Leica SP5C, Operation Manual

Page 1: ...tute 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 R...

Page 2: ...signal 12 4 4 4 Setting the slits for the spectral separation of the emitted light 13 4 4 5 Adjusting the PMT settings 14 4 4 6 Additional transmitted light channel 16 4 4 7 Saving and loading of beam...

Page 3: ...Emission Fig 1 1 3 Wait until the PC has finished booting up 1 4 Log into Windows XP using the general account TCS user which is not password protected 1 5 IMPORTANT Make sure that the objective turre...

Page 4: ...Selection of the objective lens The LAS AF software will open showing the Acquisition page Select the objective you want to use in the Beam Path window see Fig 3 IMPORTANT Never change the objective b...

Page 5: ...d mark indicates the corrective settings for 37 C Fig 4 Choose the lens you want to use by matching the refractive index RI of the immersion medium with the medium embedding your sample oil lens with...

Page 6: ...erslip you should be using should be of thickness 1 130 150 m mean thickness The configuration with the motorised stage is not particularly suitable for live cell imaging and we recommend the Zeiss LS...

Page 7: ...ragm the illuminated field will decrease to the area chosen The fluorescence light path is also fitted with a shutter that allows to block illumination of the sample and thus to protect it see the yel...

Page 8: ...ee Fig 10 and allows you to conveniently find your initial marked focal plane again IMPORTANT Never push the objective lens against the coverslip because this might damage the front lens and might req...

Page 9: ...s switched on The display at the front of the power supply see Fig 5 shows the total usage time of the present bulb If this reaches 220 hours please inform the CALM facility staff by e mail so that th...

Page 10: ...on the display at the front see Fig 10 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...

Page 11: ...Laser Excitation wavelength nm 405 Blue Diode 405 Argon 458 476 488 496 514 Helium Neon 1 543 Helium Neon 2 594 Helium Neon 3 633 4 2 Setting the USB Control Panel In the configuration tab open the w...

Page 12: ...r you can define your own settings and save them for later re use Before you start choosing your settings for a multi channel image acquisition you have to decide whether to scan the channels simultan...

Page 13: ...equential combined channel 1 and 3 in the first and channel 2 in the second sequential scan This combined method involves 2 sequential scans During the first scan channels 1 and 3 are recorded Cross t...

Page 14: ...2 Spectrum view tool In order to assist and facilitate the definition of the beam path settings the software provides a visual tool that shows the excitation lines as well as the emission spectra of a...

Page 15: ...Fig 16 S1 to 3 represent the three slits of the spectroscopic detection unit By left clicking and moving them they can be shifted to shorter or longer wavelengths without changing the slit width By cl...

Page 16: ...The accurate adjustment of each PMT with regard to each fluorophore signal is absolutely crucial for a valid image acquisition For instance gain settings that lead to saturation and thus clipping of...

Page 17: ...4 5 2 check the off set settings again and correct if necessary 4 4 5 2 Setting the PMT gain Whilst continuing to scan adjust the voltage gain setting on the control console the range is 0 1250 V and...

Page 18: ...f specificity and also contain out of focus light they often give valuable spatial information such as cell outlines or the position of the cell nucleus To access the settings click on Additional Chan...

Page 19: ...needs to be re adjusted and if you are not familiar with the condenser unit please inform any CALM staff member Fig 22 Both channels with correct settings 4 4 7 Saving and loading of Beam Path setting...

Page 20: ...d 3 spectrally different channels at the same time By activating the desired PMTs see 4 4 5 and adjusting all necessary settings for the beam paths the simultaneous scanning can be set up easily 4 5 2...

Page 21: ...ber of sequential scans To save time you can scan more than one spectral channel in one of the sequential scans as long as the excitation and emission spectra do not overlap see the third option of th...

Page 22: ...most possible combination of x y z t time and wavelength sweep are available for image acquisition in 2 3 or 4 dimensions 4 7 Setting the scanning parameters The window panel below the Acquisition mo...

Page 23: ...to be reduced However longer dwell times i e slower scan speed also increases the bleaching and this might become a problem with dim samples that are at the same time prone to quick bleaching If as i...

Page 24: ...herefore recommended not to increase averaging to more than four scans unless the fluorescent label is very stable If photobleaching still is a problem averaging should be reduced or avoided at all Be...

Page 25: ...on in the sample which increases with the thickness of the sample that needs to be penetrated Because the entire specimen illuminated by the light cone from the objective lens will suffer from photobl...

Page 26: ...ing effects of the whole specimen caused by the repeated scanning should be taken into account If bleaching is a problem avoid averaging and rather rely on removing the increased noise in the image by...

Page 27: ...y clicking the Live button Then use the dials for the Smart Gain and the Smart Off set to optimise your settings see Fig 19 Click the Live button which now shows as Stop again to stop scanning Check a...

Page 28: ...o your destination folder on the hard drive in between different scans We strongly recommend that you do this after each scan also renaming each new file accordingly 5 1 Saving images and experiments...

Page 29: ...er by annotating each file To achieve this right click the appropriate file name and choose File properties This opens a window showing all metadata of the file which are accessible at any time post a...

Page 30: ...are packages for image deconvolution and analysis are available in the bioinformatics room C2 38 You find more information about this on the CALM website www calm ed ac uk 7 Shut down procedure Please...

Page 31: ...can occur and recommendations of how they can be fixed F In any case any serious problem should be logged reported to the CALM facility staff and an error report should be generated within the softwar...

Page 32: ...Fig 11 Check whether the required lasers have been selected in the Beam Path Settings Check whether the arm that carries the tungsten light source for transmitted light is in its correct position i e...