Zeiss Lightsheet Z.1 User manual

Lightsheet Z.1
Operating Manual
February 2013
ZEN 2012 (black edition)

Carl Zeiss Copyright Lightsheet Z.1
II 000000-1790-528 02/2013
Knowledge of this manual is required for the operation of the instrument. Would you therefore please
make yourself familiar with the contents of this manual and pay special attention to hints concerning the
safe operation of the instrument.
The specifications are subject to change; the manual is not covered by an update service.
© Unless expressly authorized, forwarding and duplication of this document, and the utilization and
communication of its contents are not permitted. Violations will entail an obligation to pay
compensation.
All rights reserved in the event of granting of patents or registration of a utility model.
Issued by Carl Zeiss Microscopy GmbH
Carl-Zeiss-Promenade 10
07745 Jena, Germany
[e-mail]
www.zeiss.com/microscopy

INTRODUCTION
Lightsheet Z.1 Carl Zeiss
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Contents of this Manual
Chapter 1 Hardware
Chapter 2 Sample Preparation
Chapter 3 Quick Guide
Chapter 4 Software Operation
Annex Lightsheet Z.1 - Overview

CHAPTER 1 - HARDWARE
Lightsheet Z.1 Content Carl Zeiss
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CHAPTER 1 HARDWARE
CONTENT
Page
1MAINTENANCE AND CLEANING .........................................................................3
1.1 Maintenance of the Liquid Cooling System.................................................................5
2ERGODRIVE OPERATING PANEL .........................................................................6
3USER INTERFACES ...............................................................................................9
3.1 Installation and Deinstallation of the Detection Modules ........................................10
3.2 Adjustment – Detector Recognition ...........................................................................15
3.2.1 Adjustment – Automatic Detector Alignment..................................................................15
3.2.2 Adjustment – Manual Detector Alignment ......................................................................17
3.2.3 Adjust the Grating Focus for the Automatic or Manual Detector Alignment Tool..............19
3.2.4 Cable Connections for the Detection Module "PCO.Edge"..............................................22
3.2.5 Cable Connections for the Detection Module "Standard" ...............................................23
3.3 Assembly of the Sample Chamber .............................................................................24
3.3.1 Assembly of the Sample Chamber Windows ...................................................................24
3.3.2 Assembly of the Sample Chamber Body and the Sample Chamber Dove Tail Slide............27
3.3.3 Insertion of the Drain Connector, Luer-Lock Connectors and Blind Plugs ..........................29
3.3.4 Insertion of Accessories for Incubation ............................................................................30
3.4 Removing and Inserting the Sample Chamber ..........................................................31
3.5 Assembly of the Sample Holder .................................................................................32
3.5.1 Assembly of the Sample Holder for Capillary ...................................................................32
3.5.2 Assembly of Sample Holder for Syringes..........................................................................34
3.6 Inserting and Removing the Sample Holder..............................................................35
3.7 Installation of the Incubation Modules......................................................................37
3.7.1 Heating Components .....................................................................................................37
3.7.2 CO2-Module Lightsheet Z.1.............................................................................................39
3.7.3 Heating Device Humidity S1............................................................................................41
3.7.4 Registration of the incubation modules...........................................................................41
3.8 Switch Incubation ON and OFF...................................................................................43

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Carl Zeiss Content Lightsheet Z.1
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3.9 Illumination and Detection Optics ............................................................................. 43
3.9.1 Removing and Inserting the Illumination Optics Unit ....................................................... 45
3.9.2 Removing and Inserting the Detection Optics Unit........................................................... 46
3.10 Removing and Inserting the Reflector Turret for Emission Selection ...................... 47
3.11 Removing and Inserting the Reflector Turret for Laser Blocking Filter .................... 49
4INDEX................................................................................................................. 52

CHAPTER 1 - HARDWARE
Lightsheet Z.1 Maintenance and Cleaning Carl Zeiss
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1Maintenance and Cleaning
The maintenance to be carried out by the customer is limited to cleaning the painted surfaces, replacing
glass windows and rubber seals on the sample chamber, cleaning and disinfecting the system cavity and
sample chamber, temperature sensors and detection optics unit, replenishment of coolants for the
incubation device and liquid cooling of the detection module as necessary, as well as the replacement of
CO2sterile filters for the incubation device.
a) To clean painted surfaces proceed as follows:
•Switch the device off completely and pull the mains plug.
•Make sure that no cleaning fluid is allowed to enter the system.
•Wipe the painted surfaces with a clean cloth moistened with water to which a small amount of
cleaning agent has been added. Do not use a solvent. Dry off with a lint-free cloth.
b) To clean and disinfect the system cavity proceed as follows:
•Switch the device off completely and pull the mains plug.
•Make sure that no cleaning fluid is allowed to enter the system.
•Take care not to touch the front lens of the illumination and detection optics units. Remove these
optics as necessary.
•Remove the sample chamber for cleaning.
•Cleaning the system cavity:
Wipe the inner surfaces of the system cavity with a clean cloth moistened with water to which a
small amount of cleaning agent has been added. Do not use a solvent. Dry off with a lint-free
cloth.
•Disinfecting the system cavity:
Carefully wipe the inner surfaces of the sample chamber with a lint-free cloth soaked in 70%
ethanol or 70% isopropyl. Allow the solution to react for a few seconds. Repeat the procedure
once or twice as necessary. Carefully wipe off the inner surfaces with a lint-free cloth soaked in
distilled water.

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Carl Zeiss Maintenance and Cleaning Lightsheet Z.1
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c) To clean and disinfect the sample chamber proceed as follows:
•Remove the sample chamber and dismantle it into individual parts. Different procedures are required
for cleaning and disinfecting the various parts of the sample chamber.
−The cover glasses must be properly disposed of in the glass waste.
−The steel parts of the chamber must be cleaned and disinfected as follows:
Rinse the parts with distilled water.
Lay the parts in an ultrasonic bath containing distilled water to which a few drops of detergent
have been added.
Then rinse the steel parts thoroughly with distilled water until all detergent residue has been
removed.
The parts may be laid in a disinfecting bath of 70% ethanol (>1 hour) and then thoroughly
rinsed with distilled water.
The steel parts of the sample chamber may also be autoclaved as necessary.
−Sealing rings, hose connectors and blank plugs must be cleaned and disinfected as follows:
These parts may not be autoclaved.
First of all lay these parts in distilled water and then in a 70% ethanol solution for max. one
hour. Rinse them thoroughly with distilled water.
d) To clean and disinfect the temperature sensor proceed as follows:
•Remove the temperature sensor from the sample chamber and loosen the connections to the main
system module Lightsheet Z.1.
•Once removed, thoroughly rinse the temperature sensor with distilled water and then suspend it for
one hour in a 70% ethanol solution. Make sure that the contact plug remains dry. Complete the
cleaning process by rinsing thoroughly with distilled water.

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Lightsheet Z.1 Maintenance and Cleaning Carl Zeiss
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e) To clean and disinfect the detection optics unit proceed as follows:
•Make sure that no cleaning fluid is allowed to enter the system.
•To avoid scratches, do not wipe the front lens of the optics with dry lens paper and lens cloths.
•Remove these optics from the system cavity as necessary.
•Clean the front lens of optical elements according to general recommendations (see "The clean
microscope" www.zeiss.com/microscopy) using an optical cleaning solution (Carl Zeiss optical cleaning
solution: 85% n - hexan, 15% isopropanol. This solution is not sold by Carl Zeiss Microscopy GmbH)
and a suitable lens cleaning paper or high purity cotton wool.
•In addition to the cleaning procedure described above, for special sterility requirements the front lens
area of the detection modules can be cleaned several times with a lint-free lens cloth or lens paper
soaked in 100% ethanol. Here again, observe the general notes on cleaning the optics
("The clean microscope").
1.1 Maintenance of the Liquid Cooling System
If detection modules in your Lightsheet Z.1 are connected to a liquid cooling system, consider that the pH
value of the cooling liquid changes over time.
To prevent corrosion in the cooler of the detection module, the pH value of the cooling liquid must be
checked at least once a year and the liquid replaced as necessary. Observe the directions of the cooling
system manufacturer in the supplied operating manual.
If you have signed a service agreement with Carl Zeiss, our service staff will perform the check as part of
the maintenance procedure.
The safety data sheet for the cooling liquid should also be observed.

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Carl Zeiss ErgoDrive Operating Panel Lightsheet Z.1
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2ErgoDrive Operating Panel
1Z-axis control
2Rotation button (switches 6 to rotation drive)
3Mode button (fine / coarse)
4Axis button (switches 6 to y-axis drive)
5X-axis control
6Y-axis / Rotation control
Fig. 1 ErgoDrive operating panel

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Lightsheet Z.1 ErgoDrive Operating Panel Carl Zeiss
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With the ErgoDrive operating panel the movement
of the sample holder and therefore the sample can
be controlled. It is the manual equivalent of sliders
in the Specimen Navigator tool in the ZEN
software (Fig. 2), with which the movement of the
specimen can be controlled as well.
The function to rotate around the center of the
image, Rotate around Center of image (Fig. 2) is
only available through the software interface. Even
if chosen there, the rotation controlled by the
ErgoDrive operating panel will always rotate
around the axis of the motor drive.
The steering elements are two vertical rotary
controls for the x, y and rotation movement.
The large rotary wheel controls the z movement
(Fig. 1).
Fig. 2 ZEN software, Specimen Navigator
tool window

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Carl Zeiss ErgoDrive Operating Panel Lightsheet Z.1
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The following control elements are part of the ErgoDrive operation panel:
Buttons:
The Mode button (Fig. 1/3) changes the speed of the drive from coarse to fine and back.
The Rotation button (Fig. 1/2) changes the upper rotary control to move the rotation drive (Fig. 1/6).
The Axis button (Fig. 1/4) changes the upper rotary control to move the y drive (Fig. 1/6).
Rotation control:
Upper rotary control (Fig. 1/6), after pressing the Rotation button (Fig. 1/2):
Clockwise: clockwise rotation of the sample; the angle is reduced in the software interface
Counter-clockwise: counter-clockwise rotation of the sample, the angle is increased in the
software interface
Y axis control:
Upper rotary control (Fig. 1/6), after pressing Axis button (Fig. 1/4):
Clockwise: upward movement of the sample
Counter-clockwise: downward movement of the sample
X axis control:
Lower rotary control (Fig. 1/5):
Clockwise: right movement of the sample
Counter-clockwise: left movement of the sample
Z axis control:
Large rotary wheel (Fig. 1/1):
Clockwise: sample movement towards the detection optic
Counter-clockwise: sample movement away from the detection optic

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Lightsheet Z.1 User Interfaces Carl Zeiss
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3User Interfaces
The ports of the detection modules, upper a
nd lower system cavity doors and the upper
system door are equipped with a safety interlock system to guarantee laser safety. These
locking devices must not
be manipulated. Other interfaces not described here are reserved for
service and may only be used by authorized Carl Zeiss s
ervice personnel. The following devices
may be mounted and dismounted by, or are accessible to, the user:
−Detection modules
−Sample chamber
−Sample Holder
−Incubation components
−Illumination optics
−Detection optics
−Reflector turret.
Customized Sample Chamber
The sample chamber delivered with the Lightsheet Z.1 can be used with a multitude of media (e.g. PBS,
cell culture medium, artificial sea water) and is carefully designed to produce optimal results with the
Lightsheet Z.1 system. However, if applications demand changes to the original sample chamber, Carl
Zeiss Microscopy GmbH provides the CAD files and the corresponding technical drawing of our sample
chamber for your convenience, in accordance with the following disclaimer:
Carl Zeiss Microscopy GmbH (hereinafter “we”) hereby informs you that we will warrant the specified and
agreed performance of the Lightsheet Z.1 system only if sample chambers are applied and used that
either are delivered or explicitly approved by us.
The sample chamber design has been optimized to ensure the most established applications of Light
Sheet Fluorescence Microscopy. Exceptional applications may require a slightly modified sample chamber
design. In order to enable customized modifications of the existing sample chamber we also provide the
corresponding CAD file and a technical drawing. We explicitly advise you that already minor deviations of
the dimensions and tolerances specified in these documents will cause a significant loss of image quality
and can potentially result in a liquid leakage. Therefore, you will not hold us or one of our affiliates liable
for any damages caused by the employment of self-built or third-party-built sample chambers, the use of
such self-built or third-party-built sample chambers will be solely on your own risk. Furthermore we want
to inform you, that we will not render any assistance relating to the production and application of such
self-built or third-party-built sample chambers.

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Carl Zeiss User Interfaces Lightsheet Z.1
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3.1 Installation and Deinstallation of the Detection Modules
A cooling liquid defined as a hazardous substance is used in the Lightsheet Z.1 t
o cool the
detection modules (depending on configuration). The supplied safety data sheet with notes on
hazards and safety measures must be observed when handling the cooling liquid.
The ports of the detection modules, reflection (Cam1) (Fig. 3/1) and transmission (Cam2) (Fig. 3/2), on
the main system module Lightsheet Z.1 are equipped with a hardware interlock device. The contact rings
(Fig. 3/9) are connected to the detection modules (Fig. 3/8) and adjusted in the factory. When handling
the detection modules, ensure that neither tilting nor rotation forces are exerted on this connection to
avoid misalignment. The detection module should be gripped as close as possible to the contact ring.
A rotation guard plate (Fig. 3/7) is mounted on the contact ring of each detection module. When
mounting on both ports this must point toward the front side of the Lightsheet Z.1. It prevents rotation
of the detection module and carries the laser protection notice.
Never remove the rotation guard plates of the detection module adapters. Do not modify or
manipulate the detection module adapters.
The lock is functioning when the sensors (Fig. 3/4) of the sensor disk (Fig. 3/11) are pressed down by the
pins of the contact ring (Fig. 3/10 or 5for blind cap). If this is not the case, e.g. the space between the
two devices is too large, the laser is blocked and the system cannot be used.
If the system does not function after a detection module has been attached to or removed from a
port with a safety interlock, check the connection of the contact ring on the respective detection
module or blind cap (Fig. 3/9or 6) to the sensor ring.

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Lightsheet Z.1 User Interfaces Carl Zeiss
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1Detector port 1 (reflection, Cam1)
2 Detector port 2 (transmission, Cam2)
3 Securing screw
4 Sensors (2×) in sensor ring
5 Contact pins (2×) on blind cap
6 Blind cap
7 Rotation guard plate with laser protection notice
8 Detection module (varies)
9 Contact ring of the detection module
10 Contact pins (2×) on contact ring
11 Sensor disk
12 Sensor ring
Fig. 3 Mounting and dismounting the detection modules

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Carl Zeiss User Interfaces Lightsheet Z.1
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The exchange of the detection modules on your Lightsheet Z.1 can be divided into two parts. One deals
with the actual mounting and dismounting of the detection modules and their connections to the
system. The second part deals with the registration and alignment of the detection module using specific
software tools.
The Lightsheet Z.1 must be completely turned off before changing detection modules.
No sample, capillary or similar object must be in the beam path while the alignment and calibration
tasks are performed.
1) Dismounting and Mounting of Detection Modules
−Disconnect all cables from the detection module.
−The connection of the liquid cooling can stay in place, but the power plug for the liquid cooling
unit must be pulled when the detection module is not in use.
−Now disconnect all cables from the Lightsheet Z.1 system and the PC for system control. Even if the
detection modules are regularly switched no cable should remain on the system.
−Hold the detection module firmly in one hand.
−Loosen the securing screw with an Allen wrench (Fig. 3/3) on the sensor ring (Fig. 3/12) of the
detector port (Fig. 3/1and 2).
−Carefully pull away the detection module (Fig. 3/8) with contact ring (Fig. 3/9) using a slight tilting
motion if necessary.
−Cover the detector port (Fig. 3/1and 2) on the main system module Lightsheet Z.1 and on the
detection module with the caps provided (Fig. 3/6).
−For mounting the new detection module, proceed in the reverse order. Tighten the securing screw
(Fig. 3/3) on the sensor ring (Fig. 3/12) of the detector port (Fig. 3/1and 2) without applying force.
−With the detection module "PCO.Edge," note that part of the optics projects beyond the contact
ring. To protect it from damage and fingerprints, first push the optics into the port and then
connect the contact ring to the sensor ring.
−Connect all necessary cables to the detection module, the Lightsheet Z.1 and the PC for system
control (see section 3.2.4 Cable Connections for the Detection Module "PCO.Edge" and
3.2.5, Cable Connections for the Detection Module "Standard").
−Use the cable holder (Fig. 3/3) on the system table to connect the cables (Fig. 3/1) of the detection
modules without tension.

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−Turn on the system (see CHAPTER 4, SYSTEM OPERATION).
2) Registration and Alignment of the detection modules
−After the Lightsheet Z.1 and the PC for system control are turned on and the operation system has
booted, start the software ZEN Configuration Tool . This can be found either as a shortcut on
your desktop or in this directory: C:\ZEN\HWT as Configuration Tool 2012.exe.
−In the opening window press the Start configuring button (Fig. 5).
1Cable (e. g. of the detection modules)
2 Cable tie
3 Cable holder on system table
Fig. 4 Cable strain relief
Fig.
5 ZEN Configuration Tool Start-up window

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Carl Zeiss User Interfaces Lightsheet Z.1
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−On the Configuration tab (Fig. 6) you will find a Camera field with a drop – down menu for both
detector ports (Cam1, Cam2). Select the detection module(s) you are planning to use. When you
use the PCO.Edge, the checkbox liquid cooling will be marked automatically.
−Press the Store all Changes button and then exit the Configuration Tool. This will save the
changes you have made into the database of your system.
If you plan to exchange detection modules on a regular basis, it can be helpful to create two
databases, one for each detection module type. To do so, copy the original database with a
different name (you find it in the directory: C:\ZEN\database) and modify it with the Configuration
Tool accordingly. Make sure to always keep the original database. Name both databases, to easily
identify them. When ZEN is started open the Boot Status with the black arrow and then the
Hardware configuration database the same way (Fig. 7). Here you can choose which database
to use. The Recent… button shows all databases that have been used lately. The Choose… button
opens a window explorer window which allows one to search for a database to use.
Fig. 6 ZEN Configuration Tool, Configuration tab

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−Now start the ZEN (black edition) software
and perform the following tasks found in the
Maintain tab:
•Detector recognition
•Automatic Detector Alignment; and
if necessary
•Manual Detector Alignment.
−It might be necessary to adjust the focus
of the calibration grating (Fig. 8) for
the Automatic or Manual Detector
Alignment task. Start one of these tasks to
get a live image of the grating for evaluating
and adjusting the focus if applicable (see
section 3.2.2 Adjustment – Manual
Detector Alignment).
3.2 Adjustment – Detector Recognition
When the detection modules are exchanged or attached to the Lightsheet Z.1 system for the first time,
the system must identify which detection module is present for which channel. Pressing the button
Detector recognition will start an identification routine to do this job. A progress bar is visible during
the process. When the Cancel button is pressed, the operation is aborted without saving any result. This
routine must be conducted before the Automatic or Manual Detector Alignment.At the end of this
process you be informed if the detector recognition had to perform any changes. If so, please restart the
ZEN software before continuing.
3.2.1 Adjustment – Automatic Detector Alignment
This adjustment is necessary for one or two detection modules, when:
•detection modules have been exchanged
•reflector turrets have been removed and inserted (restart ZEN before the alignment task)
•recognition of a pixel shift between channels
•the zoom is not centered.
No sample should be present. The necessary correction plate and grating is present for each channel
within the system and needs no additional activation.
The Automatic Detector Alignment will align the image center with the detection module center (zoom
center). For two detection modules, it will additionally align both to have overlapping pixels.
After detection module exchange, first press the Detector recognition button in the Adjustment
tool window.
Fig. 7 ZEN Start-up window

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Carl Zeiss User Interfaces Lightsheet Z.1
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After pressing the Automatic Detector Alignment button, a wizard window (Fig. 8) will open that
guides you through the process. Press the Next button after each step to continue or the Previous
button to move one step back. If you press Cancel you will abort the process without saving any results.
The first step (Fig. 8) automatically positions the
necessary grating into the light path and adjusts
brightness and contrast. A progress bar is present
in the lower part of the window. You can follow
the process in the displayed live image. You find
the following text below the live image:
“This will start detector alignment for all available
detectors and filter positions in the system. Auto-
adjustment of brightness and contrast, please
wait.”
If the live image of the grating shows the
grid pattern out-of-focus, cancel the
Automatic Camera Alignment wizard and
first manually adjust the focus of the grating.
The necessary steps are described in section
3.2.3 Adjust the Grating Focus for the
Automatic or Manual Detector
Alignment Tool.
In Step 2 the movement of the correction plates is calibrated for both channels. There is no interaction
needed. Press the Next button to continue after the task is done. The following text is displayed:
“Calibration of correction plate movement, please wait.”
In Step 3 the zoom center is determined. A graphic of the grating is displayed at the left hand side of the
window (Fig. 9). Two squares are labeled with white crosses in this image. On the right hand side, a live
image of the grating is shown. Now mark the same squares as shown in the graphic in the live image by
simply clicking on them with the left mouse button. This will be repeated for a second zoom step. The
following text will be shown: “Determining the detector center. Please click on the two squares in the
right live image of the grid that are shown with the white cross within the left image display. Repeat for
both zoom settings, then continue.”
Fig. 8 Automatic Detector Alignment, first
step