Cell Biology SP5 II Instructions

Cell Biology SP5 II Instructions
STARTING the Cell Biology SP5 II system: 1‐ Turn on the fluorescent lamp 2‐ Turn on the Left – Green Button (PC/Microscope) first, then the Center – Green Button (Scanner), and last the Right – Green Button (Laser) on your console, then turn on the the Laser Key. 3‐ Wait for Windows to open. 4‐ Log into your profile. 5‐ Double Click on LAS AF icon on your computer and wait for the application window to open (LAS AF will not start if the scanner power button is not on) 6‐ Click on OK in the LAS AF window. 7‐ Wait for initialization to be completed before touching the microscope. ACTIVATE YOUR LASERS: 405 – (405nm) Argon–(458, 476, 488, 496, 514nm) DPSS – (561nm) HeNe 633 – (633nm) 1‐ Click on the Configuration tab. 2‐ Click on laser. 3‐ Activate the laser(s) needed for your experiment by checking the box(es). If you are using the Argon laser, do NOT forget to put the digital
power slider at 15-20%. You should hear the big fan come on. More laser power can be
selected if your sample requires it.
THE MICROSCOPE: 1) Select the objective and put sample on microscope 1‐ Go to Acquire (top left menu bar) 2‐ Go to Objective (center of screen in light Red) 3‐ Choose the 10X objective with the software if the 10X is not in place. 4‐ Put the slide upside down on the stage. 5‐ Pick a fluorescent filter cube using the buttons on the front of the microscope. 6‐ Open the shutter. 2) Focus sample 1‐ Unset the focal plane by pressing the upper and center z‐drive toggle buttons together until the set is removed. 2‐ Focus sample by moving the objective up with the focus knob or with the z‐drive with the toggle button. Start with coarse focus and then shift to fine focus (see Coarse and Fine on the joystick). 3‐ Re‐set the zero by holding the upper and center z‐drive toggle buttons together and it will zero the upper focal plane (see 0µ on digital readout). 3) Change to a higher power objective and add correct immersion fluid for the objective 1‐ Change to a higher power objective using the software under the Objective (center of Beam Path Window in light red) 2‐ Lower the objective. 3‐ Tilt objective on turret by hand. 4‐ Add correct immersion fluid to the objective and turn turret back into place. 5‐ Bring objective up close to focus until fluorescence blinks using fine focus with the focus knob or with the z‐drive button. 6‐ Open shutter and focus on the sample. Choose and center the area of interest. Microscope Microscope (Front) Right Side Selection Buttons Always Never Z Pressed Used Shutter Focusing Knob Fitc Tritc Dapi SETUP for ACQUISITION OF IMAGES: 1‐ With Acquire selected the acquisition will be automatically be on xyz scanning mode. 2‐ The format of your image is automatically displayed in 512x512 pixels = default setting. The speed is automatically chosen at 400 Hz and the image size as well as the pixel size is automatically calculated and displayed. More options are also available for you to select, see below. We encourage new users to try changing the scan format to select a pixel
size appropriate for the image size, objective and zoom used. Less than 200nm
pixel size is optimal (80-100nm would be a good choice). 3‐ Imaging parameters (XY Window) can be changed by opening the drop‐down window. 4‐ Click on the arrowhead. 5‐ In the opened XY window, image format and scanning speed can be changed. A better
understanding of your confocal system will
allow you to modify later scan format and speed
when appropriate. Beam Path Settings 1. Click on Visible to activate lasers (and UV if you also want Dapi) 2. Choose Leica presets appropriate for your sample. This automatically sets each laser power and beam width. You may choose to set the power level and beam width yourself of course or adjust them as necessary. 3. Click Live, adjust zoom, gain, offset. Set z‐position in acquire/acquisition/z‐stack (open with arrow) to see box 4.
to choose top and bottom. Set number of steps Set line average or frame average under XY tab (open with arrow) Choose START to collect image series. View image. Rename, save and export your image. Go to experiment; right click on series to rename. Must rename the folder if you want individual series in their own folders. Images are saved as .lif files. You may export them as .tif’s by right clicking on series to be exported. SHUTDOWN PROCEDURE: 1‐
Save your images on the drive D  Images  Your Folder. Lower the stage and remove the slide. Clean the objective (s) if you used immersion medium (oil, glycerol, or water). Change to the 10X objective with the software. If somebody is waiting for you to leave; 1‐
Exit the Leica program by going to File, then Exit. Logout of your profile, and let another begin at this point. If nobody is waiting for you to leave; 1‐
Exit the Leica program by going to File, then Exit. Go to Start, then ‐ Shutdown. Turn off the Laser Key. Turn off the Left – Green Button (PC/Microscope). Turn off Center – Green Button (Scanner). Turn off the fluorescent lamp. WAIT 10 Minutes for fans to run, then turn off the Right – Green Button (Laser). Cover the microscope and fill out the log book. Notes 1. Capture Image takes a single image. 2. Do not load your slide until after the initialization is complete. For updates, see: http://www.umassmed.edu/3dml 
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