CARV Spinning Disk Confocal Leica DMI 6000B microscope equipped with motorized X-Y stage, BD CARV-2 spinning disk confocal, Pecon/Leica cell culture incubator with temperature, humidity, CO2 control, high quantum efficiency cooled back-illuminated CCD camera (Qimaging Rolera-MGi), and integrated software control (BD IPLab). DAPI, GFP, YFP, Texas Red, Cy5 (Regular epifluorescence and confocal). Objectives: 40x/0.60 Ph2 dry and 40x/1.25 NA plan apo oil, 63x/1.40 NA plan apo oil lenses and a 63x/1.20 NA water immersion plan apo lens.

Put oil only on oil lenses and water only on water immersion lens!

Turn on the microscope. Turn on the Carv Spinning Disk fluorescence unit and the regular microscope epifluorescence unit if needed (this unit should be turned on only if you are going to look through eyepieces at your sample otherwise it can be left off).

Turn on the Carv Spinning Disk control unit, the switch is on the back right of the unit. Turn on the Rolera MGI camera, the switch is on the back of the camera. Pick your stage insert for plates or slides and line up the red-dot with the red dot on the stage and make sure it is secure (this takes some practiceangle the insert in while pushing in the springs.

To move the condenser back the top of the incubator moves backward. Objectives can be carefully moved by hand. Touch only the sides. Clean oil and water immersion objectives with Windex and lens paper after use. Some of the objectives also have correction collars that may need to be adjusted to obtain optimal image quality.

Control buttons on left side of the microscope The bottom button switches the microscope into Brightfield. Light intensity can be adjusted with the up and down buttons above this. To move the stage, change objectives, and focus the microscope use the Control pictured below. The front knob controls X &Y stage movement. The top of the knob moves the stage from the front to the back and bottom Right to left. On the right of the control are two buttons for coarse (front) and fine (back) focus. Once pressed the back knob can then be used to bring the sample into focus. Alternatively, the regular coaxial focusing knobs on the left and right side of the microscope can be used. There are also up and down buttons for objectives on the right side of the microscope. On the right of the control there are two buttons that control changing objectives.

Open IPLab software. Go to camera menu and make sure the program recognizes the Rolera MGI camera

There are two F1 icons on the right side of the screen. One is on the bottom And is titled F1 run script. Pressing this button initiates the camera. This should be the fist thing that you do upon entering the software. The other F1 icon on the top of the list entitled Adrian acquire opens up the acquisition menu (see below).

Exposure time can be adjusted between Brightfield and fluorescent filters. Capture the image by pressing snapshot. Image can be saved as a 24-bit TIF or and 8-bit TIF by using the math menu to convert. Images can be saved individually or as a group in file save all in sequential order.

To view the sample through the microscope eyepieces Press the send to eye button on the front panel of the microscope. To send it back to the computer press the send to Camera/computer button. You may also need to press the open shutter button in the center of the panel if it is closed. Left side bottom button will put the microscope in brightfield (see above) and buttons 1-4 will put the following epifluorescent filters in place. Buttons to increase the magnification of the image are on the top left. FILTER LIGHT FLUOROCHROMES A (1)

UV (BP360/40)

DAPI, Hoechst, Alexa 405

N21 (2)

Green light (BP539/45)

TRITC, Texas Red, Alexa 546

I3 (3)

Blue light (BP470/40)

FITC, Alexa 488

YP (4)

YFP

Filter settings for both regular epifluorescence and confocal can be controlled through the IPLab software by pressing the manual Illuminations Icon on the right side of the screen. The filter choices labeled non-confocal are regular epifluorescence while the ones labeled CARV are the Spinning disk settings.

IP LAB Multi Dimensional Acquire XYZ Icon on the upper right side of the screen.

Access through the Camera menu or by the Z Multi-D icon in Camera toolbar. 1. Select camera 2. Set up filter-switcher and/or focus motor using the Select Devices command (control menu). 3. Multi-wavelength acquisitions- you need the filter wheel, liquid crystal tunable filter. 4. For 3D acquisitions, you need the focus motor. Select the Multi Dimensional Acquire command and fill in the options. Begin the acquisition immediately by unchecking the Preview option. Begin the acquisition interactively, using the Preview dialog box to set up 3D acquisition and other parameters while viewing the live image. When you are ready to acquire data, click OK. *If scripting in MDA leave the preview unchecked. Timing window- IPLab will record the time after acquiring each set of colors. For 3D sequences IPLab will record a time for each Z-step.

Display After each grab- when you check this box, each frame of a multiple image acquisition will be displayed as it is captured. Don’t check if concerned about speed. Size tab Portion of CCD and how much binning used.

Full frame ROI-subregion of CCD Draw a ROI on an image around a cell or feature. Select the MDA command and click on this radio button. Select of type the name of the existing image from the drop-down box. Custom Rectangle lets you specify the exact borders of the image that you want to acquire. Binning affects both the resolution of the image (number of pixels representing the data) and the exposure time needed to acquire the image. Less exposure time when binning image.

Shutter & Filters Pick motorized shutters and filter switchers. (ie. Uniblitz shutter). To protect sample from light. Filters (FITC, GFP, etc.). Pick the right hardware. Click enables to choose filter you want to use. Exp. Time- allows you to use a different exposure time with each filter. AE – auto exposure- initial guess. AE retries- number of times IPLab will try to calculate a good exposure time.

Multi-mode imaging (both fluorescence and bright field) 1. Assign the fluorescence and bright field shutters to Shutter 1 and shutter 2. (ie. Shutter 1- fluorescence excitation shutter; Shutter 2- brightfield shutter) 2. Do not check Keep Open During Z/Preview for brightfield.

Z-steps tab 3D sequences (through focus images). Check the Use Z step box to acquire 3D sequences. Relative radio button: Start Pos. and Stop Pos. Absolute radio button measures the start and stop positions from the motor’s orgin.

Time Lapse Tab Frames, Interval, and Experiment Length. Frames: Number of frames or time points that you want to acquire. Interval: Length of time taken by one iteration. Time between starting one grab and starting to grab the next frame. Experiment length: total amount of time taken by the time-lapse experiments.

Check the Use Time Lapse checkbox. 6D Acquire Select camera Select Devices (Control menu)- Set up your filter-switcher and/or focus motor using the Select Devices command.