Open the Settings , put your connection port name in the section Stage, field Stage serial port. Close the settings, then click the Stage . If the button turns green then you have successfully connected the stage.

You can now control the stage movement by the stage control buttons (GUI image: Group 2) or the arrow keys on your keyboard. The left ⇦, and right ⇨ keys control the stage X axis, ⇧ ⇩ control the stage Y axis, and Page Up, Page Down, raise and lower the stage. Press these in combination with the Shift key to move at a slower speed.

Connect to the camera

If you have never connected to the Basler camera before, install and open the pylon Viewer. In there, you can test the connection to your camera, specify your settings such as exposure time and gain, and then export your camera setting file as .pfs. Open the Settings , put your connection port name in the section Camera, field Default camera settings. Close the settings, then click the Camera . If the button turns green then you have successfully connected the camera. You can view from the camera by pressing the live view button.

Calibrate the camera and stage relationship

Calibration is essential to translate the motion of the object of interest in the image to the compensatory motion of the stage, that centers the object.

1. Select a sample that shows a lot of structure, for example, a ruler, or a drop of fluorescent pigment on a slide.

   Fluorescent drop on a slide

   Placing the slide in the center of the objective

2. Open the Calibrate window . Navigate to the tab Camera & Stage Calibration.

3. Click Calibrate. The resulting pixel size and rotation of the camera will be shown, along with a plot display of the camera space and stage space.

   

   Camera and Stage calibration result

4. You can test if the calibration is correct by turning on the Move in image space mode option in the Settings under Stage section. After turning it on, when you move the stage using the arrow keys ⇦⇨⇧⇩, the resulting image should also be moved accordingly and intuitively, e.g. pressing ⇧ should move the image up, and pressing ⇨ should also move the image to the right.

Turn on the "Move in image space mode setting"

Dual-color calibration (optional)

(this step is only required in the dual-color configuration.) To calibrate the relationship between the two color channels, which will later allow an accurate overlay of the two images, the image-splitter needs to be calibrated.

1. Select a sample that shows either the same structure in both channels (e.g., fluorescent beads, fluorescent tape) or that has sufficient bleed-through to appear in both channels.

   Fluorescent stripes on a slide

   Placing the slide in the center of the objective

2. Open the calibration dialog by clicking on Calibrate . Navigate the tab to the section called Dual Color Calibration.

3. Click Calibrate. The calibration result will be shown in the overlay.

   

   Dual-color calibration result

Set recording file path

To pick where your image files and recording data will be stored, select a location using the file dialog in .