1. 1 QED In Vivo USB Input Output Box configuration This tutorial contains a number of instructions embedded in a great deal of explanation. Procedures that you should perform while going through the tutorial are in bold and red or they are number 1), 2), etc.

2. 2 Login and system startup status screen The system login allows you to select stored system configuration. Each configuration can have a different set of hardware selections. 5) Select the QED Exp IO configuration and click Continue. Start QED In Vivo by clicking the QED In Vivo icon on your desktop. 5) If you see this alert you are running in demo mode. All functionality will work but you will see QED watermarks in all your images.

3. 3 Initial Startup View The initial view shows the acquisition panel. The main QED In Vivo application has 4 areas of interaction. The menu items, the application toolbar and main window and the application modules. Initially no application modules are shown.

4. 4 Configuring the USB IO box for Analog Signals Below you will find the connection settings between the USB IO box and the Digital inputs and outputs controlled by QED In Vivo.

5. 5 Configuring the USB IO box for Digital Signals Below you will find the connection settings between the USB IO box and the Digital inputs and outputs controlled by QED In Vivo.

6. 6 Setup window 1) Select the Setup window to define your camera, microscope and any additional hardware configuration. Change the camera format to improve speed or actual image size. 3) Click the Full Setup options to device multiple microscope components and custom configuration of these components. 2) Select your camera from this dropdown menu. The program will perform system check and alert you if there are any problems with the camera. The calibration settings can be used to match the size of the pixels and the distance between pixels to actual metric values. Select the Setup control in the main dropdown menu which panel contains camera, calibration and microscope setup functions. Select your camera in the list of cameras. The software will initialize the camera and show a message after completing the initialization. Click on the full setup window to get access to the Microscope Configuration window where you can define your hardware configuration. The setup tool allows you to define your system configuration, including the camera and microscope hardware.

7. 7 Full Microscope Configuration window 1) Select the AOTF device as a Filter wheel and use the extern IO box to control your device through the USB box. In this section we will setup a general automated microscope with the following options: multicolor fluorescence, XY scanning stage and a Z stepper motor. This configuration will allow you to acquire individual color images or full slide scans in time and XYZ space. After selecting your controllers you will be able to define the final settings for each device. Click on the Settings button for each device to do this. The next pages will describe how to define the settings for each device. We will use the Microscope Configuration window to define the individual settings for each device. 2) Select the AOTF device as a Fluorescence shutter and use the extern IO box to control your device through the USB box. 3) Select the Piezo focus device you want to control with the USB box. Select External IO Box as the connect by option.

8. 8 AOTF and Laser Control window You can have QED In Vivo select the Emission color, Fluorescence color, Names and the corresponding wavelengths (this is done using the Spectrum Color Selection window). It also allows you to test the filter wheel and your settings. You can select the excitation and emission colors automatically by selecting a Dye, Excitation Color or Laser Line. You can name the selected filter position and also selected an abbreviated name for it. Start QED In Vivo toolbar open the AOTF and Laser tool by clicking the rainbow icon.

9. 9 Spectrum Color Selection window You can select the excitation and emission colors automatically by selecting a Dye, Excitation Color or Laser Line. You can name the selected filter position and also selected an abbreviated name for it. The colors can also manually be selected by typing the wavelengths in this section. In this window you can define individual emission and excitation colors. The window works with filter or laser lines from the various instruments. For some devices changed the emission Color also changes the settings on the device (i.e. monochromators).

10. 10 Microscope Control Window Close and Open the shutter by clicking on the shutter icon. The time it takes to open the shutter can be defined by Ctrl + click on the shutter icon. Change the postion by selecting a different filter. The number of laser lines/positions and type of filter can be defined by Ctrl + click on the filter icon. The QED Microscope Control Module provides interactive and automated control for a variety of microscope systems.Software controls include multiple shutters, filterwheels, filtercube changers, scanning stage, focus control, objective selection, etc.

11. 11 Configuring a Piezo focus device The Piezo focus device is controlled through the first Analog Output in the USB IO box. After you configure the Piezo device in the microscope configuration window you can control the Analog output from the 3D Acquire tool. 1) Click the cycle button to see a voltage cycle between 0-5 or 0- 10 Volts in Pin# 19. In the microscope configuration window you can set the travel range of the piezo. This range will correspond with a 0-5 oir 0- 10 Voltage range. 2) Change the Step size will change the step in Voltage 3) Change the number of planes will change the min and max voltage values generated.

12. 12 Imaging Modes window Save your settings as the default For future session. In this window adds easy multi-color fluorescence acquisition. It can automatically acquire up to six arbitrary color channels, each with Its own specific settings. Each channel has independent binning, gain and averaging settings. They can be adjusted interactively or automatically with on-the-fly results in the color composite. Save your settings as the default For future session. Save your settings as the default For future session. Save your settings as the default For future session. Save your settings as the default For future session. Define the number of imaging Modes/channels. Acquire the individual channel. Define the exposure time for the individual Imaging Mode/Channel. Display the associated microscope configuration. Define the gain for this Imaging Mode/Channel Define the microscope configuration associated with the Imaging Mode. Acquire all channels. Select to acquire the individual Imaging Mode.

13. 13 Time Lapse window In Time Lapse window is designed for live cell microscopy. The window includes variable speed image acquisition and playback functions. It presences up to 16 bit, high resolution, image data and is easy to use. It allows to change the acquisition settings on the fly and can be coupled to external triggers for burst acquisitions. Define the acquisition period in either time points or actual time. Enable the color acquisition during a time lapse. Includes a short cut to the color options window. Define acquisition interval, this can include custom acquisition intervals. Prepare and define the burst acquisition parameters. All images will be acquired using the Quality acquisition settings or the Imaging Modes settings. Start or append a new acquisition to an Existing one.

14. 14 Startup View after module selection After configuring the hardware and defining the various acquisition modes you should be able to create the following QED In Vivo application view.

15. 15 Trend Chart window Select the trend feature you want to calculate and plot. Export your results to Excel or other data analysis packages. Compute the trend on a previously acquired movie. Use one of the 4 trend modes. You need to define an area before you can select a mode. In Trend Chart window can display various measurements or Regions of Interest of complete images as a graphical representation. The plots can be computed on a live acquisition or on previously collected movies. The shapes of the ROI are define by the user. Selecting the draw button enables the drawing tool in the image window. Define the shape of your Region of Interest. 1) By checking off the various analog inputs the trend of these values will be plotter in the Trend Chart tool.

16. 16 Using External Triggers An external TTL signal can trigger an event in a Time Lapse acquisition. The most common one is to trigger a burst acquisition. Click the More Options button in the Time Lapse tool and select the Start Burst Acquisition on External Trigger to control the starting point of a burst by your external device.

17. 17 Using Internal Triggers QED In Vivo can trigger an event in your device. The most common one is to trigger a change is state of your device at a predefined time point. Click the More Options button in the Time Lapse tool and select the Assert Trigger at Time point to control the starting the change of state of your external device at a preset time point in your time lapse.

18. 18 Signals and Actions window In the Signals and systems tool we can define a “Feature” in a Region of Interest in our image that will trigger a burst acquisition. The different arguments are interconnected within the 3 windows. The feature can be the Ratio between 2 DAQs that, once it goes below a certain values, will trigger the event. The Signals and Action window works in conjunction with the Analog & Digital IO tool. Select the first 2 DAQs as the input and select Ratio in the Trend Tool.

19. 19 Spectral Sweep Using the USB IO box you can also control devices that take can be used to perform a spectral sweep. Refer to the Spectral Sweep documentation one how to use this tool.