Transcript
LSM Manual SWITCH ON 1. Switch on the power (grey box on the left). 2. Switch on the computer. 3. Log in
PROGRAM 4. Start program LSM-FCS (its shortcut is on the desktop). The following window will open
5. Choose Scan new images 6. Choose Expert mode
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7. The system will start initialization.
8. The new window will open:
START THE LASERS 9. First press Acquire. Then start the lasers – press Laser button – new window will open. (You should do this half an hour before starting taking pictures).
10. Choose Argon/2: first Standby and after it warms up press On. Change output to 5560% (about 6.7 A). 11. Choose HeNe1 and press On. 12. Switch on the HBO1 lamp (white box on the right).
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LOAD YOUR SAMPLE 13. In the drawer there are different types of the holders, choose the one that your sample fits the best to. 14. Place sample under microscope – to do this press Stage in the main window:
15. The new window will open:
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16. To load sample press Load (you must not open microscope without pressing the Load button), then open microscope (use the little white cylinder at the top to move the top of the microscope back). 17. Place slide and lock it with the little metal holders. Use water or immersion oil if they are required. 18. Move the top of the microscope back into position. 19. When you finish placing the sample press Work.
MICROSCOPE STARTUP 20. Press Micro in the main window:
21. The new window will open:
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22. Choose objective – to do this press Objective:
23. You should always start with 10x objective, than you can change it for the other. For 40x/1.2 W corr you should cover your specimen with water, for 63x/1.4 Oil and 100x/1.4!Oil you should use immersion oil.
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24. Next step is to use proper condensor (the condensor depends on the objective) – press Condensor. 25. The new window will open:
26. Choose VIS in the main window
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27. Press transmitted light in the Microscope Control window, and press On
28. Increase light intensity to 2-3. 29. Find your cells under microscope.
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KOEHLER PROCEDURE 30. Koehler procedure (Koehler rule: The light intensity in all fields you can see must be the same): •
Push the silver bar on the top of the microscope (just underneath the LCD display) to the back
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Center using the two thin centering knobs
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Large black knob on the right to focus the image of the iris – so that the edges are sharp
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Open with the silver bar so that the edges just disappear
31. Change objective to be able to see your cells. 32. Choose the right filter in the Microscope Control window:
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33. Press Config in the main window:
34. The new window will open:
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ONE COLOR 35. Check Single Track. 36. Press Config and the next window will open:
37. You can choose the dye that you used for staining your cells. Then press Apply. 38. To see the fluorescence of your sample press Micro in the main window. The Microscope Control window will open.
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Assure yourself that the room is dark, the darker the room the better you can see. Switch off Transmitted Light. Press Reflected Light in the Microscope Control window – this allows you to see fluorescence of your sample. Do not keep it on too long, because it causes bleaching of your sample. If you want to change objective or take picture always switch off both Transmitted Light and Reflected Light. 39. To take pictures press Scan in the main window:
40. The Scan Control window will open:
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41. First you will do pre-scanning. Press Mode and set as follows: •
Frame size – 1024
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Scan Speed – max
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Data Depth – 8 bit
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Mode – Mean
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Method – Line
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Number – 1
42. Press Channel in the Scan Control window and new window will open:
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43. Set as follows: •
Pinhole – 1 (to be perfectly confocal)
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Choose proper laser wavelength
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Detector Gain – depends on the sample, you can adjust it during pre-scanning
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Amplifier Offset - depends on sample, shows background, you should find equilibrium between sample and background
44. Press Cont in the Scan Control window – your sample will be continuously scanned, you can change Detector gain and Amplifier Offset buttons to find equilibrium between sample and the background.
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45. In the image window choose Palette. New window will open:
46. Check Range Indicator. In perfect equilibrium the image should be black and white.
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47. If there are any red fragments decrease Detector Gain, if blue – Amplifier Offset. Normally we do not use Amplifier Gain. 48. Press Palette again and choose No Palette. 49. Now you can scan your sample. Press Mode in the Scan Control window and set as follows: •
Frame size – 1024
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Scan Speed – 6-7
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Data Depth – 8 bit
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Mode – Mean
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Method – Line
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Number – 8
50. Press Single in the Scan Control window – do not touch anything during scanning. 51. To place dimension bar on your picture check Overlay in the image window, press symbol of the bar and place it on your image.
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TO SAVE THE IMAGE 52. To save your picture: •
Press Save as (next to the image)
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Find your database or create a new one.
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Save your picture
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53. If you have your picture saved you can see it in your data base.
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54. If you want to see all your pictures press Gallery:
55. You can delete the image by pressing Delete. New window will open:
56. To delete your image press Yes.
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PHASE CONTRAST 57. Taking the corresponding phase contrast image – press Config in the main window:
58. The Configuration Control window will open:
59. Un-check color channels (Ch2, Ch3) and press ChD
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60. Press Scan in the main window:
61. The Scan Control window will appear:
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62. First you will do pre-scanning. Press Mode and set as follows: •
Frame size – 1024
•
Scan Speed – max
•
Data Depth – 8 bit
•
Mode – Mean
•
Method – Line
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Number – 1
63. Press Channel in the Scan Control window and set as follows: •
Detector Gain – depends on the sample, you can adjust it during pre-scanning
•
Amplifier Offset - depends on sample, shows background, you should find equilibrium between sample and background
64. Press Cont in the Scan Control window – your sample will be continuously scanned, you should use the same settings for Detector gain and Amplifier Offset as for the fluorescence image. To do this press Reuse button. 65. In the image window choose Palette and Range Indicator. In perfect equilibrium the image should be black and white. 66. Press Palette again and choose No Palette. 67. Now you can scan your sample. Press Mode in the Scan Control window and set as follows: •
Frame size – 1024
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Scan Speed – 6-7
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Data Depth – 8 bit
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Mode – Mean
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Method – Line
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Number – 4
68. Press Single in the Scan Control window – do not touch anything during scanning. 69. Save your image.
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TWO COLORS 70. Check Multi Track in the Configuration Control window. 71. For each channel choose proper dye. Press Config and the next window will open:
72. You can choose the dye that you used for staining your cells. Then press Apply. 73. To take pictures press Scan in the main window:
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74. The Scan Control window will open:
75. First you will do pre-scanning. Press Mode and set as follows: •
Frame size – 1024
•
Scan Speed – max
•
Data Depth – 8 bit
•
Mode – Mean
•
Method – Line
•
Number – 1
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76. Press Channel in the Scan Control window and new window will open:
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77. Set for each dye chosen as follows: •
Pinhole – 1 (to be perfectly confocal)
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Choose proper laser wavelength
•
Detector Gain – depends on the sample, you can adjust it during pre-scanning
•
Amplifier Offset - depends on sample, shows background, you should find equilibrium between sample and background
78. Press Cont in the Scan Control window – your sample will be continuously scanned, you can change Detector gain and Amplifier Offset buttons to find equilibrium between sample and the background.
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79. To see each channel separately press Split x-y.
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80. In the image window choose Palette. Check Range Indicator. In perfect equilibrium the image should be black and white.
81. If there are any red fragments decrease Detector Gain, if blue – Amplifier Offset. Normally we do not use Amplifier Gain. 82. Press Palette again and choose No Palette. 83. Now you can scan your sample. Proceed as for the Single Track settings.
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TO SWITCH OFF 84. To switch off press Laser in the main window:
85. The Laser Control window will open:
86. Choose Argon/2: first Standby and after few minutes press Off. 87. Choose HeNe1 and press Off. 88. Wait 5-10 minutes to cool down the lasers. 89. Close all windows. 90. Shout down the computer. 91. Switch off HBO1 lamp. 92. Switch off the power. 93. Enter session details in the log book.
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