Guided Tour for LSM 510 and META - bio.umass.edu · Confocal microscopy Zeiss LSM 510 and Zeiss LSM 510 META Visualisation of biological structures in 3D. ... Select drive C or D:

Carl Zeiss - Training Application and Support Center

Zeiss LSM 510 META - Guided Tour

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Confocal microscopyZeiss LSM 510 and Zeiss LSM 510 META

Visualisation of biological structures in 3D



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Confocal Principle

Signals from aboveand below the planeof focus fall outsidethe pinhole and areblocked

Only signals fromplane of focus passthe pinhole and aredetected - producinga “optical section”



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Upright Zeiss LSM 510 confocal microscope

LSM 510 scan head

Axioplan 2 imaging



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Inverted Zeiss LSM 510 confocal microscope

LSM 510 scan headin base port position

Axiovert 100M



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Upright Zeiss LSM 510 META confocal microscope

Axioplan 2 imaging

LSM 510 METAscan head



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Inverted Zeiss LSM 510 META confocal microscope

Axiovert 200MLSM 510 METAscan head



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• Starting the Zeiss LSM 510 microscope, software and laserSelecting an objective and focusing the microscope

• Selecting an objective and focusing the microscope

• Configuring the laser scanning and detection for confocal imageacquisition

• Acquiring a Z- and Time - Series

• Data storage

Descriptions also include the LSM 510 META

Contents



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• Data storage


Contents



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1) First switch on the mercury lamp

2) Turn on the remote control switch

3) Wait for the computer to boot upand Login by simultaneouslypressing the Ctrl, Alt and Deletekeys

Start the Zeiss LSM 510 Confocal Microscope



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1) Double click theLSM 510 icon

2) Select “Scan NewImages”

3) Select “StartExpert Mode”

Starting the LSM 510 software



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1) In the main menu File select New database

Creating a database for acquired images

2) Select drive C or D: from pull down menu3) Create a new directory if needed



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1) Select Acquire Turning on the lasers

2) Select Laser

3) Switch requiredlaser/s to Standby

4) When status isReady, click On

5) Set Output [%] so thatthe tube current isbetween 5.5 and 6.5 A



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For laser scanning imageacquisition:

Set slider to “LSM”(pull slider out)

Change between direct observation and laser scanning

Upright Microscopes: Axioplan2 imaging and Axioskop 2 FS

For direct observation oftransmitted light andfluorescence:

Set slider to “VIS” (push it in)



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Toggle between Vis and LSM button in main menu, automatic switchingbetween direct observation and laser scanning (no slider)

Change between direct observation and laser scanning

Inverted Microscope: Axiovert 200 M



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• Data storage


Contents



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1) Select Micro (Main menu: Acquire)(For Axioskop 2 FS these settings have tobe adjusted manually)

Selecting an objective and focusing the microscope

3) Objective lens can be selected froma pull down menu by clicking onto theObjective button

2) Microscope settings can be storedand up to 8 buttons assigned for fastretrieval and adjustment



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Focusing the microscope in fluorescence mode

Fluorescence is observed byselecting the appropriate filter setin the pull down menu of Reflector

Click onto Reflected Light to openthe shutter of the HBO lamp



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Focusing the microscope in transmitted mode

Use no reflector cube in thereflector turret, chose None

Click onto Transmitted Lightand move the slider to setthe intensity of the HALillumination



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• Data storage


Contents



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Choosing the configuration

MULTI TRACK

Use for double ortriple labelling

Sequential scanning, line byline or frame by frame

When one track is active, onlyone detector and one laser isswitched on. This dramatically

reduces crosstalk.

ADVANTAGES

DISADVANTAGESSlower image acquisition

SINGLE TRACK

Use for single, double andtriple labelling

Simultaneous scanningonly

Faster imageacquisition

ADVANTAGES

DISADVANTAGESCross talk between

channels



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1) Select Config in theAcquire menu

SINGLE TRACK - lasers scan simultaneously

Configuration of the filters and storage of the track configurations

4b) The Config buttonopens the pull down menuto load/store Trackconfigurations

3) Click Excitation to selectthe laser and attenuation

4c) Transmitted light imagecan also be generated.Transmission channel isusually set to white colour.

2) Select Single Track

4a) Select theappropriate filters andactivate the Channels



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5) Chose a configuration in theTrack Configuration menu.Select Apply

If you select Store by mistake, softwarewill ask you, if you want to overwrite theconfiguration. ANSWER NO!

Each new login loads a predefined set ofcorrect configurations.

Applying a stored configuration and checking the settings

The Spectra buttonopens a window todisplay the activatedlaser lines for excitation(colored vertical lines)and channels (coloredhorizontal bars)

6) To check for correctsettings, click the Spectrabutton



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Multi Track Configuration

2) Select Config

1) Select Multi Track forsequential scanning

3) Select a stored track from thepull down menu, click on Apply

This button stores only thehighlighted single track or appliesa single track.



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Cy5-Cy3-FITC Multi TrackThree laser lines and channels activated sequentially

Excitation Detection

633 nm, usingthe METAdetector inChannel mode

543 nm

488 nm



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1) Select Scan

Setting the parameters for scanning

2) Select Mode3) Select the Frame Size as predefinednumber of pixels or enter your ownvalues (e.g 300 x 600). Use Optimal forcalculation of appropriate number ofpixels depending on N.A. and λ.

The number of pixels influences theimage resolution!



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Setting the parameters for scanning

Note: When using aAxioskop 2 FS, indicatethe Objective that is inuse in the Scan Controlwindow. This ensurescorrect calculation ofpinhole, z-stackoptimization etc.



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Adjust the scan speed - ahigher speed with averagingresults in the best signal tonoise ratio. Scan speed 8usually produces good results.Use 6 or 7 for superior images.

Adjusting the scan speed



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Select the dynamic range - 8bit will give 256 gray levels,12 Bit will give 4096 levels.Photoshop 5 will import 12and 16 Bit images.

Publication quality imagesshould be acquired using12 Bit.

12 Bit is also recommendedwhen doing quantitativemeasurements or whenimaging low fluorescenceintensities.

Choosing the Dynamic Range (8/12 Bit per pixel)



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Channel Settings - Adjusting the Pinhole

Set pinhole size to 1 Airy unit for best compromise between depth discrimination and efficiency.

Pinhole adjustment changes the “Optical slice”.

When collecting multi channel images, adjust the pinholes so that each channel has the same“Optical Slice”.

This is important for colocalization studies.

Pinhole size= 1 Airy unit



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2) Select Fast XY for continuousfast scanning - useful forfinding and changing the focus

Image Acquisition

1) Find opens new imagewindow and automatically pre-adjusts detector sensitivity

4) Stop blanks the laser beamand stops the scanning mirrors

3) Single records a single image

5) Select Cont. for continuousscanning with selected scan speed



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Minimal Pixel Size determined by Nyquist Sampling

Adjusting the field size (“XY”) to 56 µm withthe 63× lens, would produce a pixel size of0.1 µm

Field size can be adjusted by changing the objective magnification, or by optical zooming. Changingfrom 63 × to 100 × will reduce the field size, but will also reduce the amount of light available.

Brightness of image = Magnification2/NA2

Values are for scan zoom = 1.0

NA

5

10

20

40

63

100

0.15

0.3

0.5

1.3 (oil)1.4 (oil)

1.4 (oil)

1.03 µm

0.51 µm

0.31 µm

0.12 µm

0.11 µm

0.11 µm

Pixel sizeMagnification



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Optical ZoomingThe level of zoom can bechanged either by using theZoom, Rotation & Offsetcontrol in Mode menu of theScan Control, or by selectingCrop in the image menu.

The image can also berotated by selecting anddragging the bars



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Selecting gain and offset – Choosing a lookup table

1) Select Palette

2) Select Range Indicator

Red = Saturation (maximum)

Blue = Zero (minimum)



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Detector gain determines thesensitivity of the detector bysetting the maximum limit

Amplifier Offset determines theminimum intensity limit

Amplifier Gain determinessignal amplification

Scan Control – Setting Gain and Offset

Saturation at the maximum→ reduce Detector Gain

Saturation at the minimum→ increase Amplifier Offset

Gain set correctly

Offset set correctly

Gain

Offset

Amplifier Gain increases thewhole signal, and the AmplifierOffset will need to be decreased.



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Laser transmission should not be set higher than the saturation level.

• Fluorophore saturatesat 6% lasertransmission

• Photobleaching islinear

Saturation of Signal Intensity with Laser Power

2% 4% 6% 8%Argon laser 488 nm % transmission

Sign

al In

tens

ityPhotobleaching is linear!



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Adjusting the Laser Intensity

1) Set Pinhole to 1 Airy unit

2) Set Detector Gain high

3) When the image is saturated, reduceAOTF transmission in the Excitationpanel to reduce the intensity of the laserlight at the specimen

image withsaturatedpixels



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Adjusting Gain and Offset

1) Increase the Amplifier Offsetuntil all blue pixels disappear, andthen make it slightly positive.

2) Reduce the Detector Gain untilthe red pixels only just disappear.

Both Detector Gain and AmplifierOffset saturated

gain andoffset notcorrect

gain andoffsetcorrect

no blueno red



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Adjusting the Laser, Gain and Offset using a Multi Track Configuration

Each channel is selected independently byclicking on the colour button indicating thechannel i.e. Ch2-T1 (Channel 2, Track 1).The laser power and all other parametersare optimised as described in the previousslides for each selected channel.

For accurate colocalisation, adjusteach Pinhole so that each channelhas the same Optical Slice

To adjust laser, gain or offset for a single track in a multi-trackconfiguration it is possible to temporarily deactivate the othertracks in the Configuration control



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Setting up Gain and Offset - Multi Track1) Select Split XY in the Image window

2) In Palette, select Range indicator

3) Select each channel separately under Channels in the Scan controlwindow and adjust the Laser intensity, Detector Gain, and Amplifier Offsetas described previously.



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Line Averaging

2) Select Number for averaging. Themore the better for the signal to noiseratio (max 16) in this case, each line willbe scanned 4 times. But: Averagingincreases the exposure time of thesample!!

Averaging improves the image byincreasing the signal : noise ratio

Averaging can be achieved line by line,or frame by frame

1) Select Line or Frame under Mode inScan Average within the Mode panel ofthe Scan Control window



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Frame Averaging1) Select Frame

Frame averaging helps reducephotobleaching, but does not give quitesuch a smooth image. There is also alonger delay between each track whenusing “Multi Track”.

Continuous averaging has a Finish buttonwhich allows the scan currently in progress tobe completed before stopping

2) Select the Number for averaging - The more thebetter for signal to noise ratio (max 16).Continuous averaging is possible in this mode.



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Collecting an Averaged Image

1) Under Scan Average selectthe Number for the average.

In the Channels panel of theScan Control window selectSingle. An averaged image willbe collected.

Range indicator setto No Palette



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• Data storage


Contents



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Scanning a Z-Series using Mark First/Last

1) Select Z Stack

2) Start scanning using Fast XY or XY cont

3) Keep your eye on the image and move thefocus to the beginning of the Z-Series, thenselect Mark First

4) Move the focus back in the opposite directionto the end of the Z-Series, then select Mark Last

5) X:Y:Z = 1:1:1 sets the Z-interval so that thevoxel has identical dimensions in X, Y, and Z.

6) Start will initiate the acquisition of the Z-Stack. The acquisition can be stopped at anytime.

NOTEFocusing can be achieved manually (preferred),or using Stage in the LSM menu if there is amotorized scanning table.

Focus

Increment



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Using Auto Z Brightness Correction

Auto Z provides an automatic gradual adjustment of DetectorGain, Amplifier Offset, Amplifier Gain, and Laser intensitysetting between the first and last optical slice of a Z Stack.

1) After defining the Z position of the first and last optical sliceactivate Auto Z.

2) Move to the First Slice and adjust the parameter for theimage acquisition in the Channels panel for each usedchannel as described in the previous slides. Then click onSet A to store the values.

3) Repeat the procedure after moving to the Last Slice. Clickon Set B to store the parameters for the last slice.

Note: Positions A and B do not have to be the first and lastslice of a stack and can also be defined simply by focussingto the appropriate positions, adjusting the parameters andpressing Set A or Set B.

4) The parameters for image acquisition will be gradually andlinearly adjusted between the first and last slice of the ZStack. Thus signal intensity and image quality is comparablethroughout the Z Stack.



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Confocal Z SectioningNumber of Sections for correct sampling

Optical thickness d depends on:• Wavelength λ• Objective lens, N.A.• Refractive index n• Pinhole diameter P

For Z-sectioning it is optimal to have:no missing information @ minimal number of sections

Slices overlap by the half of their thickness

„Nyquist-“ or Sampling- Theorem

d ~ P n λ / (N.A)2

~ 0.5 µm @ 63x1.4

The optical slicethickness is displayedin the Scan Control



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Z Stack – Number of Slices and Increment

1) Select Z slice - the windowOptical Slice will appear

For more or less sections -adjust Num Slices

2) Select Optimal interval thecomputer will calculate theoptimum number of sections

3) Select Start toacquire thecompletestack



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Z - Series using Z Sectioning

1) Select Z Stack

2) Select Z Sectioning

3) Select Line Sel

4) Select the large arrowbutton and position theXZ cut line

XZ cutline will bedisplayed as diagramwithin the XY image



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1) Decide whether to Keep Interval(number of slices will change) or KeepSlice (Interval between slices will beadjusted)

2) Select Range and position bars todecide where the Z - Series begins andends

3) Select Start for image acquisition

Z Sectioning – Setting Range

Pull redlines toset limitsfor Z-Series

Pressing Range produces an XZ image ofselected Z-range, plus 50% above and belowthe selected stack.

Draggreen lineto changefocusposition



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Viewing a Z - Series

In the image window

1) Select xy

2) Select Slice

3) Use scroll bar toview individualsections



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Viewing a Z - Series using Gallery

1) Select Gallery

2) Select Data forscale

Use Subset toextract sections



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Viewing a Z- Series using Orthogonal Sections

1) Select Ortho

2) Select mouse(Select)

3) Using the mouse,position the cut lines.

To save orthogonalsections, selectExport and save ascontents of imagewindow.



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Selecting and Saving a Region of Interest (ROI)

1) SelectOverlay anddefine shapeof ROI

2) Extractregioncreates a Z-Stack fromthe ROI

3) Save data



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Using a ROI for faster image acquisition and data saving

1) Select EditROI from the LSM menu bar2) Select Fit Frame Size to bounding Rectangle3) Choose shape of ROI

4) Position and size the ROIin the image with the mouse

5) Start Scan

To remove ROI and overlay select blue binor deactivate ROI. Closing the window onlyremoves overlay, ROI is still active.Deactivate Use ROI in the LSM menu.



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Multiple Regions of Interest1) Un-select Fit Frame Size to bounding Rectangle, Choose shapes of ROIs

4) Position and size the ROIs with mouse

5) Start ScanTo remove ROIs and overlay select blue bin or deactivate ROIs. Closing the windowonly removes overlay, ROIs are still active. Deactivate Use ROI in the LSM menu.



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Time Series• Set up scanning parameters

for image acquisition asdescribed in previous slides

• Select TimeSeries from theLSM menu

• Enter the Number of cycles

• For a Time Delay betweenimage acquisition select min,sec or ms and set time withthe slider

• Select Start T to start imageacquisition

• Instead of using Manual youcan select Time to start andstop the series at a certainsystem time!



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Viewing a Time Series of a Z StackZ Sectionsfor any time

Time points forany Z Section

Both Z sectionsand time series



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Time Series – Physiology Experiments

1) If required, usemultiple regions ofinterest

2) Set up Time Seriesas before

3) Instead of usingStartT selectMeanROI to startscanning

View and savedata byselectingMean in theimage window



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Imaging a large area using Tile Scan

This function is onlyavailable with amotorized stage

1) Select Stage on LSMmenu

2) Enter the Tile Numbers

3) Select Start

The maximum size is4096 x4096 pixels



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Tiled ImageAny position can thenbe marked and asingle image acquiredby selecting Move toand then single



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• Data storage


Contents



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Saving Data - Using Database

1) Select Save or Save as on image window or LSM menubar

2) Enter file name and notes if required

3) Select OK



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Saving Data – Using Export

1) Select File from LSM menu

2) Select Export

3) Select Image type

4) Select Single image with raw data(No overlay or Look up table etc.is saved), Series with raw data, orContents of the image window(Saves the image as shown onthe screen)

5) Select Save as type

Tif - Tagged image File” is OK for8 bit - use “Tiff -16 bit” for 12 bitacquired images(Most other software will notrecognize 12 bit)



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Shut Down Procedure

1. Go to: Acquire in the LSM menu - Laser – and deactivate HeNe Lasers byclicking Off to switch off Lasers

2. Go to File - Exit to leave LSM 510 program3. Shut down the computer4. Wait until fan of Argon laser has switched off.5. Turn off the remote control box6. Switch off the mercury vapour lamp.

Note: To turn off the Argon laser,first click on Standby, then reduceoutput power to 25%. Select Off.



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Please note:This guided tour is intended merelyas a quick introduction into theZeiss LSM 510 software and doesnot cover all aspects of the system.

Please consult the manualfor detailed instructions!



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This guided tour is based onwork done by

Peter JordanICRF

LondonUnited Kingdom

edited and complemented byEva Simbürger, Solveig Hehl and René Hessling

Carl Zeiss Jena GmbH

Documents

Guided Tour for LSM 510 and META - bio.umass.edu · Confocal microscopy Zeiss LSM 510 and Zeiss LSM 510 META Visualisation of biological structures in 3D. ... Select drive C or D: