Workflows for setting up and applying Tiles & Positions in ... · 1 [email protected] Global Sales Support / TASC Tiles & Positions (Oct ´14) Workflows for setting up and

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[email protected] Global Sales Support / TASC Tiles & Positions (Oct ´14)

Workflows for setting up and applying Tiles & Positions in ZEN

Introduction:

Customers frequently request how to use the Tiles & Positions module in ZEN blue, ask which

powerful features it possesses and want to know which potential applications can be targeted.

(The following descriptions mainly delineate workflows for a 96 Multiwell sample carrier; however,

most steps are the same for other types of carriers, e.g. conventional glass slides.)

CONTENTS

Page

System Prerequisites 2 ‐ 4

Stage calibration and channel selection 2 ‐ 3

Define Global Support Points 3 ‐ 4

Define Tiles 5 ‐ 10

Define Positions 11 ‐ 15

Assign categories to Tile Regions and Positions 16 ‐ 17

High‐magnification imaging of selected Positions & Tiles after preview scanning 18 ‐ 23

Re‐positioning of your sample carrier after incubation 24 ‐ 26

Customize your Sample Carrier 27 ‐ 30

Additional automated options for your Tiles setup 31

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SYSTEM PREREQUISITES

[Stage calibration and channel selection]

1. Put you Sample Carrier on the stage (e.g. a Multiwell 96).

2. Go to Locate.

3. Choose a low magnification objective (e.g. 10x).

4. Press the Live button and find your focus area either using transmission or fluorescence light

(needs to be set in the Light Path tab with the light intensity and exposure time being adjusted).

5. In the Light Path tab continue to do stage calibration by clicking on , activate Show all

and then .

6. You now check for a correct alignment of your camera and joystick by dragging the cursor‐like

circle up, down, right and left and observe whether the movement of your image

corresponds to movement of the circle. In addition, check whether the image movement also

corresponds accordingly when you move the joystick.

7. When this is not the case, go to the Camera tab and click Show all followed by Model Specific. In

Orientation you can now adjust the camera orientation to the joystick orientation.

Alternatively, you can and may also invert X‐ and Y‐axis of

your stage in the MTB in order to align the joystick and the software‐controlled stage movement.

8. Now you go to the Acquisition tab.

9. Do all the prerequisites (e.g. channel and camera settings) for a Tiles & Positions experiment on

your sample. For that use Smart Setup and, if needed, Experiment Designer (not advisable for

beginners).

10. After you have defined at least one channel (e.g. EGFP), activate the Tiles module checkbox.

11. Open the Tiles section in the Multidimensional Acquisition module and activate the Show all

mode.

12. Open the Sample Carrier section in the Tiles module.

13. Select a predefined Sample Carrier template from the list (e.g. Multiwell 96) by clicking on the

Select button.

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14. Choose a predefined Sample Carrier (e.g. Multiwell 96) and click OK.

[Define Global Support Points]

15. If your sample carrier does not consist of an even flatness on the imaging surface (which most

likely will be the case ), you need to tell the software reference points, so‐called Global

Support Points, in order to approximate for an even surface flatness. The necessary amount of

Global Support Points is thereby determined by the degree of interpolation the software is

supposed to calculate. In order to know how many support points you should add in the next

step, please first go to Focus Surface in the Tiles section and click on Global (on Carrier) and

choose the degree of interpolation. As a general

rule of thumb, you choose more points if your area of interest is quite large.

16. Now, you go back to Carrier selection, open the editor window and click on Copy and

Edit.

17. Click on , choose the required amount of support points (for the desired

degree of surface interpolation) in the tab and click on

Distribute.

18. If you want to move the created Global Support Points to another preferred position, click on

them and move them to your preferred location. This may be advisable when you have a carrier

where the specimen/ cells are clustered only around a certain area. Finish this step by clicking on

OK

19. You now start the calibration of your selected Sample Carrier by clicking on Calibrate in Sample

Carrier of the Tiles tab. Now, you follow the

instructions of the Calibration Wizard. (Step 1) Set an appropriate light path for your sample; (2)

‐> it is not necessary to calibrate the stage again as it was already calibrated in 5. under Locate;

(3) Select for how many points you want to do the calibration: For slides with one coverslip or

well, there is only the option for Single Reference Point Calibration – in that case move a sample

reference point (any unique identifiable point) into the middle of the crosshairs; for a multi‐well

carrier you will have the option for 7‐ point, 4‐point, 3‐point or 1‐point calibration. Take the 7‐

point method for highest accuracy. (4)‐(6) Define the requested positions. Make use of the long

lines of the crosshair to position them at the requested sites of the wells.

You have now calibrated your carrier.

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20. As a next step, it is important to verify the Z‐positions of your selected Global Support Points.

Click on Live and go to Global (on Carrier) of Focus Surface in the Tiles section and click on

.

21. Double‐click on the point to be verified, find the desired Z‐position (either manually or by clicking

on Run Autofocus) and click on

22. Subsequently, click on Move to Next Point and repeat step 21.

23. Alternatively, you can also automate this procedure by using the different Autofocus buttons in

the Verify Global Support Points window.

24. However, when you set Z‐offsets manually, you may encounter a situation where large Z‐offsets

appear for the first support points. In that case, you may speed up your verification process by

first applying a certain offset to all the remaining points and then manually or automatically just

fine‐tune them. To do so, mark all remaining points, click on and select Apply Z‐Offset…

Enter the required offset value and press (the max. offset value to be

added is 5000 µm; for a higher value you simply have to repeat this step)

You have now successfully verified your Global Support Points

To apply your Global Support Points for your experiments, go to the Focus Strategy tab in the

Left Tool Bar Area and select Global Focus Surface

25. Open the Advanced Setup in the Tiles module.

The live mode opens in the Center Screen Area and shows the predefined well plate in the

background. If you want the Live mode to be displayed in a separated window activate the

Live in Separate Container checkbox.

To zoom in and out, use the mouse scroller. Double‐click on the any area of your sample

carrier and the stage will move to that point of interest.

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DEFINE TILES

To define tile regions in your sample carrier, you have multiple options:

I)

1. To define tile regions around your current position, go to Tile Regions in the Tiles module…

… select a contour and a number of desired tiles and

press .

II)

1. To define tile regions in different wells, open the Tile Region Setup tab below the Center Screen

Area. Activate the Show All mode.

2. In the Carrier tab (lower left side, below the Center Screen area) select

well(s) of interest by holding Ctrl and clicking on the desired wells in which you want to add and

define tile regions.

The selected wells are now bordered by a blue circle. If only one well is double‐clicked, the

stage will move to the center of that well.

3. In the Tile Region Setup select Predefined under Setup by.

4. Choose how many tiles in x and y dimension you want to have and add the predefined Tile

Regions by clicking on the button.

The predefined Tile Region is now created top left, centered or bottom right in the chosen

wells depending on the anchor position .

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III)

1. When you want to image a certain area of your wells, you open the Tile Region Setup tab below

the Center Screen Area. Activate the Show All mode.

2. Select Carrier under Setup by in the Tile Region Setup.

3. Select the individual wells for which you want to create Tile regions by holding Ctrl and clicking

the desired wells in the multi‐well template in the Carrier tab (lower left side below the Center

Screen Area).

4. Now, got back to Carrier in the Tile Region Setup (lower right side below the Center Screen Area)

and enter the size of your desired area per well in the box Fill Factor and click on + Create.

According to the selected Fill Factor, the wells will be filled with a calculated number of tiles

that are located around the center.

IV)

When you want to create tiles in a certain region of your sample carrier, you open the Tile

Region Setup tab in the Center Screen Area. Activate the Show All mode.

Select Contour under Setup by in the Tile Region Setup.

Depending on the tool and anchor position you

can select a certain area within your carrier that will be filled with tiles subsequently.

For all options:

If you want to change the positions of your tiles, just click/mark them and move them with the

mouse to a new location.

For some customers it could be important that the stage is driven in a special direction during the

experiment, e.g. Tile regions of A12 should be imaged before Tile Regions of A1. Therefore, you

need to arrange the sequence of the stage‐drive as follows:

1. In order to change the acquisition sequence of Tile Regions, uncheck the Optimize

Stage Travel check box in the Options section of the Tiles tool (Left Tool Bar Area) as

its function does not apply to multi‐well containers. When not unchecked, changes in

the acquisition order will be ignored.

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2. Now, select a Tile Region of interest and use the buttons in the Tile

Regions list under Tiles to arrange the acquisition sequence of your Tile Regions.

When it is crucial to obtain in‐focus images of all of your tiles within a tile region, you can use

different focus strategies, e.g. Definite Focus, Software Autofocus, Support Points or Absolute

Fixed Z‐Position, which you have to select in the Focus Strategy tab. Definite Focus and Software

Autofocus can be configured in the Focus Devices tab additionally. Both tabs can be found in the

Left Tool Bar Area. However, proceed as follows, when you want to apply Local Support Points

within selected tile region(s):

1. Select Local Focus Surface in the Focus Strategy tab.

2. Think about the necessary amount of support points that the software needs to

apply a certain algorithm to approximate for an even surface flatness. The necessary

amount is determined by the degree of interpolation that the software is supposed

to calculate. In order to know how many support points you should add in the next

steps, go to Focus Surface in the Tiles section and click on Local (per Tile Region) and

choose the degree of interpolation.

3. After you selected a degree of interpolation (and know how many support points you

should add), you mark those tile regions to which you want to assign the support

points either in Tile Regions under Tiles or directly in the Center Screen Area.

4. You now proceed to the Support Points tab (below the Center Screen Area) and

activate Show All. Set Columns and Rows according to the desired amount of

support points and click on Distribute. In the example below, this action would

generate 4 Local Support Points for each Tile Region that was marked. These Local

Support Points are now distributed equally within the selected tile region(s). When

you just want to have one Support Point per Tile Region click on Set One Support

Point into Center Position.

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5. Once you defined all of your Local Support Points you may still verify them in X, Y or

Z by either changing the parameters that are displayed under Properties of Selected

Support Points (see image above) or by following the path Tiles module, Focus

Surface, Local (per Tile Region) and then click on

.

6. Double‐click on the point to be verified, find the desired Z‐position (either manually

or by clicking on Run Autofocus) and click on .


8. Alternatively, you can also automate this procedure by using the different Autofocus

buttons in the Verify Local Support Points window.

9. However, when you set Z‐offsets manually, you may encounter a situation where

large Z‐offsets appear for the first support points. In that case, you may speed up

your verification process by first applying a certain offset to all the remaining points

and then manually or automatically just fine‐tune them. To do so, mark all remaining

points, click on and select Apply Z‐Offset… Enter the required offset value

and press (the max. offset value to be added is 5000 µm; for

a higher value you simply have to repeat this step).

Proceed as follows, when you want to apply Absolute Fixed Z‐Positon for your tiles:

1. Select Absolute Fixed Z‐Position in the Focus Strategy tab.

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2. Below you will now find a new tab where you are asked to select a Reference

Channel. This should be the one which you used to allocate your positions/ tiles.

3. Below the Reference Channel tab you have the option to either check the box Use Z‐

Position from Tiles Setup (which includes Z‐positions for tiles and positions) or

manually enter/ set a value for the Z‐position.

4. When your sample does not consist of an even flatness, check the box and proceed

as follows:

5. Select a newly created tile region, press Live and adjust for the focal plane, go to the

Properties tab below the Center Screen Area, activate Show All and click on Set

Current Z.

When you want to copy and paste a Tile Region setting (e.g. a certain arrangement of tiles or

local support points) from one well to other wells or even to all containers of a carrier, apply the

following workflow:

1. Select the well from where the Tile Region setting should be copied by clicking on it

in the Center Screen Area. The selected well is now bordered by a blue circle.

2. Right click within the selected well in the Center Screen Area (outside the tile region)

to open the context menu. At the bottom of the context menu select Copy Container

for replication.

3. Use the left mouse button to select the wells into which you want to paste the

copied Tile Region setting. You can select multiple wells in combination with the Ctrl

key. (This step is not needed when all wells will be chosen)

4. Right click in the Center Screen Area and select the context menu entry Paste

Replication to and either choose Selected Container or All Container

. By that, the copied

Tile Region setting is pasted into the selected wells or all the wells of the carrier with

the same relative coordinates to the center of each well.

When you want to convert a tile region into positions, apply the following workflow:

1. Select your tile region under Tile Regions of the Tiles tab (Left Tool Bar Area).

2. Right‐click on the selected tile region and select Convert to Positions.

3. You are now given the options to convert a) Each Tile of the Regions as Single

Positions, meaning that each tile of your tile region will be converted into one

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position, which will be located in the center of each former tile, b) Each Region as

Position Array, meaning that each tile of your tile region will be converted into one

position; however, these positions will be centered around the former middle tile of

your former tile region, c) Each Region as one Single Position (at the center of the

region), meaning that the whole tile region will be converted into just one position

that will be located in the center of the former tile region.

When you want to sort selected tile regions (in the your tile region list under Tile Regions)

according to their X/Y position, apply the following workflow:

1. Select one tile region under Tile Regions of the Tiles tab (Left Tool Bar Area).

2. Right‐click on the selected tile region, choose Sort and select either a) By Center

Position (Y ‐> X), meaning that the tile regions will be sorted according to their

overall Y‐position; or select b) By Center Position Position (X ‐> Z), meaning that the

tile regions will be sorted according to their overall X‐position. (Sorting by Category

will be mentioned in the chapter Assign Categories to tile regions and positions)

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DEFINE POSITIONS

To define positions in your sample carrier, you have multiple options:

I)

1. When you are interested in adding a position at the current location within your well, go to

Positions in the Tiles module…

…and press .

2. To define positions for different locations or within different wells, double‐click on any location

of your sample carrier in your Center Screen Area (you may need to zoom out before) and just

press again.

II)

1. As an alternative and faster method, you may first zoom out to have a good overview of all of

your wells, zoom into a well of interest, go to Position Setup, select Location under Setup by,

click on + and activate Keep tool.

If you now click on spots of interest in the carrier, positions will be added and displayed in

Single Positions under Positions in the Tiles module.

III)

1. When you want to generate multiple positions in a defined area of your carrier simultaneously,

you go to the Positions tab in the Left Tool Bar Area and select select Position Arrays.

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2. Subsequently, you go to the Position Setup tab (below the Center Screen Area) and click on

Array. Activate the Show All mode.

3. Either choose the rectangular or circular Contour tool, adjust the Number of required positions

and the Bias where the positions should be located. You may activate random, if desired.

Subsequently, mark the interesting area of the carrier in the Center Screen Area (by keeping the

left mouse button clicked) and the positions will be automatically generated.

IV)

1. When you want to generate a certain amount of multiple positions in defined wells of your

carrier simultaneously, you select Position Arrays.

2. Subsequently, you go to the Position Setup tab in the Center Screen Area and click on Carrier.

Activate the Show All mode.

3. Now, mark your wells of interest either in the Center Screen Area or in the Carrier tab, adjust the

Number of required positions and the Bias where the positions should be located. You may

activate Random if desired. Subsequently, click on + Create and the positions will be

automatically generated.

For all options:

If you want to change the locations of your positions, just click/mark them and move them to a

new location.

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For some customers it could be important that the stage is driven in a special direction during the

experiment, e.g. positions in A12 should be imaged before positions in A1. Therefore, you need

to arrange the sequence of the stage‐drive as follows:

1. In order to change the acquisition sequence of Positions, uncheck the Optimize

Stage Travel check box in the Options section of the Tiles tool (Left Tool Bar Area) as

its function does not apply to multi‐well containers. When not unchecked, changes in

the acquisition order will be ignored.

2. Now, select a position of interest and use the buttons in the Positions list

under Tiles to arrange the acquisition sequence of your positions.

When it is crucial to obtain in‐focus images of all of your positions, you can use different focus

strategies, e.g. Definite Focus, Software Autofocus or Absolute Fixed Z‐Position, which you have

to select in the Focus Strategy tab. Definite Focus and Software Autofocus can be configured in

the Focus Devices tab additionally. Both tabs can be found in the Left Tool Bar Area. However,

proceed as follows, when you want to apply Absolute Fixed Z‐Positon for your positions:

1. Select Absolute Fixed Z‐Position in the Focus Strategy tab.

2. Below you will now find a new tab where you are asked to select a Reference

Channel. This should be the one which you used to allocate your positions.

3. Below the Reference Channel tab you have the option to either check the box Use Z‐



4. When your sample does not consist of an even flatness, check the box and proceed

as follows:

5. Verify your selected positions in Z by following the path Tiles module, Positions,

Single Positions and then click on .

6. Double‐click on the point to be verified (Live mode), find the desired Z‐position

(either manually or by clicking on Run Autofocus) and click on

.


8. Alternatively, you can also automate the whole procedure by using the different

Autofocus buttons in the Verify Positions window.

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9. However, when you set Z‐offsets manually, you may encounter a situation where

large Z‐offsets appear for the first positions. In that case, you may speed up your

verification process by first applying a certain offset to all the remaining positions


positions, click on and select Apply Z‐Offset….Enter the required offset

value and press (the max. offset value to be added is 5000 µm;

for a higher value you simply have to repeat this step).

10. As an alternative to steps 5 to 9 or when you wish to modify the X/Y location of your

positions, you can click on your positions in the Center Screen Area, activate Live,

zoom in, go to the Properties tab (below the Center Screen Area), activate Show all

and either press Set Current X/Y/Z (e.g. after you also adjusted the X/Y location of

your position) or press Set Current Z.

When you want to copy and paste an arrangement of positions from one well to other wells or

even to all containers of a carrier, apply the following workflow:

1. Select the well from where the position arrangement should be copied from by

clicking on it in the Center Screen Area. The selected well is now bordered by a blue

circle.

2. Right click within the selected well in the Center Screen Area (not directly on a

position) to open the context menu. At the bottom of the context menu select Copy

Container for replication.

3. Use the left mouse button to select the wells into which you want to paste the

copied setting of your positions. You can select multiple wells in combination with

the Ctrl key. (This step is not needed when all wells will be chosen)

4. Right click in the Center Screen Area and select the context menu entry Paste

Replication to and either choose Selected Container or All Container

. By that, the copied setting of

your positions is pasted into the selected wells or all the wells of the carrier with the

same relative coordinates to the center of each well.

When you want to sort your positions (of your position list under Positions) according to their

X/Y position, apply the following workflow:

1. Select one position under Positions of the Tiles tab (Left Tool Bar Area).

2. Right‐click on the selected position, choose Sort and select either a) By Center

Position (Y ‐> X), meaning that the positions will be sorted according to their overall

Y‐position; or select b) By Center Position Position (X ‐> Z), meaning that the

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positions will be sorted according to their overall X‐position. (Sorting by Category will

be mentioned in the chapter Assign Categories to tile regions and positions).

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ASSIGN CATEGORIES TO TILE REGIONS AND POSITIONS

For some customers it could be important to not only display the well number together with the

acquired images (Path: Graphics ‐> Frequent Annotations ‐> Carrier Container Name) but also create

certain additional annotations for different tile regions or positions, e.g. `control condition´ or

`experimental condition 1´. For that purpose, ZEN allows you to add/ edit names and categories to

the different Tiles Regions/ Positions that have been generated.

1. In order to assign individual names to different individual positions and/or tile regions in a well

plate experiment, click on the respective Tile Region or Position and open the Properties tab in

the Center Screen Area (Tiles – Advances Setup tab). Activate the Use for Acquisition checkbox.

2. You can now edit the name for your selected Tile Region/ Position. Alternatively you can edit the

name of a Tile Region/ Position by clicking on the respective name under Tile Regions or

Positions in Tiles.

Repeat this step when you want to rename different Tile Regions or Positions.

To display the name of your Tile Region/ Position later in your acquired image(s), you

go to Graphics, Frequent Annotations, More… and select Image.Scene.Name from

the Metadata list.

3. However, to now assign or edit features/categories of your Tile Regions/ Positions, first mark all

desired Tile Regions/ Positions that should be grouped in the same category and click on the

button under Category of the Properties tab.

4. Select New… from the dropdown list

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A separated window opens. Here you can enter a name or add a description for the selected

Tile Regions/ Positions. Additionally, you can assign a color for this new category by clicking

on the color bar and choosing a preferred color.

5. After you pressed OK, choose the desired category for the selected Tile regions/ Positions from

the drop down list under Category.

The chosen category is now assigned to the selected Tile Regions/ Positions. Note that a

predefined category can also be applied to a differentiated selection of Tile Regions/

Positions from more than one well. Note also, that the assigned color is only used as a

feature in the Tiles tab (Left Tool Bar Area).

To display a Tile Region/ Position Category feature (Name and/ or Description) in your

acquired image, you go to Graphics, Frequent Annotations, More…, type “category” in the

search bar and select the desired feature to be displayed. [Although the option `color´ is

given as an option, no reasonable element will be displayed by the software]

To adjust parameters of your annotations (e.g. font size), right‐click on it and go to Format

Graphical Elements.

When you have selected several different positions or tile regions and assigned different categories,

you might wish to display them already in your tiles/positions list (Left Tool Bar Area) according to

their respective category. Therefore, you need to apply the following workflow:

Select a position or tile under Positions or Tiles of the Tiles tab (Left Tool Bar

Area).

Right‐click on the selected position/ tile, choose Sort and select By Category,

meaning that the positions/ tiles will be sorted alphabetically according to the

assigned categories.

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HIGH‐MAGNIFICATION IMAGING OF SELECTED POSITIONS/TILES AFTER PREVIEW SCANNING

For some customers, who image slice preparations or transfected cell cultures, it may be interesting

to obtain an overview image of their samples at low magnification, look at that overview and select

some regions of interest for imaging at higher magnification. E.g. a customer has a cell culture dish

and specifically wants to select and image those cells that show expression of a marker protein (e.g.

GFP).

1. You first locate the center of your sample preparation under Locate. To be quickest, use a low

magnification objective, e.g. 10x.

2. You go to Acquisition, set your Channels and Exposure Time, activate the Tiles module, go to

Tiles and click on Advanced Setup and activate Live.

3. Zoom out of the image and create a large tile region that covers all structures of your sample

preparation. To do so, go to Tile Region Setup (below the Center Screen Area), set the values of X

and Y accordingly and press .

To check whether the whole area of interest is covered by tiles, move around with the

joystick in Live mode. When you realize that you need additional tiles to cover your sample

preparation, mark your tile region, grab the short white lines of the yellow rectangle (that is

overlaid on the tiles) and drag them until a sufficient amount of tiles has been generated.

4. Now, you go to the Center Screen Area and click on Preview Scan.

5. Subsequently, you are given a choice of different objectives with which you can conduct your

Preview Scan with. For time consuming reasons, stay with a low magnification objective and

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select only one channel (Left Tool Bar Area). This channel should be the one where the customer

is most interested in and which helps the customer to identify spots of interest for subsequent

imaging at higher magnification.

6. Click on Start Preview Scan.

7. After the scan, the preview image will be displayed in the Tiles – Advanced Setup tab of the

Center Screen Area.

8. In order to hide the tiles matrix, that is overlaid on your preview image, uncheck the box in front

of the tile region name in Tile Regions under the Tiles tab.

9. You can now zoom and navigate through the image and look for any spots of interest

10. In order to create positions at a spot of interest go to Position Setup, select Location under

Setup by, click on + and activate Keep tool

If you now click on spots of interest in your image, positions will be added and displayed in

Single Positions under Positions in the Tiles module

11. However, if you want to create tile regions around a certain spot of interest (or want to add tile

regions in addition to positions), you go to the Tile Region Setup tab, select Predefined under

Setup by, click on and activate Keep tool.

12. When you now click on a spot of your image, a tile region with a certain amount of tiles is

generated around that spot according to the numbers that are set for X and Y.

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13. However, in many cases you will recognize, while screening your preview image, that not all

areas are in focus as either the tissue, the cells or even the sample carrier itself may be a little bit

tilted. As no Focus Strategy is (so far) available for the Preview Scan in the Tiles Module, a

workaround needs to be applied:

First, as described above, add positions/ tiles where the sample is in focus. Second, go to

Live and double click on an area that was not in focus. This area is now displayed in Live

mode. Zoom in and focus that area. Third, show the tiles matrix again by checking box in

front of the tile regions name in Tile Regions under the Tiles tab. Forth, uncheck all

newly created positions in Positions or newly created tile regions in Tile Regions under

the Tiles tab. Tip: Mark all created positions, right‐

click and click on Deactivate.

Fifth, click on Start Preview Scan. The new preview image will contain different areas

that are now in focus. Sixth, re‐activate all created positions in Positions or tile regions in

Tile Regions under the Tiles tab.

You can now add additional positions/ tiles at spots of interest in the same way as

described by steps 8 to 12.

To add more positions/ tiles in other un‐focused areas, just repeat steps 8 to 13.

When the customer is interested in keeping the preview images, uncheck Delete Existing

Preview Images (Center Screen Area). You can later save the

preview scans by clicking on the closing `x´ of the Tiles – Advanced Setup tab and

choosing `Yes´ in the following tab:

14. After you have defined all interesting positions/ tiles, make sure to uncheck the box in front of

the tile regions name in Tile Regions under the Tiles tab.

15. Subsequently, you go to the first tab in the Light Path Settings (Before Experiment: Before….. ‐

Left Tool Bar Area) and change to the objective, which wants to be used by the customer for the

final image acquisition (most probably: higher magnification objectives). Obey a possible change

of the immersion medium and apply the required medium e.g. oil from the side before the

objective is moved under the sample carrier (could be a bit tricky). Don´t touch the stage or the

sample carrier; otherwise the selected positions may not be accurate anymore.

16. Re‐activate all channels that the customer wants to use for image acquisition.

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17. Now, activate Live, double‐click on any area of your image (at best: close to one of your defined

positions), zoom in and adjust Exposure Time and Light Intensity for all of your channels in the

Channels tab of the Left Tool Bar Area.

18. Regarding any chosen tile region: Note, after you have changed the objective and are in Live

mode, the amount of tiles (and therefore also the data file size) is increased. Consider, whether

you want to reduce the area that is covered by the tiles.

19. If the customer is interested, activate and open the Z‐stack module, click Show all, activate Live

and define your Z‐stack parameters. Consider, whether you want to record Full Z‐Stacks per

Channel or All channels per Slice.

20. In order to acquire all positions/ tiles in a perfect focal plane, you can use different focus

strategies, e.g. Definite Focus, Software Autofocus or Absolute Fixed Z‐Position, which you have

to select in the Focus Strategy tab. Definite Focus and Software Autofocus can be configured in

the Focus Devices tab additionally. Both tabs can be found in the Left Tool Bar Area. However,

proceed as follows, when you want to apply Absolute Fixed Z‐Positon for your positions/ tiles:

a. Select Absolute Fixed Z‐Position in the Focus Strategy tab.

b. Below you will now find a new tab where you are asked to select a Reference

Channel. This should be the one, which you used to allocate your positions/ tiles.

c. Below the Reference Channel tab you have the option to either check the box Use Z‐



d. When your sample does not consist of an even flatness, check the box and proceed

as follows:

e. [Tiles] Select a newly created tile region, press Live and adjust for the focal plane, go

to the Properties tab below the Center Screen Area, activate Show All and click on

Set Current Z.

f. [Positions] Verify your selected positions in Z by following the path Tiles module,

Positions, Single Positions and then clicking on

.

g. Activate Live, double‐click on the point to be verified, find the desired Z‐position

(either manually or by clicking on Run Autofocus) and click on

.

h. Subsequently, click on Move to Next Point and repeat step g.

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i. Alternatively, you can also automate the whole procedure by using the different

Autofocus buttons in the Verify Positions window.

j. However, when you set Z‐offsets manually, you may encounter a situation where

large Z‐offsets appear for the first positions. In that case, you may speed up your

verification process by first applying a certain offset to all the remaining positions


positions, click on and select Apply Z‐Offset… Enter the required offset

value and press (the max. offset value to be added is 5000 µm;

for a higher value you simply have to repeat this step).

k. As an alternative to steps f. to j. or when you wish to modify the X/Y location of your

positions, you can click on your positions in the Center Screen Area, activate Live,

zoom in, go to the Properties tab (below the Center Screen Area), activate Show all

and either press Set Current X/Y/Z (when you also adjusted the X/Y location of your

position) or simply adjust for Z and press Set Current Z.

21. By clicking on the software will use your Z‐settings and will apply all

experimental conditions for your selected positions and start the image acquisition procedure.

All images will be put into one file. If you want to split the images into separate files, go

to Options under the Tiles tab and check .

o Note: After image acquisition, the software will display only one tab that

contains all recorded scenes. However, when you save this, the software will

additionally generate seperate .czi files that contain single scenes. These files can

23


then easily be imported to ZEN or other image processing software e.g. to do

deconvolution.

In case you selected tile regions, you can tell the software to stitch these during

acquisition. Therefore, go to Options under the Tiles tab and check

.

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RE‐POSITIONING OF YOUR SAMPLE CARRIER AFTER INCUBATION

When you want to re‐image positions/ tile regions on a sample carrier, that needed to be taken off

the stage, e.g. for incubation purposes or changes of the immersion medium, follow the following

workflow:

1. You first locate your sample preparation under Locate and run the Stage Calibration; see also the

chapter System Prerequisites [Stage calibration and channel selection] steps 1‐7.

2. Go to Acquisition, setup your desired Channels and adjust light/ camera exposure time.

3. Activate Tiles and press Show all.

4. Go to Tiles, click on Sample Carrier and then press Select.

5. Select the template of choice. For demo purposes, select a standard slide that can mimic your

test sample.

6. Regarding calibration of your template, you can customize your own carrier (see the chapter

Customize Your Sample Carrier), but for slides with one coverslip or well, there is only the option

for Single Reference Point Calibration. For Multi‐well plates, you will have the option for 7‐point,

4‐point, 3‐point or 1‐point calibration. This becomes important for adjusting for the rotation of

the sample.

7. Regarding surface adjustment, please refer to the chapter System Prerequisites [Define Support

Points] steps 15‐18.

8. However, assuming that you just use a conventional glass slide with some cells or tissue that is

positioned in the center: Press Calibrate.

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9. In the first step of the Sample Carrier Calibration Wizard, right‐click to activate Crosshairs in the

live image and move a sample reference point into the middle of the crosshairs. This reference

point can be any unique identifiable point on the slide and does not have to in the middle of the

slide.

10. In step 2 of the wizard, press Set Zero X/Y Position, press Next, go to the step Search Reference

Point and press Set Current X/Y.

‐>

11. Click on Advanced Setup in the Tiles tab and add positions/ tiles at your locations of interest (see

also chapters Define Tiles and Define Positions). Zoom in and out using the mouse scroller,

double‐click on the sample carrier in the Center Screen Area to move the stage to a point of

interest.

12. Once you have defined all your positions/ tile regions, go to the Experiment Manager tool.

13. Click on and save your experimental settings, including the lists of positions/ tile regions,

by using either Save As (setting will be saved directly in the Experiment Manager) or by clicking

Export (‐> setting will be saved to a folder of your choice).

26


14. Now, you can start your experiment and record images from your selected positions/ tile regions.

15. Remove your sample off the stage and e.g. put it back into the incubation chamber.

16. Close ZEN software

17. …[Incubation]…

18. Restart the software, go to Acquisition and Experiment Manager and load/ import your

experimental settings including your list of positions/ tile regions.

19. Subsequently, press Calibrate in the Sample Carrier tab under Tiles.

20. In the first step of the Sample Carrier Calibration Wizard, right‐click to activate Crosshairs in the

live image and move your previously chosen sample reference point (see step 9) into the middle

of the crosshairs.

21. In step 2 of the wizard, press Set Zero X/Y Position, press Next, go to the step Search Reference

Point and press Set Current X/Y.

‐>

22. Now, you still need to verify the Z‐offset of your positions. Therefore, follow the corresponding

instructions given in the chapters Define Tiles and Define Positions.

All of your selected positions/ tile regions should now be re‐assigned to the correct

X/Y/Z‐values in relation to your (unique identifiable) reference point.

23. Re‐start your experiment and record images from your selected positions/ tile regions.

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CUSTOMIZE YOUR SAMPLE CARRIER

As some customers work with sample carrier that are not listed in the template database of ZEN, you

need to apply the following workflow in order to create such a template for the software.

1. You go to the Acquisition tab.

2. Do all the prerequisites (e.g. channel and camera settings) for a Tiles & Positions experiment on

your sample. For that use Smart Setup and, if needed (not advisable for beginners), Experiment

Designer

3. After you have defined at least one channel (e.g. EGFP), activate the Tiles module checkbox

4. Open the Tiles section in the Multidimensional Acquisition module and activate the Show all

mode.

5. Open the Sample Carrier section in the Tiles module and click on the Select… button.

6. A new window opens (Select Template)

7. Please click on and choose New Template…

.

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8. A new window opens (New Sample Carrier Template)

.

9. Subsequently, you choose a Category that corresponds to the type of your carrier. Assign a

Name to your template.

10. When you select Slide, you can now configure the Width, Height of the slide and adjust the

location of a Reference Point.

29


11. However, when you select a Multislide, Petri Dish, Multiwell or Multichamber template, you can

configure and adjust additional parameters of your carrier (see images below).

30


12. In case you need to modify one of the above depicted templates even further, first select the

Category that appears closest to your carrier, go again to the Category tab and then choose

Custom.

By that, some predefined options of the template will be made accessible for further

modifications.

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ADDITIONAL AUTOMATED OPTIONS FOR YOUR TILES SETUP

For some customers it may be interesting to modify certain, already pre‐configured, automatic

settings for the Tiles module, e.g. automatic start of the Live mode when the advanced setup view is

activated. In order to know and, if necessary, change these settings, follow the path:

Tools ‐> Options ‐> Software Acquisition ‐> Tiles.

Documents

Workflows for setting up and applying Tiles & Positions in ... · 1 [email protected] Global Sales Support / TASC Tiles & Positions (Oct ´14) Workflows for setting up and