Nikon E800 Macro‐imaging Microscope Short Manual This microscope is equipped with three condensers that support 0.5x, 2x, 4x and 10‐100x objectives and thereby allow both macro‐ and micro‐imaging of samples by bright field and fluorescence microscopy. 1. Turn the power supply on. 2. Turn the microscope on using the dial on the front of the microscope. Select DIA for transmitted light and EPI for epifluorescence. If using fluorescence, turn on the power supply for FL lamp. Press ignition button firmly to ignite the lamp. When the lamp ready light is lit, the lamp is ready to use. 3. Turn on the computer. 4. For transmitted light imaging, set the lamp intensity. The lamp voltage adjustment is on the left hand side of the microscope. 1 Neutral Density filters, for reducing the light intensity, are on the far left hand side of the microscope. The lamp intensity can be “preset” or set manually. NB. Keep the ND filters and lamp intensity the same between experiments. 5. How to set‐up for Köhler illumination: a. For bright field microscopy use the DIA‐
ILL filter setting ie no filter. b. Close down the field stop on the right hand side of the microscope c. Choose the correct condenser for the objective that you are using. d. Focus the image of the field diaphragm using the condenser focussing knob. Centre the diaphragm using the centering screws on either side of the condenser lens. 2 e. Remove the eye piece and close down the aperture stop to about 2/3rds. 8. If you require macro‐imaging, use the macro lens x0.5. This will take an image the size of approx. one third of slide. NB. Ensure that you are using the correct condenser To see an image you need to push in the “macro Slider” pin under the eyepieces 9. Image acquisition: Open the Leica LAS software. 10. Pull the pin out on the top right hand side of the microscope to send the light to the camera for imaging. 11. The software will start‐up using the last archive open. Click on the setup tab, then the archive tab. Select an existing archive by double clicking on one from the list. Alternatively create a new archive by clicking on the plus icon, then choose “create a new archive”. Name the archive and ensure that it is being saved in the right place. (The archives are saved on the C:/ drive under databases). Then click on save. 3 Thumbnails of the images in the archive are displayed under the current image in the “Acquire” or “Browse” window. 12. Under the Acquire tab, go to the Mic1 tab and select the objective you are using. 13. Set the exposure Set auto exposure by clicking on the round wheel icon Alternatively you can manually adjust the exposure by dragging the slider bar or clicking on the exposure bar and then using the mouse wheel to adjust. The “Saturation” and “Gamma” can be adjusted to improve the image. The Gain should be at 1.0x for bright‐field imaging (usually grayed out). However it can be adjusted for fluorescent images. 4 14. Input option The standard frame size is 1360 x 1024 pixels. For fluorescence you can select to have the live window in a smaller, low‐resolution format, which is useful to prevent bleaching your sample. 15. Histogram The blue region on the histogram is the black level. The red region on the histogram indicates saturation or overexposure. To activate/show the dynamic range indicator tick the box “show under/over exposure” under the histogram 16. Processing ‐ Shading correction Move to a clean part of your slide or use a clean blank slide. Adjust the exposure to make sure that the image on the screen is not overexposed. Choose “create a shading reference” – at the end of the long list. When you name it use your name, the objective and your light settings, eg light ‐6, ND filter x2. Save a shading correction for each objective that you wish to use. Remember to use the same light settings. 17. Scale bar. Scale bar settings here are for display purposes only. 18. White balance: Click on the icon beside the automatic exposure icon for a whole image automatic white balance. Alternatively, draw a region of interest over an area that should be white by holding the left mouse button and dragging a box. Then select white balance. The image on the screen should be the same as you see through the eyepieces. 5 19. Click on acquire image on the bottom left of the screen. It will prompt you for the objective and the name of the image. 20. Fluorescence imaging: Turn the dial at the front of the microscope to EPI for epifluorescence. a. The pin on the right of the microscope is the shutter for fluorescence. Behind that are ND filters which can be used if the samples is bright or bleaches. b. NB. The macro slider must be out, unless you are using the 0.5 x objective. c. Choose the required filter set on the microscope – Cy5, FITC, Cy3 or DAPI. d. When setting the exposure to take a fluorescent image, a weak fluorescent signal can be enhanced by increasing the gain in the “exposure adjust” window. 21. Scale bars To make the scale bar permanent go to Process and click on Annotate. a. Select show in the scale bar window. b. Click on merge in the actions window. 6 c. Select create duplicate otherwise the original file is replaced with the image containing the permanent scale bar. d. You can drag the scale bar to any position on the image. 22. Saving Images NB. The archive saves only thumbnails in the C:\ drive. a. To save images, export to the H:\ drive by clicking on the export icon shown on the image window under browse. b. Name the image and ensure that it is saved in the right folder (H:\NIKON E800 USER\ month). c. Use the Browse tab to scroll through all the images within the archive. d. If delete thumbnails from the archive, they are not deleted from the H:\ drive. e. By selecting “Options” then “Preferences” on the tool bar a default name, file format (eg tiff) can be pre‐selected. 23. Turn off the System a. Save all your images. b. Exit the Leica LAS software. c. Turn off the computer. d. Turn off the microscope (DIA/EPI to OFF). e. If you use the FL lamp for your session, please contact the next user to confirm if FL lamp would be used before turning it off. f. Turn off the power supply. g. Cover the microscope. h. Log your instrument usage and report any problems. 7