LSM510 Confocal Microscope Standard Operation Protocol Basic Operation

Transcription

1 LSM510 Confocal Microscope Standard Operation Protocol Basic Operation Please make sure that the COMPRESSED AIR has been TURNED ON prior to the use of the equipment. Kindly inform the administrator if the gauge displays LOW level of compressed air. Turn on system Please sign on the log sheet before switching on system. Switch on main power remote control 1. Switch on HBO fluorescent lamp 2. Switch on Chameleon laser by turning the Laser Key (3) 90 clockwise to horizontal position. (Chameleon is Pulsed IR laser which is only for blue emission, e.g. DAPI or two-photon applications. Please skip this step if this laser is not needed). Press computer ON/OFF (4) switch to turn it on. (The computer is under the air table). Switch on Temperature Module S for temperature and CO2 control. (only applicable for live cell imaging, please skip this step if it is not needed). Click to log into User at the startup screen. Set the temperature and CO2 control for live cell imaging (Only applicable for live cell imaging): Double click AxioVision Rel 4.8 software icon on the desktop of WINDOWS to start the software program. Click Incubation in AxioVision software For 35mm dish: turn on Temperature channel 1 & 3 & 4 For 6-well plate: turn on Temperature channel 2 & 3 & 4 Turn on CO2 control in Atmosphere Turn on CO2 tank for CO2 supply Exit software AxioVision Rel 4.8 1

2 Starting LSM 510 software program Double click LSM 510 icon on the desktop of WINDOWS to start LSM software program. Click on Scan New Images button and Start Expert Mode button in the LSM 510 Switchboard window to initiate software and hardware connection. The LSM 510 Expert Mode Main menu appears on the screen. Turning on the lasers Click on the Laser button to open the Laser Control window. Select the appropriate Laser Unit by clicking on the name of it. For Argon Laser, click the Standby button first. Then Click On button when status changes from Warming up to Ready. (There is 2-3min is required for Argon laser to warm up!) For Chameleon, HeNe1 and HeNe2 lasers, click On button directly to switch on lasers. Click Modify to change wavelength of Chameleon laser if needed. 2

3 Setting the microscope Click on the VIS button to set the microscope for direct observation via the eyepieces. Click on the Micro button in the Acquire subordinate toolbar to open the Microscope Control window. To select an objective, open the graphical pop-up menu by clicking on the Objective button and click on the objective you want to select. (Note: Please make sure there is no oil or water at all on your slide when you are using air objective. Any Oil or water will damage air objectives) Do not change from oil lens to air lens UNTIL OIL HAS BEEN COMPLETELY REMOVED. Fix your sample (slide/dish) on the stage with coverslip facing downwards. To set the microscope for brightfield observation, click BF On button. To set the microscope for fluorescence observation, click on FITC/Rhod/DAPI. Focus the sample with Corse/fine focus knob. Locate the view of interest through stage control panel. Configuring the beam path and lasers Click on the LSM button to switch to scanning mode. Click on the Config button in the Acquire toolbar to open the Configuration Control window. Chose Single Track or Multi Track in Channel Mode for beam path configuration. Single Track Use for single, double and triple labeling; simultaneous scanning only Advantage: faster image acquisition Disadvantage: cross talk between channels Multi Track Use for double and triple labeling; sequential scanning, line by line or frame by frame Advantage: when one track is active, only one detector and one laser is switched on. This reduces cross talk. Disadvantage: slower image acquisition 3

4 Open configuration drop down list by clicking Config in Configuration control window to load or store track configurations. Single track configurations Multi track configurations For loading an existing configuration, select a defined configuration from the drop-down list and click Apply. Scanning an image Click on the LSM button if necessary. Click on the Scan button in the Acquire subordinate toolbar to open the Scan Control window. Select Channels in the Scan Control window. Adjust parameters for each track one by one by only selecting one track to be checked and highlighted in Configuration Control window. Set the Pinhole size to 1 (Airy unit) (for Argon and HeNe lasers) for best compromise between depth discrimination and efficiency. Set the Pinhole size to Max for Chameleon laser. Argon and HeNe lasers Chameleon laser Click Fast XY button to start the scanning procedure. Select Palette in the Image window of the scanned image. The Color Palette window appears In the Color Palette List panel, click on the Range Indicator item. Red = Saturation (maximum). Blue = Zero (minimum). Adjust laser power and Detector Gain to obtain few red pixels shown on image. Reduce Amplifier Offset to introduce blue pixels in background area. Click Stop to complete the preview of the sample. Repeat the above procedure for other tracks one at a time. * User is advised not to use too extreme settings on laser power and master gain (as too high laser power will induce bleaching onto the sample; and too high master gain will cause significant noise and shorten the shell life of the PMT). In any given cases, users should keep the master gain <

5 Setting the Acquisition Mode for scanning: Select Mode in the Scan Control window. Select the Frame Size as predefined number of pixels or enter your own values. 1024x1024 usually produces good results. Click on the Optimal button for best resolution depending on objective N.A. and λ. Use the slider in the Speed panel to adjust the scan speed. 6 or 7 usually produce good results. Speed 4or 5 for weak signal samples. Select the dynamic range 8 or 12 Bit (per pixel) in the Pixel Depth. 12 Bit is recommended for quantitative measurements. Select the number of average in Scan Average panel. Averaging improves the image by increasing the signal-to-noise ratio. Check all tracks and click Single to acquire an image. Creating a database and saving the acquired image: User is highly recommended to save the image immediately after acquisition. Create a folder in D drive under you name if necessary. Click on the File button in the Main menu toolbar. The File subordinate toolbar appears in the Main menu. Click on the New button in the File subordinate toolbar. The Create New Database window appears. Create a new database in your own folder. Click on Save As button in the Image window. The Save Image and Parameter As window appears. Enter file name, description and notes in the appropriate text boxes. Then click OK. The file is save in LSM format. To export image to other format, chose Export from the File menu. You can select from a number of format options (e.g. TIF, GPEG or AVI for vedio) then save file to other format. 5

6 Scanning Z stack Select Z stack in the Scan Control Window. Select Mark First/Last on the Z Settings panel. User is recommended to use one track to set First/Last. (For this purpose, please uncheck other unnecessary tracks) Click on Fast XY button to have continuously scanning. Turn the focusing knob gently to locate the starting position of the specimen to Mark First. Then locate the ending position of specimen to Mark Last. Click Stop to complete the preview of the sample. Set interval for Z-stack scanning. This digit suggests the distance between each slide. To check the optimal interval, click Z-slice button to open Optical Slice window. Select optimal interval if needed. Users are strongly suggested to set the interval within this range: 1x optimal interval <= z <=2x optimal interval. Check all tracks. Click on the Start button to start the recording of the Z Stack. Turn off system Please check if the equipment will be used by other users. Please switch off system if no one books equipment over two sessions (1h) after you. Oil lens (i.e. 40x/63x), IF USED, must be thoroughly cleaned using lens cleaning tissues (NOT KIMWIPES). Oil residue from the objective lens should firstly be removed using a DRY lens tissue. The surface is then wiped with another lens tissue with 100% ethanol. Objective lens is subsequently wiped dry with lens tissue. Switch objective to 10x and lower focus level to the lowest position. Switch off Lasers in LSM510 software. Click on the Laser button to open the Laser Control window. For Argon Laser, click the Standby button first. Then Click Off button. For Chameleon, HeNe1 and HeNe2 lasers, click Off button directly to switch off lasers. Quit LSM510 software by selecting File > Exit. There is a warning message to remind you waiting for laser fan off before switchinf off main power switch. Click OK then click Exit for closing LSM510 software. Switch off Temperature and CO2 control module (Please skip this step if temperature and CO2 control was not turned on). Turn off CO2 tank to cut down CO2 supply. Double click AxioVision Rel 4.8 software icon on the desktop of WINDOWS to start the software programs. Click Incubation in AxioVision software. Turn off all Temperature channels. Turn off CO2 control in Atmosphere. Exit software AxioVision Rel 4.8 Transfer data to Faculty Core Facility storage server and shut down PC (4). Turn the Chameleon Laser Key (3) 90 anti-clockwise to switch it to Standby Mode. Switch off HBO fluorescent lamp 2 Wait for 5 min to let laser fan off Switch off Main power remote control 1 Users are reminded to sign on the log sheet before departure. 6