Nikon A1-B Confocal Operating Manual. Start-up. Microscope

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1 Nikon A1-B Confocal Operating Manual Start-up 1. Turn on Excite 120 LED power supply. a. No need for cool down as for mercury bulb b. Open shutter by pushing down contol knob. c. Adjust intensity by turning control knob. 2. Turn on power strip to turn on a. Transmitted light b. Stage controls c. Microscope-wait for microscope to initialize. 3. Turn on Nikon A1 controller. This is the control box for the confocal. Wait for the top light to stop blinking and turn green. 4. Turn on the laser control module by turning the Laser Power key to ON. 5. Push the button for each laser line that you would like to use. Buttons illiminate. Deselect any laser lines that you do not want to use. 6. Turn on the computer and monitor. 7. Login using your uniquename and level 1 Kerberos password. 8. Activate the NIS-Elements-confocal software. (Note-microscope must be initialized before activating NIS-Elements confocal.) a. Select desired screen lighting dark or light. b. Arrange controls panels as desired. 1. Ti Pad for microscope controls 2. A1plus Compact GUI (graphic user interface) 3. A1plus Scan Area c. If any for the control panels are missing, you can find them by right mouse clicking on the screen>acquisitions Control>A1plus Compact GUI or A1plus Scan Area or TiPad. Microscope 1. Tilt back tower for easy access to the stage. 2. Objective lenses a. Three ways of turning 1) By hand 2) With toggle switch on left of scope base 3) With software Ti Pad. b. 10X dry c. 20X dry d. 40X oil immersion e. 60X oil immersion

2 f. 60X water immersion g. (40X water immersion available by special request.) 3. Mount slide. (Move only left-hand slider on stage insert.) 4. Filter cubes a. Rotate with toggle switch on right of scope base or use TiPad. 1) DAPI 2) FITC/GFP 3) TxRed 4) Analyzer (for DIC) 5) A1 (for confocal) b. See readout on front of scope. 1) Display-arrow up or down to select desired readout. Tells which filter cube is in. 2) Brightness button for display c. Open fluorescent light source using push knob on control module. Adjust intensity by turning knob. d. Shutter button on right side of microscope base is non-functional. 5. Focus three ways to focus a. Knob on microscope base--toggle to coarse, fine, or extra-fine b. Knob on stage control-- Toggle to coarse, fine, or extra-fine c. In software using mouse wheel 6. Find region of interest using stage control joystick. Twist joystick to select movement as coarse, fine, or extra-fine. 7. Focus 8. Push Reset button on front of microscope. This sets the Z reading to 0, the working position. 9. To change to load position, push Escape on right side of scope. Push up button to return to the working position. 10. Transmitted light a. Turn on halogen light with software-tipad. 1) Click on DIA rectangle. Rectangle turns from green to red. 2) Click on yellow dot. 3) Use slider to adjust light level.

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5 d. Click on range indicator dropdown list and select color for offset or black pixels. For example, you can choose blue to indicate black pixels. 13. Optimize light levels in each channel. a. Turn off all but one channel by unchecking them in GUI. b. Press Scan. c. Focus to find the channel s brightest focal plane. To focus, hover over the image and turn the mouse wheel. d. Hover mouse over HV control and turn wheel to adjust intensity up or down. e. Hover mouse over offset control and turn wheel to bring in black levels. f. Turn on next channel and turn off previous channel. g. Repeat light level adjustments for each channel. 14. Turn off range indicator. a. In top toolbar, turn off range indicator. b. Change back from grayscale to color by clicking below the images, bottom left, right mouse click on one of the channels>all color. 15. Select number of integrations. Click on integration icon to remove scan from Normal and change to Integrated mode. Click on the dropdown list to select number of scans, e.g., 4 or Capture image. Press on this icon to capture final image. Saving a single plane image 1. First, save as an ND2 Image File Format. This saves the header information needed for analysis software. a. File>Save As b. Establish path User folder>new folder titled with your unique name>new subfolder titled with today s date. c. Select file type ND2 Image File Format( *nd2) d. Type in file name e. Save all channels. (Or save selected channels if desired.) f. Save 2. Saving individual channels in Tif format. ND2 documents cannot be opened in Adobe Photoshop. This procedure automatically converts the images from 12 bit to 8 bit. a. File>Import Export>Export ND Document to tiff. b. Make sure correct path is chosen (Browse). Make Tif folder if desired. c. Select file type Tif. d. Click on Color Image for Each Channel. e. Check LUT box. f. Check convert 12 bit to 8 g. Click on Export. h. Software automatically adds a channel suffix to each image.

6 3. Saving merged image in Tif format. a. File>Import Export>ND Export to Tiff. b. Make sure correct path is chosen (Browse). Make Tif folder if desired. c. Select file type Tif. d. Click on Merged color image. e. Check LUT box. f. Check convert 12 bit to 8 bit. g. Click on Export. 4. Another way to save image as a Tif with slightly less resolution - a. Press x on the computer keyboard. This takes a snapshot of the display and automatically converts it to 8 bit. b. File>Save As c. Make sure correct path is selected (Browse). d. Select Tif format. e. Type in File name. f. Save 5. Saving display of all channels plus merged together in one panel. a. Set up display as desired with all channels plus merged image (e.g.four squares). b. Take a snapshot and save as for items 4 above. Adding a Scale Bar 1. On right side of image select scale bar icon. 2. With dropdown arrow, select properties. 3. Select desired line width, color, and font. 4. Select Burn into image or not. Setting up for a Z-Stack 1. Find ND acquisition panel. If it not already on desktop, right mouse click on desktop>acquisition Controls>ND Acquisition. 2. Deselect Save to File. (Otherwise, this would be a redundant copy of your Z-stack.) 3. Deselect time (or other) acquisition modes, select Z. 4. Define upper and then lower boundaries of the Z-stack. a. Change from Kalman to Normal scan mode. b. Press Scan.

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9 7. Raise objective turret to working position. 8. Check MIL scheduling system to see if someone will be coming onto the scope after you. 9. Log off of the computer. 10. If you are the last user of the day, shut down in reverse order of the start-up. 6) and 5) Shut down computer and turn off monitor. 4) Leave laser line buttons illuminated. Turn off Laser Power key 3) Turn off Nikon A1 Controller. 2) Turn off Power Strip #2. 1) Turn off LED 120 power supply.

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