Transcript
CALM – Confocal and Advanced Light Microscopy Facility Queen’s Medical Research Institute – College for Medicine and Veterinary Medicine University of Edinburgh
Manual for the
Leica SP5C Spectral Confocal Laser Scanning Microscope Version 1.01 – June 2009
Written and illustrated by Rolly Wiegand
We take no responsibility for the contents of this handbook. The copyright lies with Rolly Wiegand and The University of Edinburgh, © 2007-2009. Reproduction of any kind without permission is prohibited.
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Contents 1.
General properties of the Olympus Cell P microscope system...................... 1
2.
Start-up procedure......................................................................................... 1
3.
Operation of the IX81 microscope stand ....................................................... 2
4.
Shut-down procedure .................................................................................... 3
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1. General properties of the Olympus Cell P microscope system
This system is built around an IX81 semi-motorised microscope base (Fig. 1 - 1), equipped with a 10x and 20x lens. The installed stage is a XY-motorised Prior ProScan 2 (2) and the microscope is operated by a IX2-UCB controller box (3) and a Windows-based PC (4), running the Olympus software Cell P. This system is suitable for taking brightfield images, either using transmitted light or the phase contrast technique. You have to book this system via the CALM online booking page.
This system is also equipped with a heated Pecon stage insert, which can be used in conjunction with the POC or mini POC coverslip holder. If you intend to use this facility to control the control of your sample whilst timelapse imaging, you need a short training by CALM staff before you start handling the equipment.
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Fig. 1 2. Start-up procedure
Log your microscope use in the log sheet next to the system by giving the date, your name and affiliation and the start time Remove cover Switch on the Prior stage controller (Fig. 2 – 1), the IX81 controller (IX2-UCB)(2) and the PC
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Fig. 2
Start the Cell P software by double-clicking the icon on the Desktop Services To initialise the XY-motorised stage go to ‘Acquire’ > ‘Stage’ > ‘Stage manager’ (or by hitting the ‘Stage control’ button in the tool bar) and the appropriate window will open, which allows you to define parameters for your stage settings, e.g. various stage positions etc. If the stage is not initialised yet, an appropriate message will appear on the screen and prompt you to log into the system. If asked by the software, log in as ‘administrator’ with the password ’a’
Whilst this ‘stage control’ window is open, select the line ‘X-stage is not initialised’ and
the button right next to it ‘Initialise’ will become active Retract the objective lens to its lowest position, either by hitting the ‘esc’ button on the microscope base next to the left focus wheel (see Fig. 4-2) or by manually turning the focus wheel (see Fig. 4-3, coarse position). This will avoid that the stage will hit the lens whilst the initialisation Once the lens is out of the way, press the ‘Initialise’ button and the stage will be initialised automatically. Do not obstruct the stage during this calibration procedure, whilst it is moving to different positions automatically The system is ready for image acquisition now. 3. Operation of the IX81 microscope base
Fig. 3 shows the main control elements on the front of the microscope base. Once the sample is mounted onto the stage, switch on the Tungsten lamp for transmitted illumination
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(Fig. 3-3). By using the light intensity control buttons (Fig. 3-2), the intensity of the transmitted light can be adjusted. Bear in mind that less illumination of a live cell sample the better. The intensity voltage indicator gives you a read-out of the relative light intensity.
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Fig. 3 Control elements on the front panel of the IX81 microscope base 1 = Tungsten lamp voltage indicator (brightness of transmitted light) 2 = Transmitted light intensity control buttons 3 = Tungsten lamp on/off button
To focus on the desired optical plane in your sample, use the ‘focus knob’ on either side of the microscope stand (Fig. 4-1). To change between fine and coarse focussing mode, repeatedly press the ‘F/C’ button (Fig. 4-3) to switch between the alternating modes. To move the nosepiece that holds all objective lenses to its lowest position, e.g. before calibrating the XY-stage positioning, simply press the ‘esc’ button next to the ‘focus knob’ (Fig. 4-2).
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Fig. 4 Left-hand side of the microscope stand 1 = Focus knob 2 = Objective escape/return button 3 = Focus adjustment fine/coarse switching button 4 = Left camera port 4. Shut-down procedure
Remove sample and check whether the lens is clean Retract the lens by pressing the ‘esc’ button Save all your images and data to the hard drive, but bear in mind that these have to be removed asap Exit the Cell P software Shut down the PC and switch off the 2 controller boxes Put cover back on the microscope base and log your time etc.
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