LICMACROLIB Manual. Autofocus Laser Mode... 8 How to configure Laser Line and AutoFocus Track:... 8

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1 LICMACROLIB Manual LIC Macro - Quick Guide to setup an experiment... 3 LIC Macro - Manual to Setup Experiment... 5 First do a Sample Tilt Test (in Autofocus Macro):... 5 Autofocus Laser Mode... 8 How to configure Laser Line and AutoFocus Track:... 8 Autofocus Image Mode... 9 Focus Upper limit Warning Create OverView in the OverView macro, load an old OverView image and synchronize it New OverView OverView synchronisation after reload of OverView OverView Data Folder: Interactive mouse functions in the OverView image: Scan OverView Scan Slide Editor Experiment Plan - Create an experiment plan, or load one and redo/copy it Setup Experiment Plan in General Experiment plan Configure Experiment groups Plan root and grouping TileScan Experiment TracingScan in General Reference image and tracing test Tracing method Tracing method: Mass Center Tracing method: Correlation DATA Process Select source files Tile scan in Data Processing Concat Batch tools

2 Special features in Zen2010 Version 1.2 LIC Macro: Trigger method General Settings (Panel Transparency, Stage Speed, Parstage) Parstage Calibrate PMT gain calibration Tools-Scene Tools-Image Tools-Multitrack lambda Currently not yet completely documented features in the manual

3 LIC Macro - Quick Guide to setup an experiment To setup experiment, follow these steps: At the beginning of your experiment, open the ZEN software and load the LicMacro. The LicMacro will open in an additional window. 1. Perform sample tilt test in Autofocus macro (Optional) When the stage tilt test is done, it enables automatic Sample Tilt Compensation (STC) of the focus, while moving the stage around. The STC needs to be done again if the sample is touched or replaced. 2. Create OverView in the OverView macro, or load one old OverView and synchronize it. Beside of maintaining an OverView image, the OverView defines a hardware independent coordinate system for experiments created with the OverView module. 3. Scan in the OverView (Optional) First configure rough scan settings in Zen for the OverView scan. This is the same as usual (light path, lasers, tracks/channels, gain, etc), but doesn't need to be done at high-resolution, because the OverView scan will automatically adjust the scan setting (scan speed, dimension, etc) to do the fastest scan. The OverView scanning is not mandatory, you can run the Experiment macro directly after OverView creation, or make some marks in the OverView for position indication. The purpose of an OverView image is to give you a general impression of your sample layout, as well as a clear visual sign of the positions. So you can easily setup positions/tiles in the experiment by mouse clicking. 4. Create an experiment plan in the Experiment macro, or load and redo/copy a plan. Your next step is to set up an experiment plan and to configure each group in the Experiment macro. Each group has independent scan- and autofocus settings, they need to be configured and stored separately. Note: when storing scan settings, also current focus position is stored, applying scan settings will also apply the stored focus position. 5. Check all experiment settings and start experiment. You can test your experiment plan correctness by clicking "Check Plan"; this check will also be performed at the start of an experiment. 3

4 Once an experiment is started, its settings are locked, whether it is finished or aborted. The only way to unlock it is to redo an experiment, which will delete all stored experiment data. If you want to keep your stored experiment data, but redo the experiment, a new experiment has to be created with "Copy Current Experiment Plan", then the old experiment will be closed and a new experiment will be created with all settings copied from the old one. 4

5 LIC Macro - Manual to Setup Experiment To setup an experiment, follow these steps: To start your experiment, open Zen and load the LicMacro. The LicMacro will open in an additional window. First do a Sample Tilt Test (in Autofocus Macro): Sample Tilt Compensation (STC) is the process that automatically adjusts the focus while moving the stage around, to avoid a focus lost due to sample tilt. The focus adjust is the result of a calculation, so it does not cost extra time, and in some sense can be viewed as a "passive" autofocus method compared to the Laser Mode and the Image Mode. It is based on the assumption that the sample has a good enough flat cover glass and the sample layer is parallel to the cover glass. This is roughly true for many samples. To use STC, a test stage tilt needs to be done once, then the focus adjustment is calculated from the sample tilt value and the stage moving distance. This sample tilt value is always valid with different scan settings including objective changing, as long as the sample remains untouched on the stage. So after testing, do not touch the sample. If the sample is out of the original position, the test has to be done again. When STC is enabled, all macro controlled stage-moving is applied with automatic focus adjustment. 5

6 In Autofocus Macro activate Sample Tilt option. Read carefully the necessary prerequisites of the Sample Tilt test. Define the Stage Test radius to test the reference plane (cover glass surface) of four corners around the current stage position. Start the Sample Tilt Test Troubleshooting Sample Tilt Test does not start The Sample Tilt Test uses the Laser Mode so the Laser Mode needs to be correctly configured in the Laser mode sheet. Important: Obviously the STC is the fastest and ideal autofocus method for a wide area scanning, where the sample tilt is the major cause of focus loss. But in some cases, the conventional "active" autofocus is still needed: 1) Due to the fact that the cover glass surface is never an ideal flat plane, normally a um focus error per mm stage move can be expected. In experiments that need to maintain a high precision focus position, STC alone is not enough. 2) If focus loss is not due to the sample tilt, then STC cannot help. For example, long time-lapse recording may show a gradual loss of the precise focus for whatever reason: vibration accumulated mechanical error etc. Remember that STC is for sample tilt compensation only, it itself does not automatically ensure a correct focus. If the focus is not correct before moving the stage, then it will remain defocused after STC. 6

7 If a user needs to use the conventional autofocus methods, STC can still be used at the same time, because it can greatly reduce the autofocus range and frequency, thus saving time. Two modes of active AutoFocus (Laser Mode and Image Mode) are available: 7

8 Autofocus Laser Mode It is based on the detection of the sample's cover glass laser reflection (Reference Plane). This means the reference plane is the one of the cover glass surfaces with the stronger laser reflection. Best used with static samples to maintain a precise pre-defined focus position during an experiment. How to configure Laser Line and AutoFocus Track: Activate Laser Mode Sheet and define a track setting for autofocus (auto-config or customized) Activate Laser Mode Sheet Auto-Configuration using Lambda mode and laser line 633nm If the microscope has a Lambda mode, AutoFocus can use the Lambda mode for auto-configuration. The default laser line is He-Ne 633nm, which has low toxic effect on the sample due to its long wavelength and is available in most LSM system. or User Config Track Define in Zen 2010 a track for autofocus usage only in this way, that the selected laser line (i.e.633) is detectable by the track's first active channel, i.e. not filtered by beam splitters or filters, anything else (gain, alser power) is configured automatically. Initialize with current focus Before using the AutoFocus, the user needs to initialize the focus offset: first set the focus to the intended focus position, and then click the "Initialize With Current Focus" button. When the scan setting especially the objective has changed, it is better to reinitialize the focus offset, or the result may not be accurate. Note: The laser power (attenuator transmission) is adjusted automatically to get the optimum reflection. If a short wavelength laser line is used, or the sample is sensitive to toxic effects of the laser, be sure to set the "Max Laser Power" correctly to reduce the toxic effect. Always use the longest wavelength laser line if possible. Focus Search Range And Sample Thickness: The actual focus search range may not be the set "Search Range"; it may be much larger depending on different AutoFocus mode and action. Sample Thickness affects the actual search range in many ways. Normally the default sample thickness value should be OK. But for a thin sample, image mode may "miss" the sample in the fast search, if 8

9 Select the Autofocus Mode, which will be used for the experiments. this value is set too large. And for a thick sample, AutoFocus may not be able to cover all the necessary range, if this value is set too small. So it should be set to at least roughly the actual value. configured track example of user After whatever action AutoFocus has done, the original scan settings will be restored. There are certain situations where laser mode is not usable: 1) May fail if both sides of the cover glass are immersed with medium whose refractive index is very close to that of the cover glass. In such case, very weak laser reflection may be detected. 2) May not be used on a live/moving sample, as the sample may go up and down. Autofocus Image Mode Autofocus Image Mode is based on the detection of the sample's best image or the centre position along the Z axis (Reference Plane). This means in image mode, the reference plane is the best image along Z (Best Image method) or the center position along Z (Sample Center method). Activate Image Mode Sheet Initialize with current focus Before using the AutoFocus, the user needs to initialize the focus offset: first set the focus at the intended focus position, and then click the "Initialize With Current Focus" button. When the scan setting especially the objective has changed, it is better to reinitialize the focus offset, or the result may not be accurate. AutoFocus Track: Image mode uses the first active track by default, which is usually the working track of the experiment. When autofocusing, the specified track is activated and all other tracks are disabled. The autofocus track will be adjusted to scan a normal stack, i.e. the time series/tile scan/bleaching are disabled and Z-Stack is enabled. After autofocus the original state will be restored as all other autofocus action. Reference Plane Types: Best Image of Z: Finds the best contrast (standard deviation method) image along Z. This method suits for thin samples or low resolution objectives. Sample Center of Z: Finds the center of the sample by detecting the sample layer's upper and lower border along Z. When using high resolution objectives with thick samples, this method gives better results than the Best Image method. 9

10 Select Autofocus Mode, which will be used for macros Fast Scan and "Sample Thickness": Due to the fact that Image Mode is much slower than the Laser Mode, especially with a high autofocus resolution and a large range, Image Mode may firstly do a fast scan to detect the rough position if necessary, then do a fine-tune scan to reach the specified autofocus resolution. This process is decided by the macro, the only thing a user needs to know is that fast scan needs the roughly correct value of the "Sample Thickness" on the thin sample to avoid "missing" the sample with too large focus steps. BUT keep in mind: The Image mode is much slower than the Laser mode, especially with high AutoFocus resolution setting, and is not as precise and steady as the laser mode: it may give different result at different position. But in cases where the Laser Mode is not applicable, Image Mode is always available. CAUTION: FOCUS MAY GO UP, OUT OF THE UPPERLIMIT AND CAN DAMAGE BOTH THE OBJECTIVE AND THE SAMPLE! Focus Upper limit Warning During AutoFocus, focus will go up and down according to the range and offset settings in AutoFocus Panel. Normally there will be no problem, if the focus originally is at the rough sample focus position. But if the focus is originally at or very near to the upper limit position (this is very possible with a high resolution objective and is highly defocused), the focus search range may cause the focus to go beyond the upper limit, touch and squeeze the sample cover glass, and this could cause damage to both objective and the sample. To avoid this situation, AutoFocus tries to get the focus drive upper limit value from the microscope's hardware configuration and limit the focus searching range, if necessary. But in some microscopes, the hardware configuration is not correctly set, so AutoFocus may not be able to get the correct focus upper limit data. 1) Configure the focus upper limit in the microscope's hardware configuration correctly and do ZEN s Parfocal Correction, especially after installing a new high resolution objective. 2) Avoid using a large search range with a high resolution objective in AutoFocus settings, if not necessary. These both will increase the focus searching time and may pose danger to the hardware. 3) Never do AutoFocus with a large search range, while having no idea about the current focus. AutoFocus is meant to maintain focus during experiment, or fine-tune focus, when in rough focus position before experiment. But it is never designed as a "sit down and get me the focus" tool. 10

11 Create OverView in the OverView macro, load an old OverView image and synchronize it. New OverView OverView is defined as a borderless, multi-channel image plane of the sample. Images of any part of the sample can be scanned into channels of the OverView with different focus settings. One of the major features of the OverView is that it defines a microscope independent coordinate system. This system is based on the OverView image (thus it is related to the sample itself), and synchronized with the microscope through a synchronization process. Other LIC Macros - that are built on the OverView - share this feature. It enables to load and reuse the OverView and the experiment on other microscopes. When OverView is synchronized, other macros which built on the OverView (e.g. Experiment) are synchronized with the hardware automatically. Activate OverView Macro and choose new OverView function Select the resolution of the OverView and define the OverView name then Create Blank Overview. You then get an OverView image in ZEN. When a new OverView is created, it is always synchronized, a bright white rectangular, shown in the OverView, indicates current stage position and scan image size. Requirement for CREATE WITH CURRENT IMAGE: image is in a format readable by the LSM software pixel size is approximately known OR: Additional features in the macro: Create with current image. Reading of overview image from any source (other Zeiss microscopes, also of microscopes or programs from other manufacturers) and synchronization of this overview image with the slide on the actual used LSM (automatic and manual) The macro automatically scales image to the desired resolution after the foreign-overview screen is synchronized in the ZEN software. 11

12 OverView synchronisation after reload of OverView After reloading an old OverView, you have to synchronize it. In the Zen OverView image you get synchronize option Now OverView is synchronized OverView reloads after Software crash If you have restarted the LIC MacroLib or ZEN for some reason, e.g. crash, and want to resume the work with the OverView, you can load the OverView, and click "Synchronize" without defining any synchronize positions for a fast OverView reclaim. The following conditions must be satisfied for this to work: 1) The OverView was previously synchronous. 2) The sample was kept fixed all the time, so the position information of the OverView is still valid. OverView reload on a new experiment day or after sample removal OverView Synchronization is done by synchronizing multiple positions on an existing OverView. At least 3 positions (not in a line) should be synchronized for a full OverView synchronization. Otherwise only partial synchronization can be done, specifically with: One sync position: Only OV origin is adjusted (Overview shift). Two sync positions: Rotation and stage scaling are synchronized. Three sync positions: not along a line: OV tilt is fully synchronized, which does with the z coordinates. Synchronization positions can be acquired by two means: Manually set 1) Move the stage to a synchronize position by looking into the Ocular or other means. a) Good synchronization positions are positions along the border of an object or some prominent/distinct spots, which can be easily located. Synchronize positions are better scattered across the OverView, rather than located within a small area to increase the synchronize precision. 2) Adjust the focus while doing continuous/live scan until it matches with an image of the selected OV channel in the OverView. a) Because the Overview may contain multiple channels with different focus positions, the 12

13 channel which is not intended to be synchronized should be turned off in the Overview image window. b) Rotation in the scan setting can be also used to help matching, but it is not required, as the OverView rotation is not calculated from the rotation of the scan setting. c) This process can be more easily done with ZEN's image window region set to the "2 Container" mode, one for the OverView, the other for the live scanning. Or you can capture the scanned image by clicking the Capture Last Scan" button for direct comparison in "1 Container" mode. 3) Click the corresponding position in the OverView, that will add a synchronize position. Automatic image finding This method provides assistance for the synchronization position setting: 1) Move the stage to a position where it is known to be inside the scanned area of the existing OverView, and scan an image. As in the Overview scanning, the pinhole should be wide enough opened, so the record image will not be very focus-sensitive. 2) Capture the scanned image by clicking the "Capture Last Scan" button. 3) Find the matching position by clicking the "Find" button. The Macro will search in the OverView image to find the matching point with the captured image automatically. Turn off all Overview channels, which do not match with the captured image before searching the match, except the temporary channel, which contains the captured image. 4) View the matching results in a popup panel. The results are sorted by matching similarity. Click the "Add Synchronization Position" button to confirm the matching result, or click "Cancel" button and set the synchronization position by hand for fine-tune. Repeat either one of the above methods to add more synchronization positions, ideally more than 3, then press "Synchronize" Overview Synchronization- Finding Matching Image 13

14 OverView Data Folder: Each OverView creates a folder under the LIC Macro's "Data Path Root", using as the folder name the OverView name. If several OverViews are created for the same sample, they are totally independent and different OverViews without any relation to each other. The Experiment macro (or other macros that are built on the OverView) creates folders in the corresponding OverView data folder. Interactive mouse functions in the OverView image: Mouse Wheel Zoom in/out the OverView, same as selecting the "OverView Zoom 1:X" tabs. Middle Button Adjust the view to all scanned image areas, same as the "View All" toggle button. Right Button Left Button Click to center the click point and zoom to max, or drag mouse to drag & view the OverView. Perform the preselected "left button mouse function" - available at the bottom of the OverView image. When the left button is released, the specified function will be executed. To cancel an on-going preselected left button mouse function press the right mouse button or the <ESC> key, while the left mouse button is pressed down. Then the active left button mouse function will not be executed, when the left button is released. Left button mouse functions: No function in the OverView Macro User Overlay operation is only available Stage function in the OverView Macro By mouse click into the OverView image the stage will move to this stage position OV Scan function in the OverView Macro Start of the OverView Scan Mark function in the OverView Macro Using this option you can mark particular positions (set up via ocular mode) to specify the OverView scan region. Scan OverView Scan in the OverView (Optional) First configure the scan settings in Zen for the OverView scan. This is the same as usual (light path, lasers, tracks/channels, etc.), except all parameters influencing the pixel size as the OverView scan will automatically adjust the scan settings (scan speed, dimension, etc.) to do the fastest possible scan for the selected resolution. Press the OV Scan button to start OverView scanning. The OverView scanning is not mandatory, the Experiment macro can directly run after an OverView creation, or after setting some marks in the OverView to label positions of interest.. The purpose of an OverView image is to give you a general impression of your sample layout, as well as clear visual sign of the positions. So you can easily setup positions/tiles in the experiment by clicking mouse. 14

15 Save of OverView Scan settings Settings (including focus) are saved in the OverView's setting folder, and can be saved (store current scan setting) and loaded (apply to ZEN software). If not loaded, the current ZEN setting is used. Scan Mode, Frame Size, Scan Speed, Bit Depth, Line Step and the whole Scan Area settings are fully controlled by the macro; there will be no effect, if you change these settings. OV Channel Operation- OV Channels. Default name is OV1(Mapped Scan Channel), OV2(...)... By default, scan channels are mapped to OverView channels by their name. You can click the <OV Channel Operation> button to set channel mapping any way you want. For example, the ch2 channel is previously scanned to OV1 channel of OverView, later you can map it to OV2 channel, and these two channel will not interfere with each other. Or you can map ch1 channel into OV1 channel, and it will overwrite previously scanned ch2 image. OverView can have a maximum 10 OverView channels. You can click the <OV Channel Operation> button to remove unwanted OV channels. Scan Slide Editor The LIC MacroLib offers the possibility to overlay a Slide layout on the OverView image. Predefined slide layouts coming with the LICMacro can be loaded and modified or new custom slide layouts can be designed using the Scan Slide Editor.. This functionality is activated with the Slide Button at the bottom of OverView image. A slide layout in the OverviewImage can be easily manually aligned, including rotation, to match its real exact position. Activate Slide Button at the bottom of the OverView image. Click Config Slide to load a saved Layout or to create a new Slide layout To align a slide layout scan one or two small areas of your sample and move the Slide layout in the correct position. Now you can select all or only specific wells for an OverView Scan, and activate Scan Well 15

16 Experiment Plan - Create an experiment plan, or load one and redo/copy it. To start an experiment activate the Experiment Macro button and choose New Experiment. There are three options to set up an experiment plan: Create Blank Experiment Plan (default option) Create Sample Tile Scan Experiment Plan Create Sample Tracing Scan Experiment Plan Your next step is to configure each experiment group in the Experiment macro. Each group has independent scan and autofocus settings, they need to be configured and stored separately. Note: when storing a scan setting, it also stores the current focus position, applying a scan setting will also apply a stored focus position. Setup Experiment Plan in General Experiment plan The experiment plan is tree structured like a folder system. Sub groups can be added to a super group, and will be controlled by this super group. Upon experiment creation, the experiment plan contains a "root" group; the first experiment group can only be created under this "root" group. Experiment type Experiment type defines an experiment group's function. Each experiment group has some kind of experiment type. For example, a TileScan group is an experiment group of experiment type TileScan, designed to perform tile scan including scans consisting only of one tile. Different experiment types allow different types of sub groups. For example, a TracingScan group allows TileScan groups as sub groups, but does not allow other TracingScan groups as sub groups. The "root" group allows any type of experiment as sub groups, while a TileScan group does not allow any sub group. When a group is selected, the combo box beside the "Add Group" button defines, which type of sub group can be added to this group, if no sub group is allowed, the "Add Group" is disabled (greyed out). 16

17 Setup experiment plan 1) To create/rename/remove/move a group, first select a group in the plan. 2) "Add Group" - will add a group under the selected group as its subgroup, with the experiment type selected in the combo-box beside. 3) "Move Up"/"Move Down" - moves the group up and down within its super group's region, i.e. you cannot change its super group, you can only change the execution sequence along with its siblings. 4) Click the textbox under the experiment plan box to rename. Which types of experiment groups can be added as sub groups? 1) The possible types of sub groups are decided by the super group. For example, Tile Scan does not allow any sub group, while Plan Root and Grouping allow any kind of sub groups. 2) There are also some general restrictions of sub group types to prevent potential problems. For example, experiment groups with the ability to modify the scan position of the subgroups are mutually exclusively, this means a Tracing Scan group cannot be added to another Tracing Scan as sub group. Execution of an experiment group and its sub groups Once an experiment group is started, the experiment is executed first then execution of its sub groups starts. This group finishes their cycles when ALL their sub groups are finished. This means the cycle count setting effects all its sub groups. For example, if a group has cycle count of 100 and the two sub groups have cycle counts of 10 and 20, assuming the sub groups are always executed, then the first sub group will be executed 1000 times altogether and the second will be 2000 times. It also means the cycle interval of this group is the total time to complete this group and all its sub groups. Execution sequence of sibling experiment groups (groups within the same super group) The sibling experiment groups are executed in simultaneous mode, i.e. system will try to execute them by the specified interval. At the start, the first group is always being executed at once. Then, when a group is finished, system will check all sibling groups by the order they appear in the list, starting from the next group (below the finished one). The group, which has the earliest scheduled time, will be executed, if it is due. If no group is due, the system will wait. So a group with 1 second interval will be executed more frequently than the one with 2 seconds, although their actual interval is much longer because the interval settings are too short to be realistic. The only exception of the above is interval 0. If the interval is 0, this group is due when and only when the system is not busy. This actually means a group with interval 0 (except the first cycle, if it is the first group) will only have chance to be executed after a group with 1 second interval finished all its cycles, because the latter always has earlier due time and thus the system is always busy. But when the intervals of other groups are meaningful (long enough or 0), it will kick in, while the system is not busy. Configure Experiment groups When an experiment group is selected, the setting panel at the right side of the experiment plan shows the configuration of this group. The settings fall into two categories: common settings and experiment type specific settings. 17

18 Common Settings: Scan Setting and Autofocus Setting Scan setting includes the settings from Zen, which configure how the images will be scanned. In addition to ZEN it also includes the focus position. If a user wants to keep a current stored scan setting but with new current focus position, click the "Update Focus" button, and then all set z-stack positions will also be shifted correspondingly. Autofocus setting includes the settings defined in the Autofocus macro, defining which kind of autofocus will be performed. Some experiment types may also have additional settings for autofocus control in their own setting panel. "Store": "Apply": Stores current scan/autofocus settings to this group. Apply the stored scan/autofocus settings of this group to the Zen/Autofocus Macro. Some Settings in the Zen Scan Setting will be modified Generally, Zen's "Positions" will be turned off. Different type of experiment groups may also make further modifications. For example: o TileScan will turn off Zen's "Tile Scan" for the obvious reason. o TracingScan will turn off Zen's "Time Series". The rotation will be adjusted by Overview's rotation which is the result of the Overview synchronization, as well as experiment type specific adjustment. o User should be aware of the zoom settings. When zoom is less than 0.9, it will limit the rotation. If an Overview is synchronized with a large rotation or if the experiment type will generate a rotation (such as the rotation feature enabled in the TracingScan), it may cause that the scan head will not correctly rotate. Because small zoom is useful in some cases, macro does not put hard limitation on it, so the user needs to make sure, that it will not cause problems. Common Settings: Cycle Settings Cycle setting defines the number of cycles and the cycle interval of this group. Interval = 0 means no wait. These settings have also effects on its subgroups. See "Execution of experiment group and its sub groups" in help tab of "Experiment Plan". There is a "Min Interval" information at the left bottom of the panel, which indicates how long it will take approximately for this group, including all of its subgroups, to finish one cycle. It gives a hint how long the cycle interval should be set. Experiment Type Specific Settings These settings differ from one experiment type to another, but generally also include: 1) Additional autofocus control: Under which conditions to do autofocus using the stored autofocus setting. 2) Subgroup control: How to modify sub groups, how and when to start the sub groups etc. Note: Scan Setting includes Focus Position. Only when OverView is synchronized, there will be a valid focus position while using Store Scan Setting. This is also true, while using Apply Scan Setting, here also the focus position will be only applied, when OverView is synchronous. You can still store and apply a scan setting when the OverView is not synchronous or even when log-in to Zen at the start-up in the Offline mode (), and Other settings like cycles and mark tiles in Tile Scan groups, can still be set. Later when the OverView 18

19 is resynchronized, you can apply settings and (adjust focus if needed) and store them again to get the valid focus information. If Autofocus is enabled, the stored focus serves as the initial focus position to start with. Plan root and grouping Plan Root is one unique and "empty" group that does not do anything. Its sole purpose is to be the root of the experiment plan, so other experiment groups can be created under it. Grouping is another "empty" experiment type. It is used to arrange other experiment groups as its sub groups, to enable more flexible experiment plan setup. Use Grouping to set one or more experiment groups to be in "pre-emptive mode" (or "blocking mode"). Sibling experiment groups are executed in a simultaneous fashion. If one experiment group (assume group A) has a long interval and is in waiting state, other groups will be started if they are due. But if you do not want other groups to start during this group's waiting time, you can do the following: create a Grouping group, then create group A under it. Because an experiment group finishes one cycle only when all of its sub groups have finished all their cycles, the Grouping will never exit until group A finishes all its cycles, effectively setting Group A to "pre-emptive mode". If two or more groups are put under one Grouping, the same rule applies: these groups as a whole are pre-emptive - Grouping will not exit until all these groups are finished, thus other groups are blocked during this time. Among themselves, they are executed in simultaneous way as usual. Grouping cannot have scan settings nor autofocus settings. But it can have cycle settings. Grouping can have any number of sub groups of any type, including other Grouping groups. TileScan Experiment It is designed to perform multi-tile/ multi-position scans, especially for large areas. All tiles defined in one TileScan group share the same settings. After TileScan group is activated, and scan settings are stored, you can use the OverView mouse function [TileScan Mark Tiles] or [TileScan View Data]. 1) OverView mouse function [TileScan Mark Tiles]: Define tiles to be scanned on the OverView. This mouse function is visible, when the experiment is new. Tiles defined by this mouse function do not contain focus data, they use the focus stored in the scan setting. 2) OverView mouse function [TileScan View Data]: Load and view scanned experiment data. Compared to a similar function in the Data Process macro, this mouse function can only load images stored in the original experiment data folder, and load only the first image if there are many images from different cycles. Nevertheless, it is convenient to click on the OverView directly and view data after an experiment finished. This mouse function is visible, when the experiment is finished or aborted. 19

20 Root of the experiment plan Add Grouping with subgroups or directly a TileScan Group Note: A TileScan cannot have subgroups. Activate TileScan group Adjust settings in Zen Store Scan Settings for this group In OverView window: After storing scan settings for the TileScan group the OverView mouse function [TileScan Mark Tiles] becomes visible. [Loading old scanned experiment data [TileScan View Data] button will appear.] Choose Mark to specify Group Scan Area. There are different options of Alignment and Dragging 20

21 Each TileScan group has its own tiles definition. All the tiles defined in one TileScan group share the same settings. If a user wants to have tiles with different scan settings, autofocus settings or any other settings, they must be defined in different experiment groups. Focus Adjustment for tiles in one TileScan Group during experiment Although all the tiles defined in one TileScan group share the same focus, which are stored in group's scan setting, there are some possibilities to adjust the focus for each tile: 1) Sample Tilt Compensation for focus (STC): When STC is enabled, focus for different tiles are adjusted when moving the stage to the positions of the tiles, according to the Sample Tilt X/Y measured by sample tilt test. This is the fastest way of focus adjustment for large area scanning. Use STC as the main focus adjustment method instead of Autofocus, if fast tile scan is required, or in case a 2-5 um focus error is tolerable. 2) Autofocus: It has an additional autofocus control to define autofocus activating conditions. The extreme would be to activate it on each tile. STC and Autofocus can be combined together. For example, do Autofocus for the first tile only in each cycle and let STC adjust the focus for the rest of the tiles. Or do Autofocus after a certain time to prevent graduate focus lost due to accumulated mechanical error or environment vibration, and let STC take care of the rest. If both STC and Autofocus are enabled, focus is first adjusted by STC, then Autofocus is done with the pre-adjusted focus as initial focus to start with. This means even if Autofocus is done on every tile, STC can still reduce the search range for Autofocus in large area scanning. Tiles With Focus Data (Yellow colored Tiles) Tiles can also be defined from the marks of the Overview or from current state/focus position; these tiles may contain their own focus data. For these tiles, the focus position defined in the scan setting is not used, and the Stage Tilt Compensation (STC) is not activated either. However, the Autofocus (either laser mode or image mode, if Autofocus is enabled) will still be applied. In such cases the focus search starts from the focus defined in the tiles. The focus condition "Stage Moved Over xxx mm" is not applied to these tiles. The focus condition "Every xxx Cycles" is applied to every such tile, while it is only applied to the first tile of those without focus data. Note: Not all Overview marks contain focus data, thus not all tiles defined from the Overview marks contain focus data. You can tell if a mark contains focus data by checking the marks listbox in the Overview macro, or by pointing the mouse to the mark in the Overview image. If the "Z" of a mark is "None", then this mark contains no focus data. TileScan groups cannot have sub groups of any kind. Time Series and Check all experiment settings, then Start experiment. In the Experiment Macro you can set up the TimeSeries Settings. After this you can test your experiment plan correctness by clicking "Check Plan", this check will also be performed when an experiment starts. 21

22 Once an experiment is started, its settings are locked, unit it is finished or aborted. The only way to unlock it is to redo an experiment, which will delete all stored experiment data. If you want to keep your stored experiment data but redo the experiment, create a new experiment with "Copy Current Experiment Plan", then the old experiment will be closed and a new experiment will be created with all settings copied from the old one. Check Plan and Start Experiment Time Series Settings 22

23 TracingScan in General It is designed to keep track of live/moving samples, thus enables high resolution scans of them. When the TracingScan group is activated, the OverView mouse function [Tracing Position] becomes visible, for defining tracing positions on the OverView. Reference image and tracing test A reference image is pre-scanned for comparison analysis with the current tracing image scanned during the experiment. In other words, the reference image is the initial state of the traced object. The tracing method must be initialized with the reference image first by clicking "Tracing Test" before the experiment starts. When tracing position is marked/remarked on the OverView, the reference image will be scanned immediately. When tracing settings are changed, the tracing test must be done again and the reference image will be rescanned in the tracing test. When the tracing method is changed, the tracing test must be done again, but the reference image will not be rescanned. The user can tell whether a tracing test needs to be done by activating the information box at the left of "Tracing Test" button, a green colour means the tracing method is ready, yellow means the tracing test is needed, red means the tracing method is not valid. Accumulated error on reference updating Some tracing methods need to update the reference image with the last tracing image, when the traced object has changed too much. Because this updating will fix any tracing error up to this time point, which is inevitable in any tracing analysis, those systematic errors (fixed in one direction, not random errors) - caused by whatever reason - will accumulate gradually and eventually lead to a tracing failure. If this problem is expected, the updating frequency of the reference image should be reduced. Autofocus in TracingScan and the pinhole Autofocus plays a key role in the tracing scan, and since the sample is moving up and down, the image mode Autofocus is nearly mandatory. Because 3D tracing needs both long-time scanning and long-time analysis, the stock tracing method is designed to do 2D tracing and let autofocus work out the focus position. Since different focus positions will give sometimes totally different images, better Autofocus will result in tracing images, which are more similar to the reference image, thus more reliable. To minimize this focus-sensitive problem, it is strongly recommended, that the scan settings for the TracingScan use a larger pinhole to include a certain depth of the sample, therefore being less sensitive to the focus position. Depending on the thickness of the sample, commonly a pinhole around is large enough. Note the default image mode Autofocus uses the first working track; this means the Autofocus will also use a larger pinhole by default. Although autofocus precision will be reduce a little with a larger pinhole, it is generally not a big problem. Sub group control TracingScan can have sub groups of other types, e.g. TileScan. After each tracing, whatever positions are defined in the sub groups, it will be adjusted according to the tracing result, including focus position. This adjustment as a result, keeps the relative offsets and orientations defined in the 23

24 TracingScan and all sub groups unchanged. In other words, they behave like a rigid body moving and rotating as a whole. For example, if rotation is detected in the TracingScan, all the tiles defined in the TileScan sub groups will rotate around the tracing position, and their relative positions remains the same. Tracing method Because samples differ greatly, there is no easy one-for-all solution of tracing for every situation. TracingScan provides two stock methods for general purpose: Mass Center method and Correlation method. A tracing method plug-in interface is also provided to enable customized tracing for special situations. Tracing method: Mass Center The Mass Center method is the simplest, fastest yet effective way of tracing, it calculates the mass center of the image, and compares it with the mass center of the reference image. The Mass Center method is the recommended method to trace prominent objects with a clear background, which are totally inside the view, while not caring about the rotation. An excellent case is, when special fluorescent marks are labelled as "hot spots" of the sample, in such a case, it can trace the fluorescent marks forever without losing it - even with a very bad signal and focus. Add Tracing group Select Tracing Method Tracing Method needs test Scan settings have to be saved. Autofocus plays a key role in tracing scan, and since sample is moving up and down, the image mode Autofocus is nearly mandatory, selected in Autofocus window After storing the Scan Settings you get in the Overview Window the Tracing Position Button pros o Simple and fast. It does not have any additional configuration and is the default tracing method. 24

25 o o o cons o o o o Steady and never fail when tracing a prominent object with a clear background. Never needs to update the reference image, thus no accumulated error caused by the reference update. Very tolerant to noise and deformation of whatever causes: bad focus or fast sample shape/structure changing. Cannot detect rotation. This means the only correct position of sub groups is the tracing position, if the traced object rotates. It requires that the whole traced object is inside the view. Gives incorrect results if the background is not clear, especially if any other object enters into the view. Cannot work with transmission images (inversed image). Tracing method: Correlation The Correlation method is a general purpose tracing method based on image correlation analysis. It will update the reference image when the similarity between the traced image and the reference image is too low. Correlation method is designed for general purpose tracing, it performs well in most situations, but user needs to be aware of the focus and the accumulated error on reference updating. To reduce the reference updating frequency, you have to reduce the "Update Reference Image When Similarity < (%)" slider, which defines the reference updating threshold. Scan settings have to be saved Add Tracing group Select Tracing Method Tracing Method needs test Scan settings have to be saved. Autofocus plays a key role in tracing scan, and since sample is moving up and down, the image mode Autofocus is nearly mandatory, selected in Autofocus window After storing the Scan Settings you get in the Overview Window the Tracing Position Button 25

26 pros o o o o o Able to perform fast and precise rotation detection (<1 in static image test), thus can make sensible position adjustment in a wide area for sub groups, assuming these positions have the same moving pattern as the traced part. Can trace a part of the object, does not require the whole object inside the field of view. Has some tolerance for an unclear background, as long as the unintended background is not very prominent. For example, if a weaker object enters the view, it generally still can produce a correct tracing result. Has only certain tolerance on noise. Works with transmission images (inversed image). cons o o Sensitive to focus, as a different focus may give a very different image. It is strongly suggested to use a large pinhole in the scan setting for this method (Note: the sub groups of TileScan can still use small pinholes, as they use independent settings). Needs to update the reference image, if the traced object is transformed too much, thus it has a certain danger of "accumulated error on reference updating". A tracing method plug-in interface is also provided to enable customized tracing for special situations. 26

27 DATA Process Go to the Home Window and select Data Process Select Source file Additional tools in Data Process: 1. Tile Scan 2. Concat 3. Batch Tool Select source files Source files are selected by a series of filename filters. 1) General file filter It provides a standard filename wildcards filter, same as using DOS command "Dir". 2) Image Index filter It provides a convenient way to filter an image series with a numeric index, which is very often seen. For example, there are images named: Image_Tile1_T1.lsm, Image_Tile1_T2.lsm,..., Image_Tile1_T50.lsm, Image_Tile2_T1.lsm,..., Image_Tile2_T50.lsm,..., Image_Tile100_T50. So there are 100 x 50 = 5000 image files. Fill "_Tile" in the "Token in front of index", the first index get filtered. Change the value of "Index", the images with corresponding index are automatically selected. Now fill "_T" in the "Second Index", images filtered by the first index will be further filtered out. 3) Manual selection Press the"view File List -->" toggle button, the "Data Process - Selected File List" window will appear. In this window, the files can be selected manually. The selected source files are used as input file list for all data process modules. 27

28 Tile scan in Data Processing Functions here use the LICOverview coordinates. Thus the source image files can be the data of different LIC experiments, but they must be of the same Overview, or otherwise the coordinates are not consistent. Create XuvTools Profile It creates a profile (text file) which can be loaded by the XuvTools software for stitching. The profile contains original position information, thus makes the stitching process faster and easier. The profile will be created in the source image folder. If you want to copy them to another folder/computer, be sure to copy the profile along with the associated source image files with the same folder structure. It means the profile should be in the same folder as the image files, and if there are image files in sub folders, they must be in the same sub folders after copying, or else XuvTools will not be able to find the source image files. To use this function, the source files should be generated by the LICExperiment macro, or may have been processed by DataProcess macro. If they are generated from other sources, there will be no recognizable position information in the image files. Concat This function concatenates source images into one big image file. The image files are concatenated by the sorting order. The result image is generated in Zen's image window, but will not be saved automatically. The most common usage of this function is to concatenate time series images, but it is not restricted to the T dimension. For example, it can also be used to concatenate images into C dimension. In such a case images will be appended into different channels. Batch tools This is a Batch process for the selected images. The processed images are saved into the specified output folder. The Processed images retain the Meta data of the image, for example, the position data which are used by XuvTools for stitching. Most of the batch functions support "Try" method for testing purpose, before starting the timeconsuming batch process. 28

29 Special features in Zen2010 Version 1.2 LIC Macro: Trigger method In the Zen2011 compatible LIC Macro Version we support a flexible event driven trigger function for imaging triggering, which can be implemented and specified by the user themselves. Add Trigger Group for triggering Tracing Scan or Tilescan group Specify trigger settings and test Trigger method This feature will be further documented when it is finished for general use of all testers. General Settings (Panel Transparency, Stage Speed, Parstage) In this setting window you can adjust Panel Transparency, Stage Speed and activate the Parstage function. 29

30 Parstage Calibrate This calibration tool can be used for an automatic correction of different optical lens axis. This module allows a semi-automatic measurement of lens axis shift, still present focus shift and also the exact magnification (if an appropriate scale or calibration preparation is used) of all the lenses mounted in a system. The measurement is made at present semi-manually using a calibration scale. In principle any object, which remains at all magnifications in the frame, can be used for the calibration. The calculated values are written with the objective data into a text file. Select an object in Zen, apply medium if necessary. Click Scan to scan a recognizable object in the scan image. You can move the stage and focus as well as change scan zoom. After scan is completed, there will be a rectangular mark appears in the scan image. Move and resize this mark to locate the object in the scan image, then click Get Mark Position button in the macro. Note: The same object should be located and scanned for all objectives. Repeat these steps until all objectives are calibrated, then choose a filename in the Save as box and click Save. To use this data for automatic axis shift correction within the LIC macros, activate the parstage function within the settings window and open the used calibration.txt file. 30

31 PMT gain calibration (optimal or maximum useful gain adjustment) This module allows to determine semiautomatically optimal settings (PMT gain) to work with the best S / N values. Whether this macro might be useful also to the LSMs of the 700 series has not been evaluated. With the macro obtained and automatically stored images can be analysed with a MATLAB tool that was developed in collaboration with the Department of Image Processing and Pattern Recognition (Jun. Prof. O. Ronneberger and R. Bensch). Tools-Scene The Scene module allows very comfortable to save different scan configurations with preview thumbnails. All LSM settings such as standard configurations can be saved, additionally also focus values and stage positions. The user can determine when re-reading the configuration if focus and / or stage position will also be applied. Using the Scene module allows fast, complex experiments to be set-up, which contain mixed scan settings including objectives. Store scan settings and scan position in this Tool. The positions stored in this tool are raw stage/focus values, they are not related to the Overview. They are only valid when the sample is kept untouched, and there is no way to reclaim these positions, if the sample was displaced or remounted. The raw stage/focus data are dramatically dissimilar in different microscope/session, or after a microscope restart. In such cases, please never try to apply the stored position in the scene tool back to Zen, this can cause unpredictable results and/or damage the microscope or the sample! The scan settings can be re-applied at any time safely. 31

32 Tools-Image This is a collection of individual simple image manipulation functions. All manipulation functions in this tool are working also with stacks and / or series. The functions are also useful, if an Overview images, acquired by another microscope, will be used as a new Overview in the LIC macro. The functions complement further features in the Data Process Module Tools-Multitrack lambda This tool provides a multi-track acquisition in the lambda mode, which is currently not available in the ZEN software. The macro communicates closely with the ZEN software and allows setting of heterogeneous multi-channel experiments in the lambda mode in ZEN either through acquisition of settings from ZEN or by sending settings to ZEN. Whether or not this mode is available depends on the hardware. Currently only microscopes with the SP detector array are supported. Generally it is available in LSM 710 and later. 32

33 Note: This mode is unofficial and not supported by Zen, and might be changed unnoticed by later versions of Zen. Important: This function may be used with caution; we are not responsible for any fancy results. Currently not yet completely documented features in the manual Slide layout editor 33