Upright Microscope System
 
A.                     Introduction :
NOBC has 2 upright microscope systems that provide slide specimen observation and currently are equipped for brightfield, phase contrast, and fluorescence image acquisition.
 
B.                    Instrumentation :
(1) Nikon Optiphot-2 Upright Microscope with Epi-Fluorescence attachment (Objective lens: 4x/0.13, 10x/0.30, 20x/0.50, 40x/0.95, and 100x/1.40/oil)
 
Nikon DXM1200 CCD digital camera
Nikon ACT-1 imaging capture software
 
 
 
 
(2) Leica DM2500 Upright Fluorescence Microscope(Objective lens: 10x/0.30, 20x/0.50, 40x/0.75, and 100x/1.30/oil)
 
 Roper Scientific. CoolSNAP TM Cooled CCD Cameras
 Empix Northern Elipse image analysis workstations
 
   
 
 
C.         Using Guideline :
1.    The fluorescence microscopes are located in the room R2-5241 and open 24 hours for use. Prior account setting by submission of User Account Application Form to the Core research assistant Ms. Shu-Fen Hu (ext. 33702 or 33703) is required.
o    Leica Fluorescence Microscope User Account Application Form
o    Nikon Fluorescence Microscope User Account Application Form
o    NOTE: If the above links appear “not found” or “error occurred,” click the “Refresh” icon to refresh the page.
2.    Users must sign in the log book with confirmation prior to the use of microscope (no matter the mercury lamp has been turned on or not).
3.    Once the mercury lamp has been turned on, it must remain on at least 15 minutes.
4.    Once the mercury lamp has been turned off, it must remain off at least 30 minutes.
5.    After using, the lens must be cleaned by the following procedures: First, wipe off most of the immersion oil with a clean Lens paper, and then use new lens papers moistened with a bit of 95% or above alcohol to Gently re-wipe the lens in a circular motion (only letting the tissue, not your fingers, come into contact with the lens element).
6.    Shutdown procedures – seven important steps:
(1)     Adjust the stage to its lowest position (by turning the coarse adjustment knob).
(2)     Clean the immersion oil lens with certainty if used.
(3)     Rotate the objective turret into the lowest magnification objective position.
(4)     If fluorescence observation is used, unselect the filter cube, close the shutter, and switch the fluorescence illuminator into an empty position.
(5)     Adjust the Kohler illumination to the lowest intensity.
(6)     Turn off the power of light sources (including the mercury lamp).
(7)     Users must log in the using hours and the instrument using information onto the log book after use.
7.     Data saving: Set up a personal folder under D drive (Data drive) and save your data there. Any files saved outside the D drive will not be allowed.
8.     All data saved under the D drive on the system will be periodically purged on the 1st and 15th of each month to create space for newer files (If these dates are a Saturday, Sunday or public holiday, such purge will be expected on the next weekday). Users are responsible for backing up your data.
9.     Users found not in compliance with the using guideline, will be prohibited from using the instrument for at least 1 week or up to 1 month.
10. Prior to your use, if you perceive the instrument was performed improperly, please contact the technical research assistant (Ms. Shu-Fen Hu) immediately. If this unreported improper use was discovered after your use, the liability for this improper use may directly transfer onto you.
 
NOTE: To facilitate all users having a convenient operation environment at NOBC, do not leave behind any personal items, including trashes, and also do not take away anything that belongs to NOBC (such as immersion oil, lens paper, and pens). Thanks for your cooperation.
Had any question or comment regarding the service of Optical Biology Core Laboratory, please contact Ms. Shu-Fen Hu at ext. 33702 or 33703.
 
D.                   Operation :
 
Instrument:
Nikon Optiphot-2 Upright Microscope with Epi-Fluorescence attachment
 
Fluorescence observation operating procedure:
1.     Switch on the fluorescent lamp power supply unit, the green light will be on. Must keep pressing the button for 5 10 sec until seeing the orange light on or glittering and then becoming off. If this step cannot be accomplished, switch off the power and restart the step again. Remember must see the orange light on and off before letting go the button pressing. Wait for about 10 sec, the orange light will be on again and getting stable. Please wait for another 10 minutes to start using.
2.     Adjust the brightfield mercury lamp to the lowest intensity. According to the fluorescence marker used to select an appropriate fluorescence filter cube. Open the shutter to the O position to observe.
3.     Once the fluorescent lamp has been turned on, it must remain on at least 30 minutes. Once it has been turned off, it must remain off at least 30 minutes.
4.     Be aware of the light intensity when using fluorescent UV filter. If the light is too strong, use ND filter.
Microscope operating procedure:
1.     Switch on the light power supply unit, put the specimen on stage, and then set the light intensity.
2.     Use the 4x objective to observe first and focus on the specimen using the coarse adjustment knob. The condenser top is swung out for objective magnifications < 10x.
3.     Adjust the interpupillary distance of the oculars.
4.     Vision adjustment: close left eye to adjust right side and close right eye to adjust left side.
5.     Select the condenser ring diaphragm to objective N.A. value = 70% - 80% position.
6.     Adjust the condenser to its highest position.
7.     Steps of optical axis adjustment: (while a microscope has been moved)
(1)   Focus the specimen using the 10x objective.
(2)   Close the field diaphragm to its minimum.
(3)   Raise or lower the condenser with the condenser height adjuster until the edge of the field diaphragm is sharpest.
(4)   Open the field diaphragm until approaches the field of view.
(5)   If the field of view of the condenser is not centered, it must be moved into the middle of the field of view with the help of the centering blots.
(6)   Open the field diaphragm just enough for it to disappear from the field of view.
8.     Always begin with the lower power objectives. The coarse adjustment knob can be used to adjust 4x and 10x objectives, but must only use the fine adjustment knob to adjust 40x and 100x objectives. This will avoid getting objectives and slide damaged while focusing.
9.     The 40x objective has a correction collar, which can be rotated to adjust image definition.
10. 100x Objective is an oil immersion objective that requires an immersion oil droplet to use. It must be cleaned by using lens papers moistened with 95% or above alcohol to wipe off the lens several times after use.
11. If the immersion oil is used on slide, do not use the 40x objective to observe to avoid the oil rubbing off on the lens. If it does happen, clean the lens with 95% or above alcohol moistened lens papers several times.
 
Image acquisition procedure:
1.     Center the desired image in the field of view and turn the oculars 90 degree clockwise direction to switch on camera.
2.     Turn on the computer and log in to your account. Open the ACT-1 software to start image acquisition.
3.     ACT-1 software user manual.
 
 
 
Instrument:
Leica DM2500 Upright Fluorescence Microscope
 
Fluorescence observation operating procedure:
1.     Switch on the fluorescent lamp power supply unit, the green light of Temp and Safety will be on. Wait for about 5 10 sec until the lamps orange light is on.
2.     Adjust the brightfield mercury lamp to the lowest intensity. According to the fluorescence marker used to select an appropriate fluorescence filter cube. Open the shutter to the O position to observe.
3.     Once the fluorescent lamp has been turned on, it must remain on at least 30 minutes. Once it has been turned off, it must remain off at least 30 minutes.
4.     Be aware of the light intensity when using fluorescent UV filter. If the light is too strong, use ND filter.
Microscope operating procedure:
1.     Switch on the light power supply unit, put the specimen on stage, and then set the light intensity.
2.     Use the 10x objective to observe first and select a corresponding light ring in the condenser when swiveling in the objective for phase contrast. Focus on the specimen using the focus wheel.
3.     Adjust the interpupillary distance of the oculars.
4.     Vision adjustment: close left eye to adjust right side and close right eye to adjust left side.
5.     Select the condenser ring diaphragm to objective N.A. value = 70% - 80% position.
6.     Always begin with the lower power objectives. Select a corresponding light ring in the condenser when swiveling in a suitable objective for phase contrast.
7.     The coarse adjustment knob can be used to adjust 10x objective, but must only use the fine adjustment knob to adjust 20x, 40x, and 100x objectives. This will avoid getting objectives and slide damaged while focusing.
8.     100x Objective is an oil immersion objective that requires an immersion oil droplet to use. It must be cleaned by using lens papers moistened with 95% or above alcohol to wipe off the lens several times after use.
9.     If the immersion oil is used on slide, do not use the 40x objective to observe to avoid the oil rubbing off on the lens. If it does happen, clean the lens with 95% or above alcohol moistened lens papers several times.
 
Image acquisition procedure:
1.     Center the desired image in the field of view and pull out the beam splitter to switch on camera.
2.     Turn on the computer and log in to your account. Open the Northern Elipse software to start image acquisition.
3.     Northern Elipse software user manual.

 

公告日:2011/07/14





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