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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 1.11 – September 2008
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, © 2008.
Reproduction of any kind without permission is prohibited.
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Rolly Wiegand – CALM Leica SP5 manual
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Summary of Contents for Leica SP5C
- Page 1 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 1.11 – September 2008 Written and illustrated by Rolly Wiegand We take no responsibility for the contents of this handbook.
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Page 2: Table Of Contents
Using the ROI tool ....................24 4.10. Optimising settings via the USB control panel............25 Saving and exporting data files................26 Post-acquisition data handling and analysis............27 Shut-down procedure ..................... 28 Trouble shooting ..................... 29 _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 3: Starting Up The Sp5
1.6. Start the LAS AF software by double clicking the icon on the desktop. The following window will appear (see Fig. 2A). Choose ‘ok’ without going into the configuration or microscope stand setup. Fig. 2A _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... - Page 4 DRY – air (never apply any fluid to theses lenses) OIL – immersion oil only (use the provided Zeiss immersion oil) WATER – water only (MQ water) _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual...
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Page 5: Operation Of The Dmi6000 Inverted Microscope Base
2. Operation of the DMI6000 inverted microscope base 2.1. The microscope should be already switched on via the TCS control panel (see above section 1.2.). _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... - Page 6 TL/IL button on the left hand side of the microscope stand (see Fig. 6). The system is not equipped for the use of any contrast method (PC, DIC, etc). _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual...
- Page 7 It should also be mentioned that the given filter cubes are prone to spectral cross-talk and thus care should be taken regarding conclusions from the observed fluorescent signal. _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual...
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Page 8: Rules For The Use Of The Mercury Vapour Arc Lamp
If in doubt, contact the previous user. 3.2. Once the lamp has been switched on, it has to be left on for a minimum of 30 minutes. _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... - Page 9 Z-drive X-drive Z-drive Fine/coarse Y-drive Fig. 8 SmartMove remote control In Fig. 8, the arrow on the right indicates the selection button for the Z-focus mode (‘coarse’ or ‘fine’) _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual...
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Page 10: Image Acquisition Via The Las Af Software
Fig. 10 Display on the front of the microscope stand You can find more details about the DMI6000 microscope stand in the Leica handbook, and a copy of the pdf file can be found on the CALM website. 4. Image acquisition via the LAS AF software 4.1. -
Page 11: Setting The Usb Control Panel
3). Note: Please do not change any other setting in the configuration tab. If you need more detailed information about the objective lenses, open the appropriate windows, but do not change anything. _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 12: Defining The Beam Path Settings
To avoid spectral cross-talk, sequential image acquisition is recommended. This allows the sequential recording of up to 7 channels, each of them can be defined individually. Fig. 13 _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... - Page 13 A = Laser control module B = Objective selection tool C = Spectral view tool D = Slit settings E = PMT settings F = Control for saving/loading of settings Fig. 14 ‘Beam Path Settings’ windows _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual...
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Page 14: Generating New Beam Path Settings
Note: This is just an auxiliary tool that has no impact on the actual settings. 4.4.3. Spectroscopic separation and detection of the emitted fluorescence signal The Leica SP5 uses a sophisticated filter-free technology to separate the emitted light from the fluorescent sample into different spectral bands. In brief, the entire emitted signal is spectrally dispersed by passing the light through a prism (see Fig. -
Page 15: Setting The Slits For The Spectral Separation Of The Emitted Light
Right-clicking the sliders will open a little window that indicates the settings of each slit in nm (see Fig. 16). This can also be used to set the spectral separation. _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 16: Adjusting The Pmt Settings
1 in Fig. 20. Then change the look-up table (LUT) to glow scale (GloOU) by clicking the button indicated by the circle and arrow in Fig. 20. By clicking this button repeatedly, it will change through LUT-GloOU-grey scale, consecutively. In the GloOU _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... - Page 17 (see Fig. 22). Once you have adjusted the PMT settings for one channel, proceed with adjusting all other channels in the same way. _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual...
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Page 18: Additional Transmitted Light Channel
PMT settings (C, and see above). Fig. 20 Ch1 – off-set too low, Ch2 – off-set correct Fig. 21 Ch1 – PMT gain correct, Ch2 – PMT gain too high, saturated _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 19: Saving And Loading Of Beam Path Settings
D:. This function saves the activated laser lines, laser power settings, slit positions and PMT settings. Fig. 23 Setting of the transmitted light channel _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 20: Scanning Mode
405 nm diode laser for excitation of fluorophore 1 and the second the 488 nm Argon laser line, the UV laser must be switched on in channel 2 although its laser power is set to 0%. _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... - Page 21 If you intend to also record a transmitted light scan, remember that this is using the 488 nm laser line and thus should be defined as a separate channel or simultaneously scanned with the channel that uses the same laser line. _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual...
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Page 22: Setting The Acquisition Mode
This is helpful when the object to be imaged is smaller than the whole field and scanning speed is important. The less lines and pixels are being scanned the shorter the scan. _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 23: Pinhole Size
(pixels per line), the NA and magnification of the lens and the excitation wavelength. Based on the zoom, resolution settings and lens properties, the system automatically calculates the pixel dimensions, which are shown in the same panel together with the _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 24: Averaging
Programming the acquisition of a Z-stack requires to define the start and end section to be imaged as well as the Z-step (or Z-interval), which defines the stepping of the Z-galvo between the different optical sections. _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... - Page 25 Z-position the system is at. Now find the last (‘End’) position of the sample to be imaged, which usually is pretty close to the coverslip, and set _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual...
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Page 26: Using The Roi Tool
ROI by left-clicking the top left corner of the position of the frame and pulling the frame to the desired size, i.e. the _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 27: Optimising Settings Via The Usb Control Panel
Bear in mind that a high resolution image stack recorded at Nyquist rate with a several-fold averaging can easily take 20-30 minutes. Fig. 32 ROI tool _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 28: Saving And Exporting Data Files
Data file organisation A well thought-through nomenclature for your file names will tremendously facilitate your post-acquisition file handling. An example for an explanatory nomenclature is: 2008 08 08 RW Exp001 S002 Z _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 29: Post-Acquisition Data Handling And Analysis
‘Delete file’ function allows you eliminate unwanted files you do not want to save with your experiment. Bear in mind that the saved ‘.lif’ file is the Leica standard file format and usually contains a selection of different files (called experiment). For post-acquisition handling of the .lif file, your software package needs an appropriate file opener that can deal with this format. -
Page 30: Shut-Down Procedure
Do not switch off the power for the laser unit on the front panel before the fans switch off. Only this guarantees that the lasers are cooled down sufficiently before they are switched off. _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... -
Page 31: Trouble Shooting
F: Check the setting of the PMT gain (the default setting is ‘zero’ V), normal settings vary in the range between 600 and 1200 V. See whether laser light is coming through the objective, if not: _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual... - Page 32 A: This occasionally happens and you have to restart the system. However, do not switch off anything at the front panel (see Fig. 1) or in the laser module (the lasers should be left running). _____________________________________________________________________________________ Rolly Wiegand – CALM Leica SP5 manual...