Guide To The Confocal Microscope at LSHTM and Codes of Practice.
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This is a guide for using the Zeiss LSM510 Confocal Microscope. Do not attempt to use this microscope without the proper training. If you have any queries or problems please contact either Rachel Gregory (2804) or Liz Brooks (2063). Please read through the transfer procedure before booking or shutting down the system.
1. Starting up the microscope
Switch on the remote control box-this will provide power to the system
(you will also need to switch on computer)
Switch on the mercury lamp (if needed) and fill in the log book
Log on to computer by pressing [ctrl+alt+del]
Type in username and password
Double click on the LSM510 start icon:-
The LSM510 start menu will appear-
Select ‘scan new images’ for acquiring new images
or ‘use existing images’ if using previously saved
Select ‘start expert mode’. This will start up the
software and initialise the computer/microscope
2. Switching on the lasers
From the LSM510 tool bar, select ‘Acquire’ from the main menu and ‘laser’ from the sub folder
There are four lasers connected to the confocal
microscope which are used for the wavelengths
When operating this laser system, these
precautions should be followed;
Do not look directly into the beam
Do not disable any of the safety features on the microscope.
Knock before entering a room with this type of laser.
Do not use any type of reflective surfaces when operating this instrument.
Laser Diode: 405nm i.e. DAPI
Argonlaser: 458, 488, 514nm i.e.FITC/Alexa 488
HeNe1laser: 543nm i.e. rhodamine/CY3/Alexa 546
HeNe2laser: 633nm i.e. CY5/Alexa 633
To turn the Argon laser on, first highlight the laser name in the control window and click the ‘stand-by’ button. When the laser has warmed up a ‘Ready’ message will appear, and the laser may now be turned on, select ‘on’. The laser output (%) will need to be adjusted to create a tube current of 6.1A (see diagram). Laser output is usually adjusted to around 55%.
The HeNe and laser diode lasers can be turned on directly by first highlighting the laser names individually and then clicking the ‘on’ buttons. There’s no need to adjust the laser output (%).
3. Set Track/Laser configurations
Select ‘config’ from the tool bar subfolder
Choose the required laser path settings from the pre-programmed configurations-
You can toggle between scanning mode and visual mode using
the LSM toolbar
From LSM510 toolbar select ‘Micro’
This window allows you to:-
Switch on/off transmitted light and adjust
Select the objective to be used
Switch on/off reflected light
Select the filter to be used
Select the objective you require to image your sample. If the objective needs it, add a drop of oil onto your slide.
Place the slide ‘upside down’ and clamp into place.
It is normally best to focus using transmitted light in case the fluorescence labelling has been unsuccessful.
Switch on the transmitted light by selecting the ‘transmitted light’ in the microscope control menu. Light brightness is usually set to 5-6, (but you can go brighter if you wish). Once you have focused on your sample you can check for fluorescence.
Switch off the transmitted light and select ‘reflected light’. Choose the appropriate filter for your sample from the ‘reflector’ drop down menu.
N.B. you cannot visualise far red (i.e. Alexa 633 or CY5) with the filters, these can only be visualised by scanning.
Remove your sample and clean oil from all the lenses used.
Ensure the objectives and reflective filters are left in ‘empty’ position.
If you have been contacted by the next user to leave the microscope on, do so. If you have not been contacted (please check your emails) then continue with the rest of the shut down procedure.
Switch off the lasers:-
in the laser control window, select lasers individually
HeNe1 and HeNe2 lasers may be switched off directly-select ‘off’.
The Argon laser must be allowed to cool down-select ‘standby’,then select ‘off’.The status will show ‘cooling’. The status will then change to ‘connected’ (this will take up to 5mins)the remote control must not be switched off before this time.
Switch off the mercury lamp, and fill in the log book
Exit from LSM510 and shut down the computer.
When the computer has fully shutdown, switch off the remote control
Re-cover the microscope with blue dust cover and plastic sheets
Dispose of all used tissues and other general mess in the autoclave
N.B. This tin is not for glass coverslips!
Ensure room is left locked if there are no other users present and return key to the Log book.