OPERATING INSTRUCTIONS Nikon TiE Deconvolution Microscope CCRB 1-220G

Transcription

1 OPERATING INSTRUCTIONS Nikon TiE Deconvolution Microscope CCRB 1-220G Conventions Through these notes bold font refers to a software button and italics refer to a hardware switch. 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. A. Turn on procedure Everything is connected to power strips that are connected to power strips etc. Turn on the power strip (#1) next to the computer Start PC (#2) and WAIT until you get to the Windows desktop Turn on Ibidi 37 C/CO 2 environmental controller if needed (note: system requires ~30 min to equilibrate). Install the correct stage insert for your sample. 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 Getting Started with Elements: Load the appropriate optical configuration (OC) from the top menu bar or under Calibration> Optical Configurations. Designing an OC is beyond the scope of these notes; please contact UIC staff to create your OC B. Specimen location Place sample on stage Place proper immersion media on the coverslip as required. Select the appropriate OC from selection across the top menu.

2 Select the appropriate objective. Be sure to adjust the correction collar on 20x and 40x objectives. Click live image button to activate light source. Select E100 to find your sample by eye or Click the Eye button on the front of the microscope Focus and compose sample. Move stage with joystick Select L100 port to use Camera Make adjustments to binning, gain and exposure time as needed under Zyla Settings. Click capture image for single picture. Save as.nd2 format or.tif format. C. ND Multidimensional acquisition Acquiring more than one image, wavelength, 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 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.

3 XY: allows imaging of different positions within the sample. Clear all preexisting coordinates. To select a FOV coordinates press +. You can change the imaging order ( ), Save the coordinates or Load previously saved coordinates (encoded stage). Once you are done clear the coordinates from the XY tab. Custom: can be used to scan predefined patterns such as multi- welled plates. 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 concatenating different OC to image the same FOV or separate FOV when combined with XY. 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. D. Scanning (tiling) of large images This option allows scanning of large areas, including whole histology slides. Select a representative FOV in contrast and brightness. If using Shading Correction this must be done before opening the Scan Large Image screen. To set up shading correction, find an area in the FOV that is relatively clear of fluorescence and go out of focus. Under Acquire> Shading Correction> Capture Correction Image click Next and move the stage to another blank area. Click Next again to capture the next image. When done with Shading Correction click Finish. This will equalize intensities of the large image so the lines between the individual images aren't as obvious. PFS (Perfect Focus) must also be turned on before entering Scan Large Image. This can be used in any objective other than the 10x and is located in the Ti Pad. Under PFS click Offset and the Z plane coordinates will be locked in. The On button should turn green. To change the coordinates, the PFS Offset Controller has to be used instead of the stage controls.

4 Acquire> Scan Large Image opens a navigation window. Zoom in and out of the FOV scrolling the mouse. Select the desired OC (typically current) and Objective (same for Macro Image and Scanning). The area to be imaged can be selected by number of fields in X and Y or by defining the edges of the area (recommended) in the dropdown box under Area. Press the green Play button in the lower left hand corner to view the live image. Navigate with the Nikon Ti- S- Ejoy to the right edge of the desired area. Press the black right arrow in the Area panel. Repeat for left, top, and bottom edges. The Stage Overview shows a grid with the individual images to be captured. Double click on any frame to move to that area and confirm your coordinates. Adjust as necessary. Select Overlap: 15% and Stitching via: Blending. You can save individual images as TIFF files and/or create a large image in the lower left hand corner of the pop up window. Select Save Large Image to file, Keep file opened, and select a saving path and Filename for the large image. If your sample is not flat or parallel to the focal plane the system will rapidly lose focus. You can Focus manually every few frames or define a Focus surface (minimum three points). For Multichannel check the appropriate boxes. Press Scan. The microscope acquires and stitches the individual FOV into a large image. NOTE: Before and after you use any of the ND configurations make sure to clear any coordinates in the XY tab. Failing to do so can result in permanent damage to your sample (?), the stage ($$$$) and the objective ($$$$$). E. Data handling and export The orginal data format for Nikon Elements is a proprietary software extension (.nd2) and the files are directly readable by Imaris and ImageJ/FIJI. Files can also be exported as TIFFs. Nikon offers an Elements reader software package that allows visualization, LUT adjustment, and export of files to TIFF. Elements Viewer and FIJI are vailable in Apple OS and Windows versions from our website. All screen views (Captured, frozen ) can be exported as screen shots under 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 between the key frames and creates an.nd2 animation file that can then be saved as a Quicktime (.mov) or WMP (.avi) file. In Settings define total movie length and uncheck Consider movie as a loop.

5 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. For your computer Download FIJI. F. Turnoff procedure Make sure to clear any coordinates from the XY tab and Focus surface. 1. Transfer saved files to removable media, or server. Do not leave your files on the computer. NEVER save any files to the C: drive. 2. Close all applications. Select shutdown in reverse order from Start Menu. This will also log you out of ilab. 3. Become rich and famous!