Zeiss LSM 510 META Manuel d'utilisateur

Confocal microscopyZeiss LSM 510 and Zeiss LSM 510 METAVisualisation of biological structures in 3D

Selecting an objective and focusing the microscope1) Select Micro (Main menu: Acquire)(For Axioskop 2 FS these settings have to be adjusted manually)3

Focusing the microscope in fluorescence modeClick onto Reflected Light to open the shutter of the HBO lampFluorescence is observed by selecting the ap

Focusing the microscope in transmitted modeClick onto Transmitted Lightand move the slider to set the intensity of the HAL illuminationUse no reflecto

Contents•Starting the Zeiss LSM 510 microscope, software and laser•Selecting an objective and focusing the microscope•Configuring the laser scanning a

Choosing the configurationSINGLE TRACK MULTI TRACKSimultaneous scanning onlyUse for single, double and triple labellingUse for double or triple labell

Configuration of the filters and storage of the track configurationsSINGLE TRACK - lasers scan simultaneously1) Select Config in the Acquire menu2) Se

Applying a stored configuration and checking the settingsIf you select Store by mistake, software will ask you, if you want to overwrite the configura

Multi Track Configuration3) Select a stored track from the pull down menu, click on ApplyThis button stores only the highlighted single track or appli

Cy5-Cy3-FITC Multi TrackThree laser lines and channels activated sequentiallyExcitation Detection633 nm, using the META detector in Channel mode543 nm

Setting the parameters for scanning1) Select Scan3) Select the Frame Sizeas predefined number of pixels or enter your own values (e.g 300 x 600). Use

Inverted Zeiss LSM 510 confocal microscope

Setting the parameters for scanningNote: When using a Axioskop 2 FS, indicate the Objective that is in use in the Scan Control window. This ensures co

Adjusting the scan speedAdjust the scan speed - a higher speed with averaging results in the best signal to noise ratio. Scan speed 8-9 usually produ

Choosing the Dynamic Range (8/12 Bit per pixel)Select the dynamic range - 8 bit will give 256 gray levels, 12 Bit will give 4096 levels. Photoshop 5 w

Channel Settings - Adjusting the PinholePinhole size = 1 Airy unit0.8 “Airy units” produces the best signal : noise ratioPinhole adjustment changes th

2) Select Fast XYfor continuous fast scanning - useful for finding and changing the focusImage Acquisition1) Findautomatically pre-adjusts detector s

Minimal Pixel Size determined by Nyquist Sampling×NA5102040631000.150.30.51.3 (oil)1.4 (oil)1.4 (oil)1.03 µmPIXEL SIZE0.51 µm0.31 µm0.12 µm0.11 µm0.1

Optical ZoomingThe image can also be rotated by selecting and dragging the barsThe level of zoom can be changed either by using the Zoom, Rotation &am

Selecting gain and offset – Choosing a lookup table1) Select Palette2) Select Range IndicatorRed = Saturation (maximum)Blue = Zero (minimum)

Scan Control – Setting Gain and OffsetDetector gain determines the sensitivity of the detector by setting the maximum limitAmplifier Offset determines

Saturation of Signal Intensity with Laser Power 2% 4% 6% 8%Argon laser 488 nm % transmissionSignal IntensityPhotobleaching is linear!•Fluorophore satu

Contents•Starting the Zeiss LSM 510 microscope, software and laser Selecting an objective and focusing the microscope•Configuring the laser scanning a

Adjusting the Laser Intensity1) Set Pinhole to 1 Airy unit2) Set Detector Gain high3) When the image is saturated, reduce AOTF transmission in the Exc

Adjusting Gain and OffsetBoth Detector Gain and Amplifier Offsetsaturated1) Increase the Amplifier Offset until all blue pixels disappear, and then ma

Adjusting the Laser, Gain and Offset using aMulti Track ConfigurationEach channel is selected independently by clicking on the colour button indicatin

Setting up Gain and Offset - Multi Track1) Select Split XY in the Image window2) In Palette, select Range indicator3) Select each channel separately u

Line Averaging2) Select Number for averaging. The more the better for the signal to noise ratio (max 16) in this case, each line will be scanned 4 ti

Frame Averaging1) Select Frame2) Select the Number for averaging - The more the better for signal to noise ratio (max 16). Continuous averaging is pos

Collecting an Averaged ImageIn the Channels panel of the Scan Control window select Single. An averaged image will be collected.Range indicator set to

Contents•Starting the Zeiss LSM 510 microscope, software and laser•Selecting an objective and focusing the microscope•Configuring the laser scanning a

Scanning a Z-Series using Mark First/LastNOTEFocusing can be achieved manually (preferred), or using Stage in the LSM menu if there is a motorized sca

Using Auto Z Brightness Correction Auto Z provides an automatic gradual adjustment of Detector Gain, Amplifier Offset, Amplifier Gain, and Laser inten

Contents•Starting the Zeiss LSM 510 microscope, software and laser•Selecting an objective and focusing the microscope•Configuring the laser scanning a

Confocal Z Sectioning Number of Sections for correct samplingOptical thickness d depends on:• Wavelengthλ• Objective lens, N.A.• Refractive index n•

Z Stack – Number of Slices and Increment1) Select Z slice - the window Optical Slice will appear2) Select Optimal interval the computer will calculate

Z - Series using Z Sectioning1) Select Z Stack2) Select Z Sectioning3) Select Line Sel4) Select the large arrow button and position the XZ cut line

Z Sectioning – Setting Range1) Decide whether to Keep Interval (number of slices will change) or Keep Slice (Intervalbetween slices will be adjusted)2

Viewing a Z - SeriesIn the image window1) Select xy2) Select Slice3) Use scroll bar to view individual sections

Viewing a Z - Series using Gallery1) Select Gallery2) Select Data for scaleUse Subset to extract sections

Viewing a Z- Series using Orthogonal Sections1) Select Ortho2) Select mouse (Select)3) Using the mouse, position the cut lines.To save orthogonal sect

Selecting and Saving a Region of Interest (ROI)1) Select Overlay and define shape of ROI2) Extract region creates a Z-Stack from the ROI 3) Save data

Using a ROI for faster image acquisition and data saving1) Select EditROI from the LSM menu bar2) Select Fit Frame Size to bounding Rectangle3) Choose

Multiple Regions of Interest1) Un-select Fit Frame Size to bounding Rectangle, Choose shapes of ROIs4) Position and size the ROIs with mouse5) Start

Starting the LSM 510 software1) Double click the LSM 510 icon2) Select “Scan New Images”3) Select “Start Expert Mode”

Time Series1)Set up scanning parameters for image acquisition as described in previous slides2)Select TimeSeries from the LSM menu 3)Enter the Number

Viewing a Time Series of a Z StackZ Sections for any timeTime points for any Z SectionBoth Z sections and time series

Time Series – Physiology Experiments1) If required, use multiple regions of interest2) Set up Time Series as before3) Instead of using StartT select

Contents•Starting the Zeiss LSM 510 microscope, software and laser•Selecting an objective and focusing the microscope•Configuring the laser scanning a

Saving Data - Using Database1) Select Save or Save as on image window or LSM menu bar2) Enter file name and notes if required3) Select OK

Saving Data – Using Export1) Select File from LSM menu2) Select Export3) Select Image type4) Select Single image with raw data (No overlay or Look up

This guided tour is based on work done by Peter JordanICRF LondonUnited Kingdomedited and complemented byEva Simbürger and Solveig HehlCarl Zeiss Jena

Creating a database for acquired images1) In the main menu File select New database2) Select drive C or D: from pull down menu3) Create a new director

1) Select Acquire3) Switch required laser/s to Standby or OnArgon power should be set to about 6.1ATurning on the lasers2) Select Laser

Change between direct observation and laser scanningInverted Microscope: Axiovert 200 MToggle between Vis and LSM button in main menu, automatic swit

Contents•Starting the Zeiss LSM 510 microscope, software and laser•Selecting an objective and focusing the microscope•Configuring the laser scanning a