Zeiss ZEN3.1 User Manual

Software Manual

ZEISS ZEN 3.1 (blue edition)

ZEISS ZEN 3.1 (blue edition)

Original Manual

Carl Zeiss Microscopy GmbH

Carl-Zeiss-Promenade 10

07745 Jena

Germany [e-mail] www.zeiss.com/microscopy

Carl Zeiss Microscopy GmbH

ZEISS Group

Kistlerhofstr. 75

81379 München

Document Name: ZEISS ZEN 3.1 (blue edition) Software Manual

Revision: 1

Language: en-US

Effective from: 10/2019

© 2019 This document or any part of it must not be translated, reproduced, or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information or retrieval system. Violations will be prosecuted. The use of general descriptive names, registered names, trademarks, etc. in this document does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Software programs will fully remain the property of ZEISS. No program, documentation, or subsequent upgrade thereof may be disclosed to any third party, unless prior written consent of ZEISS has been procured to do so, nor may be copied or otherwise duplicated, even for the customer's internal needs, apart from a single back-up copy for safety purposes.

ZEISS reserves the right to make modifications to this document without notice.

​Content​	ZEISS

Content

1 General information ........................................................................		12
1.1	Opening the Online Help ............................................................................	12
1.2	Safety Notes and Safety Labels ...................................................................	13
1.3	Text Conventions........................................................................................	13
1.4	Legal Notes ................................................................................................	14

2 First Steps		.......................................................................................	16
2.1	Starting .................................................................................the Software		16
2.2	User Interface.............................................................................................		16
	2.2.1 .........................................................................................	Title bar	17
	2.2.2 ..............................................................	Workspace Configuration	17
	2.2.3 ......................................................................................	Menu bar	18
	2.2.4 .........................................................................................	Tool bar	18
	2.2.5 ................................................................................	Left Tool Area	18
	2.2.6 ........................................................................	Center Screen Area	19
	2.2.7 ..............................................................................	Right Tool Area	20
	2.2.8 ...............................................................................	Document bar	21
	2.2.9 ......................................................................................	Status bar	21
2.3	Setting .........................................................................the User Language		23
2.4	Acitvating ....................................................................the Show All Mode		24
2.5	Configuring .........................................................Microscope Components		24
2.6	Acquiring ..................................................................a First Camera Image		26
2.7	Adding ................................................................Annotations to an Image		28
2.8	Adjusting .....................................................................Live Image Settings		28
2.9	Creating ..........................................................................a Manual Scaling		30
2.10	Closing ..................................................................................the Software		30

3 Basic Concepts ................................................................................		31
3.1	Introduction ...............................................................................................	31
3.2	Image Acquisition.......................................................................................	31
3.3	Image Processing .......................................................................................	31
3.4	Image Analysis ...........................................................................................	31
3.5	File Format .................................................................................................	32
3.6	Extensions ..................................................................................................	32

4 Managing Users and Groups ...........................................................		33
4.1	Introduction ...............................................................................................	33
4.2	Creating a new user ...................................................................................	33
4.3	Adding users to a group.............................................................................	34

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​Content​				ZEISS
	4.4	Managing access rights for user groups......................................................		36
	4.5	Options	......................................................................................................	38
5	Image Acquisition............................................................................			40
	5.1	Using Smart Setup with Airyscan LSM 980 and LSM 900............................		40
	5.2	Using the Sample Navigator with LSM 980 and LSM 900 ...........................		41
	5.3	Acquiring Confocal Images.........................................................................		48
	5.4	Acquiring LSM 900 images with Airyscan 2 multiplex modes......................		53
	5.5	Acquiring LSM 980 images with Airyscan 2 multiplex modes......................		55
	5.6	Acquiring Multi-Channel Images with Cameras...........................................		61
		5.6.1	Set up a new experiment ...............................................................	62
		5.6.2	Variant 1: Smart Setup...................................................................	63
		5.6.3	Variant 2: Channels Tool................................................................	65
	5.7	Acquiring Z-Stack Images ...........................................................................		65
		5.7.1	Configuring a Z-Stack automatically...............................................	66
		5.7.2	Configuring a Z-Stack manually (First/Last Mode)...........................	67
		5.7.3	Configuring a Z-Stack manually (Center Mode) ..............................	68
	5.8	Acquiring Time Series Images .....................................................................		69
	5.9	Acquiring EDF Images with ZEN lite ............................................................		70
		5.9.1	Introduction ..................................................................................	70
		5.9.2	Prerequisites ..................................................................................	72
		5.9.3	Acquiring EDF image with Timer mode ..........................................	72
		5.9.4	Acquiring EDF image with F12 Key mode ......................................	73
		5.9.5	Compare Images using Split Display...............................................	74
	5.10	Working with Focus Strategies ...................................................................		76
		5.10.1	Introduction ..................................................................................	76
		5.10.2	Using Software Autofocus .............................................................	77
		5.10.3	Using Definite Focus in Time Series Experiments ............................	78
		5.10.4	Using Local or Global Focus Surfaces .............................................	79
	5.11	Using the Dye Editor...................................................................................		80
		5.11.1	Introduction ..................................................................................	80
		5.11.2	Dye Editor Menus ..........................................................................	81
		5.11.3	Dye Editor Dialog...........................................................................	82
		5.11.4	Creating a Custom Dye ..................................................................	82
		5.11.5	Adding a New Dye to the Data Set ................................................	85
	5.12	Displaying and adapting a grid in the image area .......................................		85
	5.13	Setting an experiment as startup default ....................................................		87
	5.14	Creating default experiments as templates automatically............................		89
	5.15	Applying an experiment template...............................................................		91
	5.16	Acquiring a Panorama Image Automatically ...............................................		91
	5.17	Adding Annotations to Images or Movies...................................................		94
	5.18	Editing Annotations in Images or Movies....................................................		95
6	Image Processing ............................................................................			96

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​Content​				ZEISS
	6.1	Image Processing Workflow .......................................................................		96
	6.2	General Settings .........................................................................................		96
	6.3	Performing Deconvolution Using Default Values.........................................		97
	6.4	Performing Configurable Deconvolution.....................................................		101
		6.4.1 Step 1: Load input image for deconvolution ..................................		102
		6.4.2 Step 2: Set parameters for deconvolution ......................................		103
		6.4.3 Step 3: Perform deconvolution ......................................................		105
		6.4.4 Step 4: Info View and re-using Deconvolution parameters from a
			processed image............................................................................	108
	6.5	Measuring the PSF using subresolution beads.............................................		109
	6.6	Extracting Individual Fluorescence Images of a Multichannel Image............		110
	6.7	Applying Batch Processing..........................................................................		114
	6.8	Cropping a ROI...........................................................................................		117
	6.9	Creating an EDF Image...............................................................................		121
	6.10	Creating image subset and split dimensions ...............................................		124
	6.11	Using Analyze to Label Image.....................................................................		124
	6.12	Image Processing Functions........................................................................		126
		6.12.1	Deconvolution ...............................................................................	126
		6.12.2	Adjust............................................................................................	139
		6.12.3	Edges ............................................................................................	146
		6.12.4	Geometric......................................................................................	148
		6.12.5	Morphology...................................................................................	160
		6.12.6	Sharpen.........................................................................................	162
		6.12.7	Smooth .........................................................................................	164
		6.12.8	Time Series ....................................................................................	167
		6.12.9	Arithmetics ....................................................................................	171
		6.12.10Segmentation ................................................................................		175
		6.12.11Binary ............................................................................................		178
		6.12.12Utilities ..........................................................................................		181
		6.12.13Export/Import ................................................................................		199
		6.12.14Image Analysis...............................................................................		216
		6.12.15Batch Processing Functions............................................................		220
7	Importing/ Exporting Images ..........................................................			224
	7.1	Workflow Export/Import.............................................................................		224
	7.2	Exporting images........................................................................................		227
	7.3	Exporting movies........................................................................................		227
	7.4	Exporting Multichannel Images ..................................................................		228
	7.5	Exporting Multiscene Images......................................................................		231
	7.6	Importing Z-Stack images...........................................................................		233
8	Customizing the Application............................................................			238
	8.1	Selecting a Screen Design...........................................................................		238
	8.2	Customizing Toolbar ..................................................................................		238

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8.3	Adjusting the Workspace Zoom..................................................................		238
8.4	Showing/Hiding Workspace Areas..............................................................		239
8.5	Undocking/Docking Tool Windows.............................................................		239
9 Software Modules & Extensions ......................................................			240
9.1	Advanced Processing..................................................................................		240
	9.1.1	Experiment Feedback.....................................................................	240
	9.1.2	Workflow Experiment Feedback ....................................................	241
9.2	APEER Connector .......................................................................................		244
	9.2.1	Introduction ..................................................................................	244
	9.2.2	Apeer Connection Settings ............................................................	245
	9.2.3 How to connect to APEER..............................................................		245
	9.2.4	Starting Workflows........................................................................	246
	9.2.5	Uploading Files ..............................................................................	247
9.3	Automated Photomanipulation ..................................................................		248
	9.3.1 Using Automated Photomanipulation settings ...............................		248
	9.3.2 Performing an Automated Photomanipulation experiment.............		249
	9.3.3	Functions & Reference ...................................................................	250
9.4	Confocal Topography .................................................................................		252
	9.4.1	Acquiring Topography Images .......................................................	252
	9.4.2	Measuring Layer Thickness ............................................................	256
	9.4.3	Functions & Reference ...................................................................	261
9.5	Correlative Array Tomography (CAT)...........................................................		268
	9.5.1 Basics of Array Tomography ..........................................................		269
	9.5.2	Sample Preparation........................................................................	269
	9.5.3	Pre-Settings (Light Microscope)......................................................	270
	9.5.4	Experiment Settings .......................................................................	270
	9.5.5	The CAT Workflow ........................................................................	271
	9.5.6	Functions & Reference ...................................................................	300
9.6	Deconvolution............................................................................................		319
	9.6.1	Basics of Deconvolution.................................................................	319
	9.6.2	Deconvolution (defaults) ................................................................	320
	9.6.3	Deconvolution (adjustable) ............................................................	320
	9.6.4 Creating a PSF - With Wizard and Without ....................................		331
	9.6.5 Image types suitable for deconvolution..........................................		333
	9.6.6 Deconvolution Methods in ZEN......................................................		335
	9.6.7 Table of Default Parameter for Deconvolution ...............................		338
	9.6.8	Creating Deconvolution Settings....................................................	340
9.7	Direct Porcessing........................................................................................		340
	9.7.1	Direct Processing ...........................................................................	341
	9.7.2 Connecting the acquisition computer with the processing com-
		puter .............................................................................................	341
	9.7.3 Defining the communication path..................................................		344
	9.7.4 Setting up your PC as discovery proxy............................................		345
	9.7.5 Defining settings in the Auto Save tool ..........................................		345
	9.7.6 Using Direct Processing with Airyscan............................................		345
	9.7.7 Using Direct Processing with deconvolution...................................		347

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	9.7.8 Using Direct Processing with advanced deconvolution settings		......348
	9.7.9	Functions & Reference ...................................................................	349
9.8	Guided Acquisition .....................................................................................		351
	9.8.1 Licensing of Guided Acquisition .....................................................		352
	9.8.2 Preliminary work to Guided Acquisition .........................................		352
	9.8.3 Performing a Guided Acquisition ...................................................		353
	9.8.4 Using Guided Acquisition settings..................................................		354
	9.8.5	Functions & Reference ...................................................................	355
9.9	Image Analysis ...........................................................................................		357
	9.9.1 Creating a new image analysis setting ...........................................		358
	9.9.2 Creating an image analysis setting from an analyzed image ...........		360
	9.9.3 Measuring Fluorescence Intensity in a Multichannel Image ............		360
	9.9.4 Counting Number of Fluorescence Signals per Nuclei.....................		366

9.9.5Measuring Mean Fluorescence Intensity on a Ring around the Pri-

		mary Object ...................................................................................	374
	9.9.6	Counting the number of Objects in a Ring around the Nucleus ......	381
	9.9.7	Performing an interactive analysis ..................................................	384
	9.9.8	Creating a measurement data table ...............................................	385
	9.9.9	Charts and Tables of the Analysis View ..........................................	385
	9.9.10	Measurement Features ..................................................................	389
9.10	ImageJ	.......................................................................................................	402
	9.10.1 ..................................................................................	Preparations	402
	9.10.2 ..............................................................	Activate ImageJ Extension	403
	9.10.3 ..............................................................	Send and Retrieve Images	403
	9.10.4 .....................................................................	Use ImageJ Methods	404
	9.10.5 ..........................................	Image Type Send/Retrieve Conventions	406
9.11	Intellesis .....................................................................................................		406
	9.11.1 ......................................................................................	Fact Sheet	408
	9.11.2 ...........................................................................	FAQ/Terminology	409
	9.11.3 ........................................................................	Operating Concept	411
	9.11.4 ................................................................	User Interface - Training	411
	9.11.5 .......................................................................	Workflow Overview	416
	9.11.6 ...................................................................	Creating a New Model	417
	9.11.7 ...............................................................................	Editing Classes	421
	9.11.8 ................................................	Importing Labels from Binary Mask	422
	9.11.9 ...............................................	Converting segmentations to labels	423
	9.11.10Renaming ........................................................................a Model		423
	9.11.11Deleting ...........................................................................a Model		424
	9.11.12Cloning ............................................................................a Model		424
	9.11.13Creating ...............................................................Analysis Setting		425
	9.11.14Exporting .........................................................................a Model		426
	9.11.15Exporting ...................................................................with Images		426
	9.11.16Importing ..........................................................................Models		427
	9.11.17Using ...................and downloading neural networks for Intellesis		428
	9.11.18Using ..................................a Trained Model for Image Processing		429
	9.11.19Using ......................................a Trained Model for Image Analysis		431
	9.11.20Using ............................................................Intellesis within OAD		434
	9.11.21Feature ..........................................................................Extractors		435
	9.11.22Remarks ..............................................and Additional Information		438
	9.11.23Image ...........................................................Processing Functions		439

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​Content​			ZEISS
9.12	Macro Environment....................................................................................		439
	9.12.1 Running an existing macro.............................................................		440
	9.12.2	Recording a macro ........................................................................	441
9.13	Panorama...................................................................................................		442
	9.13.1	Prerequisites ..................................................................................	443
	9.13.2 Acquiring a Reference Image .........................................................		444
	9.13.3 Settings for the Panorama Experiment ...........................................		444
	9.13.4 Acquiring the Panorama Image......................................................		446
	9.13.5 Processing the Panorama Image ....................................................		449
	9.13.6	Functions & Reference ...................................................................	456
9.14	Physiology..................................................................................................		457
	9.14.1 Workflow MeanROI View (offline) .................................................		458
	9.14.2	Workflow Physiology Experiments .................................................	464
	9.14.3 Sample Experiment Fura-2 with DG4/5 ..........................................		471
9.15	Shuttle & Find.............................................................................................		473
	9.15.1 Settings and Image Acquisition with the Light Microscope.............		474
	9.15.2 Shuttle & Find Sample Positions at the Electron Microscope...........		483
	9.15.3 Correlating Two Loaded Images ....................................................		487
	9.15.4 Correlation of Live Image and Loaded Image ................................		489
	9.15.5 Shuttle & Find with an EVO 10.......................................................		489
	9.15.6	Functions & Reference ...................................................................	491
9.16	Software Autofocus ...................................................................................		503
	9.16.1	Terminology & Abbreviations.........................................................	503
	9.16.2 When is focus the "right" focus?....................................................		507
	9.16.3 Software Autofocus in ZEN ............................................................		508
	9.16.4	FAQ...............................................................................................	509
	9.16.5	Functions & Reference ...................................................................	510
9.17	Tiles & Positions .........................................................................................		514
	9.17.1	General Preparations .....................................................................	514
	9.17.2 Calibrating Stage and Selecting Channel ........................................		515
	9.17.3 Setting Up a Simple Tiles Experiment .............................................		520
	9.17.4 Setting Up a Simple Positions Experiment ......................................		521
	9.17.5 Tiles & Positions with Advanced Setup ...........................................		522
	9.17.6 Copying a Tile Region or Position...................................................		531
	9.17.7	Adjusting Z-Values.........................................................................	532
	9.17.8 Local and Global Focus Surfaces ....................................................		536
	9.17.9 Assigning Categories to Tile Regions and Positions ........................		544
	9.17.10Re-positioning Sample Carrier after Incubation ..............................		547
	9.17.11Using Sample Carriers ....................................................................		552
	9.17.12Exporting Tile Images.....................................................................		554
	9.17.13Functions & Reference ...................................................................		557
9.18	ZEN Connect ..............................................................................................		586
	9.18.1 Licensing of ZEN Connect ..............................................................		586
	9.18.2 Introduction to the User Interface..................................................		587
	9.18.3 ZEN Connect 3D View ...................................................................		588
	9.18.4 Opening the Correlative Workspace...............................................		589
	9.18.5 Opening images in ZEN Connect 3D view ......................................		589
	9.18.6 Project and Image Management....................................................		589
	9.18.7	Import and Export .........................................................................	595

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	9.18.8	Handling of images........................................................................	598
	9.18.9	Functions & Reference ...................................................................	610
10 Software Functions & Reference .....................................................			624
10.1	Menus........................................................................................................		624
	10.1.1	File Menu ......................................................................................	624
	10.1.2	Edit Menu......................................................................................	628
	10.1.3	View Menu....................................................................................	629
	10.1.4	Acquisition Menu ..........................................................................	630
	10.1.5	Graphics Menu ..............................................................................	631
	10.1.6	Macro Menu..................................................................................	632
	10.1.7	Tools Menu ...................................................................................	636
	10.1.8	Window Menu ..............................................................................	656
	10.1.9	Help Menu ....................................................................................	656
10.2	Main Tabs ..................................................................................................		656
	10.2.1	Locate Tab.....................................................................................	656
	10.2.2	Acquisition Tab..............................................................................	660
	10.2.3	Processing Tab...............................................................................	673
	10.2.4	Analysis Tab...................................................................................	674
	10.2.5	Applications Tab ............................................................................	674
	10.2.6	Extensions Tab...............................................................................	675
10.3	Tools ..........................................................................................................		675
	10.3.1 Tools on Locate Tab.......................................................................		675
	10.3.2 Tools on Acquisition Tab................................................................		691
	10.3.3 Tools on Processing Tab ................................................................		783
	10.3.4 Tools on Analysis Tab ....................................................................		783
	10.3.5 Tools on Applications Tab..............................................................		810
	10.3.6 Tools in Right Tool Area.................................................................		811
10.4	Dialogs.......................................................................................................		821
	10.4.1 Stage/Focus not calibrated Dialog..................................................		821
	10.4.2	ApoTome Dialog............................................................................	822
	10.4.3	ApoTome Settings Dialog ..............................................................	822
	10.4.4 Add Dye or Contrasting Method Dialog .........................................		823
	10.4.5 Connected Processing PCs Dialog ..................................................		824
	10.4.6	Download Model Dialog................................................................	824
10.5	Image views ...............................................................................................		825
	10.5.1	General image views......................................................................	825
	10.5.2	Specific image views......................................................................	845
	10.5.3	General View Options....................................................................	891
10.6	View Modes ...............................................................................................		913
	10.6.1	Full Screen mode ...........................................................................	913
	10.6.2	Exposé mode .................................................................................	914
	10.6.3	Splitter mode.................................................................................	915
10.7	File Browser ...............................................................................................		916
	10.7.1	Tools Tab.......................................................................................	917
11 Systems, Applications & Components..............................................			918

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​Content​			ZEISS
11.1	LSM 980 ....................................................................................................		918
	11.1.1	Polarization Imaging ......................................................................	918
11.2	ApoTome.2 ................................................................................................		923
	11.2.1	Introduction ..................................................................................	923
	11.2.2 Principle of imaging using fringe projection ...................................		923
	11.2.3	Optimum acquisition conditions ....................................................	927
	11.2.4 List of recommended objectives.....................................................		928
	11.2.5	Preparation: Phase calibration........................................................	931
	11.2.6 Step 1: Define channels using Smart Setup ....................................		931
	11.2.7 Step 2: Grid focus calibration.........................................................		933
	11.2.8 Step 3: Perform ApoTome experiment ...........................................		936
	11.2.9 Step 4: Process the resulting image................................................		938
	11.2.10Step 5: Perform Z-stack acquisition ................................................		941
	11.2.11Step 6: Perform ApoTome deconvolution ......................................		943
11.3	Celldiscoverer 7 ..........................................................................................		946
	11.3.1	Introduction ..................................................................................	946
	11.3.2 Sample Tab (Interactive mode).......................................................		947
	11.3.3 Sample Tab (Automation mode) ....................................................		950
	11.3.4	Magazine View..............................................................................	954
	11.3.5	Navigation View ............................................................................	957
	11.3.6	Navigation tab...............................................................................	958
	11.3.7	Celldiscoverer Tool.........................................................................	959
	11.3.8	Dispensing Tool .............................................................................	960
	11.3.9	Perfusion .......................................................................................	961
	11.3.10Celldiscoverer Options ...................................................................		962
	11.3.11Performing a Celldiscoverer calibration ..........................................		964
	11.3.12Mixed Mode Settings.....................................................................		966
	11.3.13Calculating the disparity map for mixed mode acquisition..............		970
	11.3.14Airyscan HS ...................................................................................		975
	11.3.15Airyscan MPLX HS .........................................................................		976
	11.3.16Smart Setup...................................................................................		977
	11.3.17Auto Immersion for Celldiscoverer .................................................		977
11.4	Slidescan ....................................................................................................		981
	11.4.1	Introduction ..................................................................................	981
	11.4.2 Working with ZEN slidescan...........................................................		982
	11.4.3	Slidescan Wizards ..........................................................................	1030
	11.4.4	Functions and Reference................................................................	1076
12 Service / Maintenance .....................................................................			1088
12.1 Creating a Service Report ...........................................................................			1088
12.2 System Maintenance for LSM 980 ..............................................................			1089
	12.2.1	Collimators Adjustment ................................................................	1089
	12.2.2	Pinhole Adjustment .......................................................................	1090
	12.2.3	Adjusting Readback Signals ...........................................................	1090
	12.2.4	Airyscan Detector Adjustment........................................................	1091
13 FAQ	.................................................................................................		1096
13.1 What can I do If my image is too dark? ......................................................			1096

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13.2	How can I balance my images color? ..........................................................	1096
13.3	How can it be that my image has dust or a shadow, although my speci-
	men is clean?..............................................................................................	1097
13.4	Why my image seems to look that something have burned in? (i.e. a
	shadow of a previous specimen?) ...............................................................	1097
13.5	How can I fix a color gradient cast?............................................................	1098
13.6	What can I do if my live image is of a low quality and looks pixelated?.......	1098
13.7	What can I do if my live image is slow? ......................................................	1098
13.8	What can I do if my live image is mostly red/blue?......................................	1099
13.9	What can I do if my live image is still black or white after setting the expo-
	sure? ..........................................................................................................	1100
13.10 Why my live image shows extreme colors in comparison to what I see in
	the eyepieces?............................................................................................	1100
13.11 Why is my image resolution lower than the given camera specification?.....1100
13.12 What can I do if I do not see a focused live image? ....................................		1101
13.13 Why is my image color not the same that I see through the eye pieces? .....1101
Glossary...........................................................................................		1103
Index ...............................................................................................		1113

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1 General information | 1.1 Opening the Online Help	ZEISS

1 General information

1.1Opening the Online Help

If you need help on a specific area, tool, or section within the software simply press the F1 key to open the related help topic.

As it is difficult sometimes for the software to know on which topic you want help for when clicking the F1 key, you can use the Question Mark symbol alternatively. Simply click on the question mark symbol in the Title bar. The courser will then appear as a question mark symbol now. Click on an area within the software where you want to get help for. If there’s a related help topic available it will open directly.

Online Help User interface

The following screenshot indicates the main elements of online help user interface:

1		2		3		4			5

6

1Topics

Contains the structure tree with a list of all the topics in the Online Help

2Index

List of keywords to help you find topics and content quickly

3Search

Search through the entire text

It supports partial strings but not wildcards.

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1 General information | 1.2 Safety Notes and Safety Labels	ZEISS

4Structure tree

Enables you to navigate through topics sequentially. A + indicates a topic has subtopics.

5Content panel

6Related links

Further information relevant for the current topic.

1.2Safety Notes and Safety Labels

The display of safety notes in the documentation and software follows a system of risk levels, that are defined as follows:

CAUTION

Risk of personal injury

CAUTION indicates a potentially hazardous situation which, if not avoided, may result in minor or moderate personal injury.

NOTICE

Risk of property damage

NOTICE indicates a property damage message. In addition, NOTICE is used for data loss or corrupt data as well.

The safety icons / labels on the device or in the documentation refer to potential dangers or information that are defined as follows:

Icon / Label	Name	Description
	Crushing	This icon warns you of a potential risk of crushing fin-
	Fingers	gers.

1.3Text Conventions

The following text conventions are used in this documentation:

Format	Description

Format "bold" The format "Bold" within text is used for

§Clickable user interface elements, e.g. buttons and icons

Example: Click on Save.

§Hardware buttons on the microscope

Example: On/Off button

§ Non-clickable user interface elements, e.g. name of a dialog

Example: Image dialog

Format "italic" Italics highlights the following:

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1 General information | 1.4 Legal Notes	ZEISS

Format	Description
	§ text to be entered by the operator
Format for keyboard	Text in bold + brackets is used for keyboard commands. E.g.
text	Example:
	Press [F1] to open the online help.
Format for a path in	Description of a path within the software.
the software	Example:
	Go to Tools > Options > Acquisition
Format for program-	Used for programming code, e.g. macro code as well as for anything
ming code	that you would type literally when programming, including keywords,
	data types, constants, method names, variables, class names, and in-
	terface names.
	Example: Integer
Format for links (in-	Links to further information internally or in the web.
ternal or web links)	Example: Link
Format for proce-	A numbered procedural instruction is used for actions which are per-
dural instructions	formed by the user. The steps must be performed in the given order.
	Optionally there are pre-requisites which have to be fulfilled in ad-
	vance. At the end of the instruction normally the result of the proce-
	dure is presented.
	Example:
	1. Action step which has to be actively performed by the user
	à Intermediate result
	2. Another step within the procedure.

Additional information is indicated as follows:

Info

Helpful additional information, e.g. about necessary additional actions.

1.4Legal Notes

ZEISS draws the User's attention to the fact that the information and references contained in these documentation may be subject to technical modifications, in particular due to the continuous further development of ZEISS products. The documentation enclosed does not contain any warranty by ZEISS with regard to the technical processes described in the documentation or to certain reproduced product characteristics. Furthermore, ZEISS shall not be held liable for any possible printing errors or other inaccuracies in this documentation, unless proof can be furnished that any such errors or inaccuracies are already known by ZEISS or that these are not known to ZEISS due to gross negligence and that furthermore ZEISS has for these reasons refrained from eliminating these errors or inaccuracies appropriately. ZEISS hereby explicitly draws the User's attention to the fact that this documentation only contains a general description of the technical processes and information, the implementation of which in any individual case may not be appropriate in the form described here. In cases of doubt, we recommend the User to consult ZEISS service and support.

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1 General information | 1.4 Legal Notes	ZEISS

This documentation is protected by copyright. ZEISS has reserved all rights to this documentation. It is prohibited to make copies, partial copies, or to translate this documentation into any other language, except for personal use.

ZEISS explicitly draws attention to the fact that the information contained in this documentation will be updated regularly in compliance with the technical modifications and supplements carried out in the products and furthermore that this documentation only reflects the technical status of ZEISS products at the time of printing.

Safety

Refer to the safety notes and instructions in the documentation of all necessary devices (e.g. microscope peripherals, cameras, computers, computer accessories, etc.) before installing and using the software.

Disclaimer

The author is not responsible for any contents linked or referred to from his pages - unless he has full knowledge of illegal contents and would be able to prevent the visitors of his site from viewing those pages. If any damage occurs by the use of information presented there, only the author of the respective pages might be liable, not the one who has linked to these pages. Furthermore the author is not liable for any postings or messages published by users of discussion boards, guest books or mailing lists provided on his page.

Please note that this software contains an extension that enables you to connect it with the third party software ImageJ. ImageJ is not a ZEISS product. Therefore ZEISS undertakes no warranty concerning ImageJ, makes no representation that ImageJ or derivatives such as Fiji or related macros will work on your hardware and will not be liable for any damages caused by the use of this extension. By using the extension you agree to this disclaimer.

Notice of the Producer

This software product was designed, realized, verified, validated and released in a certificated process environment. The quality management system is certified following the rule of DIN EN ISO 13485.

The fields of application of the Software are common tasks and applications in microscopy respectively imaging (so called “Off-The-Shelf Software”). Though the user acknowledges that in any kind of use the end user of the software is responsible for the validation of the Software for the end user’s dedicated intend of use considering all requirements of law and standards (e.g. FDA/21 CFR part 11, IvDD, etc.). If necessary the end user has to establish, to document, to implement and to maintain a special process to fulfill all the requirements to be conform with the validate rules of law and standards. It is pointed out that displayed measure values (e.g. length measurement) may not be used directly as analytical values for diagnostic results.

CARL ZEISS DOES NOT WARRANT THAT THIS SOFTWARE IS USABLE FOR SPECIAL PURPOSES OTHER THAN IN THE FIELDS OF APPLICATION DEFINED ABOVE.

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2 First Steps | 2.1 Starting the Software	ZEISS

2 First Steps

2.1 Starting the Software

Prerequisite ü You have installed ZEN (blue edition) on your computer.

1. Double click on the program icon on your desktop.

2.Alternatively click on Start | All Programs | Carl Zeiss Microscopy | ZEN | ZEN (blue edition) entry (blue icon).

à The software starts. After a while you see the login screen.

3.Click on the button of the application you want to work with. The available applications depend on your licenses and system (e.g. if you work with an LSM, only ZEN system and Image Processing is available). Make sure that the hardware components you use are switched on and are ready for operation.

à The software starts. During the program start the hardware settings will be initialized.

You successfully started the software.

Info

For using pre-recorded images when starting the software, in the menu Tools | Options |

Startup, the Reload Last Used Documents checkbox must be activated.

2.2 User Interface

The software user interface is divided into three main areas. Via the tabs in the Left Tool Area

(1) you can access all the main tools for microscope control (Locate tab), acquisition (Acquisition tab), image processing (Processing tab) and image analysis (Analysis tab). The Center Screen Area (2) is used to display your images with several image views available. In the Right Tool Area (3) you find the Images and Documents Gallery, the Objective Selection and the Stage and Focus controls. Additionally system specific tools can be available here (e.g. Definite Focus.2 controls).

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Fig. 1: User interface

2.2.1 Title bar

Parameter	Description
Help icon	Activates the "drag & drop“ help function. A question mark ap-
	pears beside the mouse pointer. Move the mouse pointer to a
	place in the software where you need help. Left-click on the de-
	sired location. The online help opens.
Minimize	Minimizes the program window.
Maximize Over 2	Maximizes the program window across 2 screens if available.
Screens	This option is only possible if you are working with 2 screens
	with the same resolution.
Maximize	Maximizes the program window to the main screen.
Restore Down	Reduces the program window to any selected size.
Close	Closes the program window.

2.2.2 Workspace Configuration

Fig. 2: Workspace Configuration

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Here you find settings to adjust your workspace. Select Light/Dark Design of the user interface or enlarge the screen with Workspace Zoom slider. You can also save and reload all personal

settings in a workspace configuration. With the Dock all tool windows button in the top right corner you can easily dock all undocked tools by one click.

2.2.3Menu bar

Fig. 3: Menu bar

The menu bar contains all the menus you need to manage, edit and view your projects, see

Menus [} 624].

See also

2 Edit Menu [} 628]

2 View Menu [} 629]

2 Acquisition Menu [} 630]

2 Graphics Menu [} 631]

2 Macro Menu [} 632]

2 Tools Menu [} 636]

2 Window Menu [} 656]

2 Help Menu [} 656]

2.2.4Tool bar

Fig. 4: Tool Bar

Here you gain quick access to important functions, e.g. saving or opening files. Further right you find more workspace settings, e.g. Design and Workspace selection. Read how to customize the Tool bar in chapter Customizing Toolbar [} 238].

2.2.5Left Tool Area

This area contains the main tabs for microscope and camera settings (Locate tab), image acquisition (Acquisition tab), image processing (Processing tab), and image analysis (Analysis tab). The main tabs are organized in an order which follows the typical workflow of experiments in bioscience or material science.

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Fig. 5: Left Tool Area

See also

2Locate Tab [} 656]

2Acquisition Tab [} 660]

2Processing Tab [} 673]

2Analysis Tab [} 674]

2Extensions Tab [} 675]

2.2.6Center Screen Area

The Center Screen Area is structured in 4 areas. The Document bar (1) is on top. Down the left side of the displayed image you find the tabs for the general and specific Image Views (2). In the middle of Center Screen Area is the Image Area (3), images, reports and tables were shown here. Under the image area you find the General - and Specific View Options (4) organized on tabs. View specific control tabs are flagged with a blue corner.

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1 2

3

4

Fig. 6: Center Screen Area

1Document bar, learn more [} 21].

2Image Views (organized on tabs), learn more [} 825].

3Image Area

4General and specific view options, learn more [} 891].

2.2.7Right Tool Area

This area contains mainly the tools for image and file handling (e.g. Image Gallery) and hardware control (e.g. Stage / Focus tool). Depending on your system configuration, other tools can be available. The tools are described in the corresponding chapters of the online help.

See also

2 ZEN Connect Tool [} 619]

2 Stage Tool [} 812]

2 Focus Tool [} 814]

2 Incubation Tool [} 816]

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2Microscope Tool [} 812]

2Images and Documents Tool [} 811]

2Macro Tool [} 818]

2.2.8Document bar

Fig. 7: Document bar

Here you see tabs of all opend documents. Click on a tab to view the image/document. On the right end of document bar you find buttons to switch view mode from Expose to Splitter mode and further view options (View menu).

Info

A asterisk (*) next to an image/document title indicates that unsaved changes have been made to this document. Save your pictures/documents from time to time in order to avoid data loss.

2.2.9Status bar

The status bar shows important information on the system status:

Parameter	Description
Scaling	Displays which lateral scaling is currently being used. If you click on
	the arrow, the Scaling dialog [} 640] will be opened. There you have
	access to advanced scaling settings and the scaling wizard.
System Informa-	Always shows the latest, currently active process that the system is
tion	performing.
Progress bar	Displays the progress of the currently active process. Each new
	process added supersedes older still active processes. If you click on
	the arrow button, a window opens with a list of all processes in
	chronological order. You can stop a process that is running using the
	Stop button.
Performance indi-	In this group you will see an overview of the performance of individ-
cators	ual computer components:
	§ Free RAM indicates how much physical memory is still available.
	§ Free HD indicates how much space is still available on the hard
	drive onto which the next image is to be acquired (see Extras/Op-
	tions/Save).
	§ CPU indicates the usage of the Central Processing Unit.
	§ The small status bar provides an overall assessment of the system
	usage.

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Parameter	Description
	Info: Double-clicking in the Performance Indicators area opens the
	Windows Task Manager.
Frame Rate	Indicates the current frame rate in frames per second (fps) used by the
	active camera for producing new images. Please note in most cases
	that at speeds greater than 100 frames per second, this value cannot
	always be accurately determined.
Pixel Value	Displays the gray value to the image at the current position of the
	mouse pointer. In the case of multichannel images the gray value/
	channel is displayed for up to 4 channels.
Position	Displays the X/Y position (in pixel coordinates) of the mouse pointer in
	the image.
Information (i)	If you click on the icon, a window opens with a System Messages
	[} 22].
Storage Folder	Displays the location where new images are automatically saved. This
	path can be changed in the menu Tools | Options | Saving.
Status: Airyscan	If you click on the arrow the Alignment Tool window opens. See
Detector Align-	Airyscan Detector Adjustment [} 1091].
ment
User	Shows the Windows user name of the logged in user.
Time	Shows the current Windows system time.

2.2.9.1System Messages

If you right click on a system message the Copy button will appear. Left click on Copy button to copy the message to clipboard. Then paste it into a text file or an E-Mail. The idea behind is that you can easily send error messages to your support team for example. This copy/paste function works for all upcoming system messages or error messages within the application as well.

Parameter Description

System information that arises during normal operation. This system information does not lead to an interruption of the workflow. The in-

formation window is not displayed automatically.

Information

Information that requires input from the user, e.g. a prompt to change a manual microscope component. This information leads to

the information window being shown briefly. However, it closes again

Warnings

after a few seconds.

Error messages indicate a malfunction by the system. In this case the information window opens and remains open. The system requires in-

put from the user in order to continue.

Errors

Info

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2 First Steps | 2.3 Setting the User Language		ZEISS

Hundreds of messages can accumulate in the course of a session. A maximum of 300 messages are displayed. To display messages for a certain category, activate or deactivate the corresponding checkboxes.

2.3 Setting the User Language

Prerequisite ü You have successfully started the application.

1.Click on menu Tools | Options.

à The Options dialog opens. The General entry in the Software group is selected.

2.Deactivate the Select Automatically checkbox if you want to set the language manually.

3.Select user language from the Fixed Language dropdown list.

àA message appears to restart the application.

àNote, that the availability of additional languages can differ between software versions.

4.Click on OK.

àThe Options dialog closes.

5.Exit and restart software.

You have successfully set the user language.

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2 First Steps | 2.4 Acitvating the Show All Mode	ZEISS

2.4Acitvating the Show All Mode

1.With the Show All mode deactivated (default setting), only the basic functions of tool windows or view options are shown.

2.To show the advanced settings or expert functions of tool windows or view options, click on the Show All button.

2.5Configuring Microscope Components

This chapter refers to the manual configuration of the microscope components in ZEN lite. All microscope components definitions will be stored in the meta data of the acquired image.

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2 First Steps | 2.5 Configuring Microscope Components	ZEISS
Prerequisite ü You have selected the Camera tab.

1. Click to the blue header of the Microscope Components tool.

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2 First Steps | 2.6 Acquiring a First Camera Image	ZEISS

à The tool will open. Consider that the button Show all is activated.

2.Under Objective select that objective you will use for your acquisitions.

3.Select all other microscope components you eventually will use (i.e. Optovar, Reflector, etc.).

You have successfully configured your microscope components.

Info

If you have activated the Select automatically button in the status bar under Scaling (standard settings), the scaling will be calculated on the basis of your definitions. If you want to perform a manual scaling, read the chapter Creating a Manual Scaling [} 30].

See also

2Creating a Manual Scaling [} 30]

2.6Acquiring a First Camera Image

This topic guides you through acquiring your first camera image with the software.

Prerequisite ü	You have connected and configured a microscope camera (i.e. Axiocam 305 color/mono) to
	your system.
ü	You have started the software.

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üYou have configured the microscope components (e.g. objective, camera adapter) und you are using the automatic or manual scaling.

üYou are on the Camera (ZEN light only) or Locate tab.

üYou see your microscope camera available in the Active Camera section. If not, select the camera from the list.

1.Position your sample on the microscope and adjust the microscope to see a focused image through the eyepieces.

2.Adjust the tube slider of the microscope to divert the image to the camera (e.g. 50% camera and 50% eyepieces).

3.Click on Live button.

àThe Live Mode will be activated. In the Center Screen Area you will see the camera live image. By default the live image shows a cross hair helping to navigate on the specimen. In the chapter Adjusting Live Image Settings [} 28] you will learn how to optimize live image display.

4.Click on Set Exposure button.

à The exposure time will be automatically determined and set.

5.Click on Snap button.

You successfully acquired your first image. Save the image in the file system via the File menu |

Save as.

Info

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2 First Steps | 2.7 Adding Annotations to an Image		ZEISS

If you do not see a focused image please refocus the specimen on the microscope. You may activate the focus bar as an additional aid. Right click in the Center Screen Area to open the context menu. Select the entry Focus Bar. The focus bar will be shown.

See also

2Document bar [} 21]

2.7Adding Annotations to an Image

Annotations are the generic term for all the graphics (e.g. rectangle, arrow, scale), measurements, texts or other metadata (e.g. recording time) that you can add to your image.

Prerequisite ü You have acquired or loaded an image.

1. In the Center Screen Area select the Graphics tab.

2.Click on the Scale Bar button.

àThe scale bar will appear directly in the image.

àTo edit an annotation (e.g. color, line width) you can right click on the annotation in the image and select Format Graphical Elements from the context menu.

3.Click on the Draw Arrow button.

àNow you may draw an arrow into your image.

You added the annotations Scale Bar and Arrow from the toolbar to your image.

See also

2Adding Annotations to Images or Movies [} 94]

2.8Adjusting Live Image Settings

Prerequisite ü You have started the Live mode via the Live button and see the camera’s live image in the

Center Screen Area.

üUnder the image area you see the general view options on Dimensions tab, Graphics tab and Display tab.

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2 First Steps | 2.8 Adjusting Live Image Settings	ZEISS

1.In the Dimensions tab activate the Range Indicator checkbox. This will mark overexposed (too bright) areas in the live image in red and underexposed (too dark) areas in blue.

2.On the Display tab click the 0.45 button. The display curve will be adapted to a gamma value of 0.45. This will set the optimum color presentation. If you do not see this button, activate the Show all mode.

3.Move the controls under the display curve left and right in order to directly adjust the values for Brightness (White), Gamma, and Contrast (Black) in the live image.

1 Contrast (black point) control

2 Gamma control

3 Brightness (white point) control

Info

With the settings above the display of the live image will be adapted. These settings will also be transferred to your acquired image. This will not change the camera settings.

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2 First Steps | 2.9 Creating a Manual Scaling	ZEISS

2.9 Creating a Manual Scaling

Prerequisite ü You have an object micrometer oriented horizontally on the microscope stage.

üYou have selected all definitions for your microscope correctly in the Microscope Components tool (ZEN lite only). In our example we use an objective with a 10x magnification.

1.Acquire an image (see Acquiring a First Camera Image [} 26]) of the scale in your object micrometer using the objective to be scaled manually.

2.In the bottom status bar click on the arrow in the Scaling area. In the Scaling dialog deactivate the Select Automatically checkbox.

3.In the Create new scaling section, click on the Interactive Calibration... button.

à The calibration wizard will appear in the image area.

4.Click on single Reference Line button (selected as default) and activate the Automatic Line Detection button (activated as default).

5.Draw in the reference line along the scale.

6.Enter the true distance between both scale lines in the calibration wizard. In our example this is 500 micrometer.

7.Enter a name for the scaling (i.e. Obj 10x) and click the Save Scaling button.

You performed a manual scaling for your objective. Repeat this sequence for all objectives you will need a manual scaling for. Always ensure that you did select the correct objective in the tool Microscope Components and for this performed and selected the matching scaling in the status

bar.

Info

§The function Automatic Line Detection calculates the theoretical maximum of the reference line‘s both end points to the closest scale lines in the image. Thus the distance will be calculated with sub-pixel accuracy.

§If you defined manual scalings for your available objectives, and you click in the status bar in the Scaling area to open the Scaling dialog and to activate the checkbox Select Automatically again, the system will use the measured scalings instead of the theoretic ones.

You will recognize this via the label "Measured" instead of "Theoretic" beside the pixel size.

2.10Closing the Software

1.Click on File | Exit. Alternatively you can use the short cut ALT+F4 or click on the Close icon in the program bar.

Info

If you haven’t saved your files the Save Documents dialog will open before the program closes. Select files you want to save or unselect files you don’t want to save.

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