Live_Data_Mode_SP8_AppLetter_EN
Contents
1. O Focus Offset Live Data Mode Configuration LDM Pattern x ob 1 pause 1 55 Job 2 Experiments Acquisition Load Save single setting Leica Settings gt v Acquisition Mode W2 v 0 00 0 00 XY 51251 400 Hz 4 00 0 69 AU 7 Stack 1 Steps v Autofocus Best Visible OEE Settings z Galvo Focusdrive Focus Modes FirstChannelOnly Objective CPLAN Analyze Type Contrast Based Meth Capture Range microns specimen Figure 19 Autofocus Best Focus window 5 Define hardware settings for a fast Best Focus job Scan mode with z dimension faster scan speed smaller pixel format etc For a very fast Best Focus job one can use the Analyze Type Intensity Based Method in combination with a small pixel format high zoom and low laser power e g 64x64 zoom factor 6 12 low Ar laser power set the Speed Accuracy slider to a middle position 6 Link a data acquisition job with the Best Focus job This will assign the focus position of the Best Focus job to the data acquisition job Therefore right click on the data acquisition job Select the option Use Focus and choose the job that is defined as the Best Focus job here Job1 Figure 20A Link other jobs in the same way 7 A linked job is indicated with a sign Figure 20A red arrow Undo the link by choosing None Use Focus of Focus Offset Figure 20 Linking a data acqu2. at 4 gt CATION LET m LICATIONLETTER 7 i 5 4 4 r kd ut gt a A a x 7 Ta sa Ni a DS F Lp L amp D 4 ptt An A Contents 1 4 4 2 43 44 Titel page Introduction 3 How to get started 4 Definition of an experiment 5 Continuous data acquisition 6 Tools in detail 7 The main tool set Insert Add Load Save Loop View jobs without hardware change 7 Moving and deleting record elements 9 Defining Pauses and Triggers 9 Compensation of focus drift 11 Execution of an LDM experiment 14 Data handling 15 Small images Heart muscle cell cardiomyocyte labeled with Fluo4 for Ca imaging short part of a complex time series Courtesy Leif Hove Madsen Catalan Cardiovascular Institute Barcelona Spain Observe user manual Follow the safety notes and instructions in the user manual WARNING Risk of permanent eye and skin damage from laser radation Skin and eye damage can occur while using lasers if safety precautions are not taken Pay particular attention to the laser safety A GUIDE THROUGH THE LIVE DATA MODE LIVE DATA MODE WITH LEICA TCS SP8 1 Introduction The Live Data Mode LDM tool is designed for live cell ex
3. sequently an experiment can be composed by several jobs Every job can be individually defined to accommodate the different imaging conditions during a complete experiment All parameters like scanning format speed zoom factor excitation laser selection of detection channel etc can be specifically assigned to a job LDM Pattern New Confocal Job New Pause New Pattem New Sequential Job Reference to Existing Record Record from File voguisition LDM Pattem pattern 3 FY EB OQ 8 Figure 5 A second pattern was defined A job or a pattern that works well for a certain application can be saved for further experiment sessions For complex experi ment conditions a pattern can also be integrated into another pattern thereby containing some jobs Figure 5 Thus experi ment patterns can be easily re assembled and re combined This will save the time for the redefinition of a new pattern To add a pattern into the time line click the gt button and choose New Pattern from the upcoming list The added pat tern is shown as a record element in the time line Figure 6 For every new pattern a tab appears next to the LDM Pattern tab that starts with Pattern 3 By clicking on the respective tab a job can be defined Note As long as a pattern does not contain any job there is no access to the hardware settings and the beam path window remains empty Job 2 Figure 3 LDM Pattern Options to add records
4. Load Save single setting job pause pattern User Settings gt ka a 5 5 2 8 gt Figure 4 Displayed hardware settings for Job left and Job2 right LDM Pallern Pattern 3 FH 566166 x x Job 1 Pattern 3 Figure 6 Definition of a new pattern inside the LDM Pattern Possible experimental designs just a few examples e Timelapse xyt slow speed timelapse xyt fast speed e Timelapse xyt slow speed pause timelapse xyt fast speed e Timelapse xyt stack xyz timelapse xyt 3 1 Continuous data acquisition is a valueable tool to obtain significant information of changes in the sample during a time lapse experiment This could be the behavior of cells treated with a specific drug with unclear effects move ments of cells within a tissue tumor embryos or a group of cells that appear to yield interesting information at a cer tain time point For above described applications a tool for continuous da ta acquisition in a time lapse would be helpful On the other hand the possibility to assign other scan settings at a certain time point like higher scanspeed and higher zoom factor to catch all details can be required The Live Data Mode can handle such a request by a job that works especially for continuous data acquisition in the Acquire until stopped mode This so called Default job is useful when observing a sample continuously over time Whenever the
5. formats TIFF JPEG QuickTime AVI etc In order to analyze all jobs of a sequence together in a quantification chart 26 click the respective sequence in the projects tree www leica microsystems com etca MICROSYSTEMS The statement by Ernst Leitz in 1907 With the User For the User describes the fruitful collaboration with end users and driving force of innovation at Leica Microsystems We have developed five brand values to live up to this tradition Pioneering High end Quality Team Spirit Dedication to Science and Continuous Improvement For us living up to these values means Living up to Life Leica Microsystems operates globally in three divisions where we rank with the market leaders LIFE SCIENCE DIVISION The Leica Microsystems Life Science Division supports the imaging needs of the scientific community with advanced innovation and technical expertise for the visualization measurement and analysis of microstructures Our strong focus on understanding scientific applications puts Leica Microsystems customers at the leading edge of science INDUSTRY DIVISION The Leica Microsystems Industry Division s focus Is to support customers pursuit of the highest quality end result Leica Microsystems provide the best and most innovative imaging systems to see measure and analyze the microstructures in routine and research industrial applications materials science quality control forensic science investig
6. to correct focus first using the software based Best Focus second using the hardware based Adaptive Focus Control AFC or third using a combination of both Upright systems and MP systems do not provide the hard ware based AFC Here instead Best Focus is used Best Focus always finds cells due to intensity or contrast chang es no matter if the cells have been moved in the mean time In the following paragraphs focus drift compensation using Best Focus as well as using AFC are described 4 41 Working Principle of Best Focus A very fast scanning Best Focus job is defined Image acqui sition jobs can be linked to the Best Focus job to assign the corrected focus position to these jobs Follow the steps below to take full advantage of this functionality 1 Define a pattern with at least one job in the time line 2 Insert or define a second job that should be used as a focus job 3 Click on the Autofocus panel Figure 19 Now the Autofocus window Figure 19 opens and the Best Focus function is assigned to the job indicated by a symbol Figure 19 4 Define these settings inside the Best Focus dialog Focusdrive z galvo or z wide Focus Modes Ana lyze Type Speed Accuracy and No of Steps Fur ther options are available for time series upon a click on the Timelapse button Figure 21 Trigger Rename Leica Settings 3 efine Loop Save Remove ft Delete a D Use Focus of
7. Demand Mode and wish to acquire z stacks as part of an experiment follow these steps Configure the settings for acquiring a z stack in the z stack dialog At the end click the Move To Center button This position center of the z stack is the focus position for the AFC function it will be applied to the experiment workflow via Set Experiment Position WARNING Autofocus APC Best Focus AFC Best Focus Best Focus Adaptive Focus Control AFC AFC Best Focus Focus System AFC Pos Objective 10x Max Operation Mode On demand Mode Set Experiment Position L Store AFC Pos Figure 23 AFC control AFC is active and the experiment position is set The experiment position is also indicated within the focus range Risk of permanent eye and skin damage from laser radation From this time on laser radiation may be present in the specimen area of the laser scanning microscope Make sure to follow the safety notes for operation of the system 5 Execution of an LDM experiment The following function buttons for the execution of the LDM experiment are located at the bottom of the acquisi tion window Autofocus Live Start Record Start Pattern 1 9 6 Figure 24 Functions for excecuting an LMD experiment Autofocus finds the focus of a selected job with Start Record executes a single job by first clicking the Focus Mode selected in the Autofocus the respective job element and t
8. al XyZ positions z positions or even objective lenses without any x y and z movement of the motorized xy stage or the 7 galvo stage Further the objective lens will not be changed when the View jobs without hardware change function is active Click the View jobs without hardware change button go to another job to check the settings and this job is now highlighted Finally deactivate the View jobs without hardware change button by another click Now the frame that marks a job automatically jumps back to the job from which the View jobs without hardware change button was activated A GUIDE THROUGH THE LIVE DATA MODE LIVE DATA MODE WITH LEICA TCS SP8 4 2 Moving and deleting record elements To move a record element to a different position within the time line select the element keep the left mouse button pressed and move the element horizontally along the time line to the desired position Move at least over the middle of the record you want to jump over All records can either be removed or deleted A removed record vanishes from the time line but Is still in the record items list To remove a record right click on the respective element and choose Remove in the context menu Figure 14 Click ing the x in the upper right corner of the record elements will remove the record from the time line Delete takes the record off the record items list too Trigger Rename Define Loop gle setting Leica 5 Save Re
9. ation and educational applications MEDICAL DIVISION The Leica Microsystems Medical Division s focus is to partner with and support surgeons and their care of patients with the highest quality most Innovative surgical microscope technology today and into the future Leica Microsystems an international company with a strong network of worldwide customer services Active worldwide Tel Fax Australia North Ryde 61 28870 3500 2 9878 1055 Austria Vienna 43 1 486 80 500 1 486 80 50 30 Belgium Diegem 32 279098 50 2790 98 68 Canada Concord Ontario 1 800 248 0123 847 4050164 Denmark Ballerup 45 4454 0101 4454 0111 France Nanterre Cedex 33 811 000 664 156 05 23 23 Germany Wetzlar 49 64 41 29 40 00 6441 29 41 55 Italy Milan 39 02 574 861 02 574 03392 Japan Tokyo 81 3 5421 2800 35421 2896 Korea Seoul 82 251465 43 251465 48 Netherlands Rijswijk 31 70 4132 100 70 4132 109 People s Rep of China HongKong 852 2564 6699 2564 4163 Shanghai 86 21 6039 6000 21 6387 6698 Portugal Lisbon 351 21 388 9112 21 385 4668 Singapore 65 6779 7823 6773 0628 Spain Barcelona 34 93 494 95 30 93 494 95 32 Sweden Kista 46 8 625 45 45 8 625 45 10 Switzerland Heerbrugg 41 717263434 717263444 United Kingdom Milton Keynes 44 800 298 2344 1908 246312 USA Buffalo Grove lllinois 1 800 248 0123 847 405 0164
10. e for defining the focus position cannot be found Pos gray No focus position set Pos red Focus position defined but not held Pos green Focus position defined and held Operation modes Continuous Mode The Continuous Mode can be used to maintain a speci fied focus position until the z position is manually changed e g via the focus wheel on the microscope stand the remote control module SmartMove or the Smartlouch Panel STP6000 Once you have configured the focus posi tion for your experiment activate Continuous Mode with Hold Current Position The specific focus position is then held and indicated by Hold in the graphic display of the operating range Note When using the Continuous Mode in an imaging experiment the AFC performs the focus search prior to the Image acquisition RESOLUTION TRIGGERING WITH LEICA TCS SP8 AND DM6000 CFS Operation modes On Demand Mode The On Demand Mode integrates the AFC into an ex periment workflow During the process of an experiment the focusing is only carried out at certain points of time and or at certain positions which can be defined in the dialogs Execution of Focus in Timelapse and or Exe cution of Focus in Mark Find Once you have config ured the focus position enable Set Experiment Position to apply it to the experiment workflow The focus position is Indicated by Exp in the graphic display of the operating range Figure 23 Note If you are working in On
11. ecting Trigger the Trigger Set tings window opens Figure 18 In this dialog input triggers Trigger IN and output triggers Trigger OUT are selected Different options are available including triggering within xt scans A defined trigger is automatically assigned to the selected job For more information about triggering refer to Confocal Application Letter 45 Triggering with Leica TCS SP8 and DMI6000 CFS An Application Guide 1593104032 June 2014 e ause4 55 Figure 17 Trigger selection Trigger IN on Frames Trigger OUT on Function Start Trigger OUT on Function End Trigger Line None Trigger Line Nine JS Record in Event List SY Record in Event List Show in Graph Show in Graph Description Description Trigger OUT on Frames Trigger Line w Record in Event List SY Show in Graph Description Trigger Line Nine JS Record in Event List Show in Graph Description Figure 18 Settings dialog for input Trigger IN and output trigger Trigger OUT E ANTA Trigger Rename Define Loop Save Remove Delete A GUIDE THROUGH THE LIVE DATA MODE LIVE DATA MODE WITH LEICA TCS SP8 4 4 Compensation of focus drift Long term experiments usually need a focus correction over time to compensate for any focus drift Live Data Mode allows focus corrections to be integrated into the time line which enables a fast focus correction during a running experiment There are three ways
12. eference to the cover glass of the sample which 15 generated by means of an LED generated reflex at the boundary surface Using the AFC drifts of the whole sample in the z direction which for example are induced by temperature changes are corrected by following these movements with the objective focus Note Movements of objects within the sample such as migrating cells cannot be corrected For this purpose Best Focus can be specified In addition AFC and Best Focus can be performed in a sequence The AFC keeps the reference position within the specimen while the Best Focus searches for the z position of cells of interest From this time on laser radiation may be present in the specimen area of the laser scanning microscope Make sure to follow the safety notes for operation of the system Figure 22 Potential color combina tions displaying different states of the AFC in the software AFC states The AFC function can be activated by choosing Adaptive Focus Control or AFC Best Focus from the Focus Systems list Two indicators AFC and Pos show the Status of the AFC in the software Figure 22 and 23 and at the front of the microscope The colors of the display Figure 22 have the following meaning AFC gray Inactive AFC green Active AFC orange Water immersion objective in use two bound ary surfaces exist between water immersion and cover slip or between coverslip and medium it is possible that a reference plan
13. ent eye and skin damage from laser radation From this time on laser radiation may be present in the specimen area of the laser scanning microscope Make sure to follow the safety notes for operation of the system A GUIDE THROUGH THE LIVE DATA MODE LIVE DATA MODE WITH LEICA TCS SP8 4 10015 in detall 41 The main tool set Insert Add Load Save Loop View jobs without hardware change LDM Pattern Load Save Loop View jobs without hardware change Figure 9 Tools for designing experiment patterns The new user interface facilitates the design of complex and long term experiments The most important tools are located directly above the time line Figure 9 The follow ing section explains the tools in detail Insert and Add Insert lt Integrates a new record job pause and pattern into the time line on the left side of the selected record element Add gt Appends the new record at the end of the time line Every newly added job will contain the same settings as the job that was selected during the addition For example if the element of Job1 is selected the settings of Job1 are automatically applied to the new job Upon a click on Insert lt or Add gt the following op tions are available for inserting and adding into the time line Figure 3 1 New Confocal Job creates a job for an experiment without sequential scan mode 2 New Camera Job creates a job for an experiment w
14. experiment makes it necessary a different hardware setting from a second job with a defined duration can be added Figure 7 Furthemore whenever needed for the experiment it is possible to define and execute several additional jobs with different hardware settings To get access to the Default job all displayed patterns need to be deleted Then the user interface shows the Default job Figure 7 Default e Timelapse xyt timelapse xyt triggered by external device for stimulation drug delivery Multiposition xy xyz xyxt at different z position for each timelapse e Multiposition xy xyz xyxt with objective change at different positions Continuous data acquisition fe Acquire until stopped Time Interval Minimize Acquire Until Stopped Duration Frames Figure 8 Acquire Until Stopped is active in the default setting At any time during the running of a Default job the addi tional job can be selected and executed by a click on Start Record When this job is accomplished the system auto matically switches back to the Default job and continues with the data acquisition For this purpose the Default job is set to the option Acquire Until Stopped within the t dialog Figure 8 Note The option Acquire Until Stopped is only available for the Default job The default setting can be freely changed but cannot be deleted Figure 7 A second job was added to the Default job WARNING Risk of perman
15. hen choosing Start Best Focus dialog Record The beam path window displays the Live starts a live scan with the settings of the hardware settings of the selected job selected job for image optimization Start Pattern executes the pattern selected from Pause interrupts data acquisition and restarts the the list of tabs experiment when the Pause button Is clicked again Skip Job skips the currently executed record Leica Infobar shows important process notifi Stop general stop function that stops the experiment cations Job and Pattern WARNING Risk of permanent eye and skin damage from laser radation From this time on laser radiation may be present in the specimen area of the laser scanning microscope Make sure to follow the safety notes for operation of the system RESOLUTION 6 Data handling LDM Pattern p gt Pause 1 Projects Acquisition Load Save single setting Leica Settings 4 Project 1 6 MB aBSequence 786 KB Job 1 001 262 KB xyt Job 2 002 262 KB xyt Job 3 003 262 KB xyt 4 GySequence_001 786 KB Job 1 004 262 KB xyt Job 2 005 262 KB xyt Job 3 006 262 KB Figure 25 Projects tree Figure 26 Quantification chart Each experiment is saved as a file named Sequence containing the executed jobs Figure 25 Click on the jobs to view the corresponding image series Data can be exported in the most common file
16. ile the shift key is pressed Finally click the Define Loop button above the time line or choose Define Loop in the context menu opened by right clicking Figure 11B Now the loop is defined and indicated by a line The com plete count of executions is indicated in the left corner of the line Figure 12 To customize the count double click on the number to open an edit line To delete the loop click the cross in the right corner of the loop line Job 3 Trigger Rename Define Loop Save Remove Delete Figure 13 View jobs without hardware change View jobs without hardware change some experiments require viewing and changing hardware settings in different jobs without applying them to the hardware at the same time The function View jobs with out hardware change is designed for such requirements When the View jobs without hardware change button is activated Figure 13 all data acquisition buttons are inac tive As the changes have to be carried out without active scanning offline viewing of job settings is done inde pendently from the hardware settings that means without any hardware changes Now the hardware settings of jobs can also be modified However those modifications do not affect the current hardware settings This makes it possible to view and modify hardware set tings laser intensity number of detection channels dura tion of time lapse etc of jobs containing also individu
17. isition job to a Best Focus Job A and defining an offset B x pause 2 5s 000 0 00 626 0 00 0 00 0 00 0 00 000 0 00000 498 4 6 488 501 514 520 S68 641 WARNING Risk of permanent eye and skin damage from laser radation Offset for Best Focus adjustment To ensure the stability of the focus with respect to a certain structure within a cell an offset can be set The offset is automatically set when focusing on the structure of interest e g the cell nucleus The offset is indicated Figure 20B and the z position of the offset is displayed on the control panel Best Focus job within a time lapse experiment The execution frequency of a Best Focus job during a time lapse experiment xyt xyzt can be defined by clicking the Timelapse button Figure 21 A cycle represents one frame in a xyt experiment or one z stack in a xyzt experi ment The Timelapse button is active after a t series has been defined T Autofocus Best Focus Focus System Best Focus Settings Timelapse Execution of Focus in Timelapse First Cycle Only Every Cycle Every 10 th cycle Figure 21 Setting the execution frequency of the Best Focus job 4 4 2 Adaptive Focus Control AFC Using the hardware based AFC a focus position can be specified that is maintained while manually searching for the sample and or throughout the course of an automated imaging experiment e g setup via LMD The focus position is held in r
18. ith camera detection Only available for systems equipped with a digital camera 3 New Pause definition of a pause between jobs or patterns 4 New Pattern definition of a new pattern for further definition of jobs 5 New Sequential Job creates a job for an experiment using sequential scan mode When this option is selected the sequential scan dialog appears automati cally in the Acquisition tab on the left side x Pause 1 10s Job 2 Oo Select Record Items y Jobs 3 Job 1 Job 2 Job 3 SeqJobs 0 Patterns 0 w Pauses 1 Pause 1 Insert Delete 1 40 OIL Figure 10 Example for a reference Job 1 in the list is the reference of both Job 1 records in the time line 6 Reference to Existing Record opens a list of record items showing all defined records for the recent LDM session Figure 10 Each of these records can be inserted into the time line by selecting the respective record and clicking Insert Each inserted record from the list is a reference to the already existing one e g Job 1 Thus changes in one Job 1 record are con ferred automatically to all other Job 1 records This provides an easy method of multiplying a record Note Job records with the same name but not linked as references get the extension 01 02 etc in order to differentiate them This happens when a job is loaded that has already been saved or when a job has been renamed In this ca
19. move 0 00 0 00 Figure 14 Removing a record 4 3 Defining Pauses and Triggers Pauses some experiments require pausing of data acquisition for a defined time e g for drug delivery to cells or to pass the time of inactivity in cells or developing embryos To pause the data acquisition for a defined time click on Insert lt or Add gt and select New Pause Figure 15 LDM Pattern New Confocal Job New Pause New Pattem New Sequential Job Reference to Existing Record Record from File Figure 15 Definition of a pause Right click on the inserted pause A context menu now ap pears that shows the pause duration at the bottom Figure 16 Double click the edit line to change the duration Pauses can be defined in several milliseconds up to days During a pause no data Is recorded and memory space is saved Trigger Rename Define Loop Save Remove Delete Figure 16 Changing of the duration of a pause Triggers Trigger functions can be used to synchronize the scan pro cess with external devices such as patch pipettes and pumping devices A prerequisite for these applications is that the system be equipped with the trigger unit by Leica Microsystems The Trigger option can be found with a right click on a job or pause To assign a trigger to an individual job or pause select the respective element in the time line A subsequent right click opens a context menu with several options Figure 17 By sel
20. periments and dynamic investigations such as cell physiology The ability to predefine experimental settings within time lapses for more advanced applications is crucial Manipula tion of the living specimen by the scientist e g application of a drug data acquisition start triggered by external devices or the pause of an experiment is a prerequisite to good experimental control To optimize live cell applica tions with a confocal microscope It is beneficial to have online signal intensity information from the specimen Furthermore the ability to optimize and interact with hard ware control settings such as adjusting laser power scan ning speed or other imaging parameters can Improve experimental conditions and prolong cell viability The LDM tool fulfills all these requirements It allows the scientist to define an experiment before data acquisition These experiments can be composed of several time lapses jobs A life clock and frame counter record the progress and duration of the experiment Input and output triggers are adjustable and ROI scanning is available for bleaching experiments For a fast overview online ROl based data evaluation is also available The software simplifies the general handling and in partic ular makes the definition of complex experiments easy to achieve This document guides you through the functions of Live Data Mode and gives you a fast understanding of how to use the tool The following
21. se changes will not transfer to the other records 7 Record from File a job pattern or pause saved on the hard disc can be added or inserted into the time line Trigger Rename Define Loop Save Remove Delete 0 00 0 00 Figure 11 Save and define loop buttons Job 2 Figure 12 Setting the execution count for the loop Job1 Job3 Load A click on this button opens the load dialog for loading saved jobs or patterns These records will be integrated into the time line to the left of the highlighted record Save This function opens the save dialog for saving currently selected jobs or patterns Choose Selected Record in the context menu to save a selected record from the time line choose Pattern to save the pattern currently selected in the list of tabs Alternatively right click the respective record element in the time line and choose Save in the context menu Figure 11A To save a pattern right click the respective tab and choose Save Loop In live cell imaging it might be important to repeat the complete experiment or just a part of the experiment one or more times This can be done in LMD by defining loops A loop definition can be freely chosen for a single job pause or pattern or for a group of records To define a loop a single record or a group of records have to be selected Multiple selections can be performed by selecting the first record and then clicking the last record of the loop body wh
22. terms are used to describe the LDM tool An experiment is designed within a Pattern that can be composed of one or several parts A job describes one part of the experiment with a specific hardware setting time lapse or stack etc A pattern can contain several jobs and pauses no data acquisition Features of Live Data Mode e Easy design of complex experiments composed of time lapses image stacks jobs e Focus drift compensation in time lapse experiments by hardware software based autofocus e Synchronization of scanning process with external devices input and output triggers e Intuitive user interface control by multiple selections for deleting and removing loop definition repositioning of jobs by drag and drop renaming of jobs pauses and patterns e Free time definition of pauses from milliseconds up to days e Data acquisitions parameters for each job can be viewed without changing the hardware 2 Howto get started A How can the LDM be accessed Use chosen hardware a instrument parameter settings in the LAS AF beampath window focus on the specimen and begin imaging by Experiments pressing the Live button Next open the Live Data Mode tool by clicking Live Data Mode in the list of wizards Figure 1 TCS SP8 ElectroPhysiology By entering the Live Data Mode tool the user interface shows an LDM Pattern tab An experiment can be XY 512x512 Ge RAE designed using the tool but
23. tons Inside the LDM Pattern za o ope menu all subsequently defined jobs pauses and patterns are displayed in a time line Figure 2 All jobs pauses and Time Interval patterns can be freely adapted to the experiment condi is FRET AB tions Note The LDM Pattern can be freely changed Live Data Mode Configuration Acquire TOCESS Quantity Load Save single setting User Settings gt oc ROL PY OFF Set Background OFF Bleachpoint 1 Figure 2 Live Data Mode time line the job elements are located within the time line A GUIDE THROUGH THE LIVE DATA MODE 3 Definition of an experiment some experiments can be more complex and require differ ent hardware settings at a certain time within the course of the experiment For such experiments Live Data Mode 15 the tool of choice First of all hardware parameters for Job 1 like scan mode and speed laser line and power detector etc should be set To add an additional job into the time line click the gt but ton and choose New Confocal Job from the upcoming list Figure 3 All added jobs are displayed as record elements in consecutive order in the time line For each selected job the hardware settings are displayed within the beam path window and Acquisition tab Figure 4 A job can be defined as a time lapse experiment using all available time scan modes xyt xzt xt xyzt Furthermore a job can just be one frame or an image stack xyz xzy Con
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