OPERATING INSTRUCTIONS Nikon C2si Confocal Microscope Conventions Through these notes bold font refers to a software button and italics refer to a hardware switch. • The NIS Elements window is flexible, with many different panels available for acquisition, visualization and analysis. The control sections can be minimized to free up image space. All panels can be moved around, docked to each other and closed. To restore them use right click. At the minimum you will need panels C2plus compact GUI, C2Plus Scan Area, and Ni-‐E Pad to image in confocal mode. A. Turn on procedure Everything is connected to power strips that are connected to power strips etc. • Start PC (#1) and wait until you get to the Windows desktop • Turn the power strip (#2) on next to the computer • Push power button on C2 controller (#3) • Press the buttons to turn on only the lasers needed. WAIT for microscope stand to complete initialization • Log in Windows using UIC User account. Pswd: uic • Log in with your iLab UIC account user name and password • Start NIS-‐Elements from Desktop icon • To view by Eye -‐Load the appropriate Optical Configuration (OC) from the top menu bar of either Eye-‐DAPI, Eye -‐GFP or Eye -‐Texas Red to find your sample by eye. Designing an OC is beyond the scope of these notes; please contact UIC staff to create your OC. B. Specimen location • Focus on your sample and move x/y stage to find your area of interest. C. Adjusting basic illumination settings for confocal imaging The software makes extensive use of right click and long click to access settings, when in doubt start there. “X” anywhere resets to the appropriate default. • Select an appropriate confocal OC (numbers only) for your needs. (405-‐488-‐561 for example) • Every OC has one or more channels (laser/filters/detector combination), check the channels you will use, and include TD if desired (it will use the longest laser wavelength when in Ch Series). • Start with a full FOV (no zoom, no cropping) by hitting the red X icon in the C2plus Scan Area panel. Select Pixel Dwell Time (1.9) in a medium size frame (512). • Unselect Averaging and Sequential scanning of the signal. • Frame size (64 to 2048 pixels), dwell time (1.9-‐44.2 µs), scan directionality (uni or bi), and image averaging/integration determine the speed of acquisition in frames per second. • Press Scan, the confocal image appears in the live window. • Search for your field in Z using the Ni-‐E Pad or scrolling the mouse over the active acquisition window (forward away from the sample or towards the Top of the stack, backwards deep into the sample or towards the Bottom of the stack). • Use the sliders in each channel to adjust laser intensity, detector HV (sweet spot 80-‐ 120, noisy >150, a good start is obtained with autogain button, AG). • If there is cross talk between the channels (DAPI into 488 channel is always an example), select Sequence Scanning. This will result in sequential scanning of each channel. • Adjust the frame Size and Zoom to your resolution needs in the C2plus Easy GUI using your brightest channel, keeping in mind Nyquist resolution requirements. Crop out undesired field areas. • You may need to choose Averaging to obtain the appropriate S/N ratio and reduce noise to acceptable levels. Average lines (2x-‐32x), the decrease noise in the image at a cost of acquisition speed. • Press Capture. The single optical section image is acquired but not saved. D. Multidimensional acquisition Acquiring more than one optical section, field or time requires the use of ND acquisition (Nth dimension) software. This software also allows concatenation of several OC that can be applied to the same or different fields. If a simple time lapse or Z-‐series is desired there is no need to use ND Acquisition. • Check Save to File box so acquired images will be automatically saved • • Update the folder to which files will be saved in Path Enter a Filename that will be used as prefix for all acquired files • Experiments that require photo manipulation are performed with the ND Stimulation module. Check Save to File box so acquired images will be automatically saved Update the folder to which files will be saved in Path Enter a Filename that will be used as prefix for all acquired files Select your field using Scan and set up illumination parameters Open Simple ROI Editor Draw a ROI of the appropriate shape and size Right click and choose Use as Stimulation ROI Open ND Stimulation module. The Time schedule window indicates that photomodification and imaging will use the same scanner. This panel allows definition of the Phases or stages of the experiment, with each Phase having its Interval (time between images), Duration (total phase time) and Loops (recycling the duration and interval n times). In addition each phase has a function: Acquisition: regular imaging following the OC Stimulation: Bleaching: Time: allows time lapse image acquisition. Each Phase can have its own Interval (time between images), Duration (total phase time) and Loops (recycling the duration and interval n times. Z: allows imaging different optical sections of a FOV. To define your range you can select the Top and Bottom of your stack. Range gives the number of sections. Select the spacing between the sections using Step. Check the box for Close active Shutter during Z Movement. Lambda: allows combining different OC to sequentially image the same FOV. All of these options can be combined to generate exceedingly complex protocols. Order of Experiment determines the order in which the different modes are implemented and Timing provides an estimate of total acquisition time. Click Run now to perform the Acquisition. E. FRAP & Photoactivation • • • • • • • • • • • • • • • Waiting: In a typical FRAP experiment Phase #1 Acquisition collects baseline followed by Phase #2 bleaching and Phase #3 Acquisition. Once the basic protocol has been programmed open C2Plus Stimulation panel. Up to three ROIs can be bleached differently. Select the lasers and power to stimulate the ROI. In the ND Stimulation module press Apply Stimulation Settings. Press Run Now to launch the experiment. F. Data handling and export The Nikon Elements proprietary software files .nd2 are readable by Imaris and ImageJ/FIJI. Files can be exported as TIFFs. Nikon offers an elements reader software package that allows visualization, LUT adjustment and export of files to TIFF. Available in Apple OS and Windows versions. FIJI is recommended to open the .nd2 formatted images directly. See UIC staff for assistance. All screen views (Captured, frozen…) can be exported as screen shots (Edit>Create View Snapshot). Three dimensional volume renderings can be made into movies showing different angles, in which key frames are assigned. The software interpolates the move magnification and rotations. The Movie creator (easier to use in Director’s cut mode) is a time line movement between the key frames and creates an .nd2 animation file that can then be saved as a n .avi file. • In Settings define total movie length and uncheck Consider movie as a loop. • Select the starting frame and press Mark. • • Select the next time, move the object and mark the key frame. Repeat until all time is used. Remember: No loop, Time-‐Move-‐Mark. G. Turnoff procedure • Transfer saved files to removable media, or server. Do not leave your files on the computer. NEVER save any files to the C: drive. • Close all applications. Select shutdown in reverse order from the Start Menu. • Be sure to record your time completely using the online reservation calendar. • Become rich and famous!