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Byeong Cha
Enter you name, the date, the time, and the account number in the user log book.
Things to check before start-up.
• Make sure that your sample slides are clean and sealed. Use Windex and cotton balls or Kimwipes to clean
coverslips. Fixed samples need to be sealed with nail polish.
• Check the objectives. The 10x and 20x objectives are DRY objectives, and should NEVER have oil on them!
There are 40x and 63x OIL immersion objectives, and a 63x WATER immersion objective also on this
microscope. If oil immersion objectives have oil on them, wipe the lens gently only with lens paper. With
the microscope controller
at the front of the microscope. The order of
the objective lenses in the lens turret is:10X, 20X,
40X Oil, 63X Oil, and 63X Water.
• The 63X objective has aperture collar adjustment
rings that control the size of aperture opening. It
should be wide open for most imaging condition.
If your sample looks dim or the whole view field
is not illuminated evenly when you look through
the eyepieces, turn the collar to open the aperture.
• The field diaphragm and aperture diaphragm in
the excitation path of the mercury lamp are
controlled by round, black dials on the left side
toward the back of the microscope, and control
the fluorescence illumination from the mercury
lamp. For all imaging with the mercury light
source, these should be set wide open by turning all the way counter-clockwise.
Setting up for bright-field and fluorescence
viewing.
Basic Operation of Leica DM IRE2 Microsocpe.
Turn on the microscope controller
Turn on the mercury arc lamp
Bright-field viewing:
Place your sample slide with coverslip (preferably #1.5
thickness) facing down to the objective on the stage.
For bright-field viewing, turn on the microscope light by turning the wheel
toward you and turn the VIS-SCAN switch
switch back to Scan position for confocal scanning, see below)
Start with low magnification objectives (10X or 20X) first to find and focus
onto your specimen. The information of the selected objective and their z-
Page 1
Manual for Leica SP2 Confocal Microscope
ON, you see the information of the objective in position on the reading panel
.
.
to VIS position (Note:
11/24/2009
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Summary of Contents for Leica SP2
- Page 1 Byeong Cha Page 1 11/24/2009 Manual for Leica SP2 Confocal Microscope Enter you name, the date, the time, and the account number in the user log book. Things to check before start-up. • Make sure that your sample slides are clean and sealed. Use Windex and cotton balls or Kimwipes to clean coverslips.
- Page 2 Byeong Cha Page 2 11/24/2009 position can be read on the readout panel at the front of the scope. You can place the desired objective in position by turning the lens turret hand or by pressing the objective change button on the left side of the microscope.
- Page 3 Byeong Cha Page 3 11/24/2009 Setting up for Confocal Imaging. Turn on the Scanner/Laser He/Ne switch (the scanner should be on for about 1 min before starting the LCS program). Turn on the PC/Stand switch and turn on the computer.
- Page 4 Byeong Cha Page 4 11/24/2009 LCS Imaging program setting Upon LCS program start, the basic image acquisition menu will appear. The “Acquire” button should appear pressed. Laser power level Dichroic beamsplitter setting Detector bandwidth and range Detector selector & ...
- Page 5 Byeong Cha Page 5 11/24/2009 Press Z-scan button and select z-Wide in order to use Z POS knob move the objective for focusing (Default is z-Galvo, which is used with Galvo stage adaptor). Click MicCtrl button to switch to “Visual” mode, in which you can examine your sample visually at the microscope.
- Page 6 Byeong Cha Page 6 11/24/2009 Working with Images. Single button– puts up full screen of one fluorescent channel. Ch1, 2, 3, 4 buttons– activate the channel on the screen. Tiled button– views up to 3 channels simultaneously. Ovl button – overlays (merge) multiple channels into one.
- Page 7 Byeong Cha Page 7 11/24/2009 To scan sequentially: • Choose the beam setting parameter for your fluorophores (i.e. GFP and DsRED) by selecting laser lines, dichroic mirror, and PMTs. • Set up a condition for one fluorophore by only activating and adjusting laser level and PMTs. Save this parameter set up as your own setting (i.e.
- Page 8 Byeong Cha Page 8 11/24/2009 • Click the Seq button in the beam window. Add the fluorophore acquisition setting one at a time by selecting them in the beam window and then click Add button in the Sequential scan settings window. Keep the scan mode at “between lines”...
- Page 9 Byeong Cha Page 9 11/24/2009 Click Sect button to select the predetermined number of optical section, or to select Others … It brings up a z-configuration window to determine the number of sections and spacing (step size) between sections.
- Page 10 Byeong Cha Page 10 11/24/2009 Saving Images. This is a very important for your effort and data. Computer crash or power outage will result in loss of unsaved data! SAVE FREQUENTLY!! To save the imaging data files, click “Save” button at the top of the window. Select D drive and users folder. Create your folder, rename the Experiment# and save.
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