Operating Manual
February 2013
ZEN 2012 (black edition)
Lightsheet Z.1
Carl Zeiss Copyright Lightsheet Z.1
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
microscopy@zeiss.com
www.zeiss.com/microscopy
II 000000-1790-528 02/2013
INTRODUCTION
Lightsheet Z.1 Carl Zeiss
Contents of this Manual
Chapter 1
Chapter 2
Chapter 3
Chapter 4
Annex
Hardware
Sample Preparation
Quick Guide
Software Operation
Lightsheet Z.1 - Overview
02/2013 000000-1790-528 III
CHAPTER 1 - HARDWARE Lightsheet Z.1 Content Carl Zeiss
CHAPTER 1 HARDWARE
CONTENT
Page
1 MAINTENANCE AND CLEANING ......................................................................... 3
1.1 Maintenance of the Liquid Cooling System ................................................................. 5
2 ERGODRIVE OPERATING PANEL ......................................................................... 6
3 USER 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 CO
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
02/2013 000000-1790-528 1
-Module Lightsheet Z.1 ............................................................................................. 39
2
CHAPTER 1 - HARDWARE
Carl Zeiss Content Lightsheet Z.1
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
4 INDEX ................................................................................................................. 52
2 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Lightsheet Z.1 Maintenance and Cleaning Carl Zeiss
1 Maintenance 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
CO
sterile filters for the incubation device.
2
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.
02/2013 000000-1790-528 3
CHAPTER 1 - HARDWARE
Carl Zeiss Maintenance and Cleaning Lightsheet Z.1
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.
4 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Lightsheet Z.1 Maintenance and Cleaning Carl Zeiss
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.
02/2013 000000-1790-528 5
CHAPTER 1 - HARDWARE
Carl Zeiss ErgoDrive Operating Panel Lightsheet Z.1
2 ErgoDrive Operating Panel
1 Z-axis control
2 Rotation button (switches 6 to rotation drive)
3 Mode button (fine / coarse)
4 Axis button (switches 6 to y-axis drive)
5 X-axis control
6 Y-axis / Rotation control
Fig. 1 ErgoDrive operating panel
6 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Lightsheet Z.1 ErgoDrive Operating Panel Carl Zeiss
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
02/2013 000000-1790-528 7
CHAPTER 1 - HARDWARE
Carl Zeiss ErgoDrive Operating Panel Lightsheet Z.1
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
8 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
nd lower system cavity doors and the upper
system door are equipped with a safety interlock system to guarantee laser safety. These
be manipulated. Other interfaces not described here are reserved for
ervice personnel. The following devices
Lightsheet Z.1 User Interfaces Carl Zeiss
3 User Interfaces
The ports of the detection modules, upper a
locking devices must not
service and may only be used by authorized Carl Zeiss s
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.
02/2013 000000-1790-528 9
CHAPTER 1 - HARDWARE
o cool the
Carl Zeiss User Interfaces Lightsheet Z.1
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
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 5 for 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. 3/9 or 6) to the sensor ring.
10 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Lightsheet Z.1 User Interfaces Carl Zeiss
1 Detector 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
02/2013 000000-1790-528 11
CHAPTER 1 - HARDWARE
Carl Zeiss User Interfaces Lightsheet Z.1
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. 3/1 and 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. 3/1 and 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. 3/1 and 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.
12 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Fig.
Lightsheet Z.1 User Interfaces Carl Zeiss
1 Cable (e. g. of the detection modules)
2 Cable tie
3 Cable holder on system table
Fig. 4 Cable strain relief
− 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).
5 ZEN Configuration Tool Start-up window
02/2013 000000-1790-528 13
CHAPTER 1 - HARDWARE
Carl Zeiss User Interfaces Lightsheet Z.1
− 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.
Fig. 6 ZEN Configuration Tool, Configuration tab
− 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.
14 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Lightsheet Z.1 User Interfaces Carl Zeiss
− 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).
Fig. 7 ZEN Start-up window
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.
02/2013 000000-1790-528 15
CHAPTER 1 - HARDWARE
Carl Zeiss User Interfaces Lightsheet Z.1
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. Autoadjustment of brightness and contrast, please
wait.”
If the live image of the grating shows the
grid pattern out-of-focus, cancel the
Fig. 8 Automatic Detector Alignment, first
step
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.”
16 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Lightsheet Z.1 User Interfaces Carl Zeiss
Step 4 is the final step in which the alignment for
all filters and detection module channels is
performed. A progress bar is present at the bottom
of the window. When the procedure is finished the
Finish button becomes available, press it to save
all adjustments and leave the wizard. This text is
displayed: “Detector Alignment for all available
filters in progress, please wait.”
If you still encounter pixel shifts between the two
channels after the Automatic Camera Alignment
tool, you can fine tune settings using the Manual
Detector Alignment tool, see section 3.2.3 Adjust
the Grating Focus for the Automatic or
Manual Detector Alignment Tool.
Fig. 9 Automatic Detector Alignment,
third step
3.2.2 Adjustment – Manual Detector Alignment
This adjustment is necessary for one or two
detection modules when you recognize a pixel shift
between channels, or when the zoom is not
centered.
The Automatic Detector Alignment
should always be performed first since it
might already solve the problems.
Furthermore it defines the settings for the
combination Channel 1 (Cam1) and
position 1 of the Emission Selection Filter,
which should be used as the reference
during the Manual Detector Alignment.
The Zoom in the Acquisition tool window
is fixed to 1 while performing the Manual
Detector Alignment.
Fig. 10 Manual Detector Alignment
window
02/2013 000000-1790-528 17
CHAPTER 1 - HARDWARE
Carl Zeiss User Interfaces Lightsheet Z.1
No sample should be present when performing this task. When pressing the Manual Detector
Alignment button, a window will open (Fig. 10). A grating is automatically inserted into the light path
and a live image of it is shown in a new image container within the ZEN software. The Set Exposure
button is activated automatically to adjust brightness. All components of the light path can be controlled
in the Manual Detector Alignment window. The light source is the LED white light source to ensure
the visibility of the grating independent of the filters in the light path. All other controls for adjusting
imaging settings should be controlled in the tool windows of the main software.
The activated (blue) Automatic button loads the
settings which have been previously defined with
the Automatic Detector Alignment for each light
path.
After pressing the Continuous button in the
Manual Detector Alignment window or in the
Main tool tabs a new image will open with a live
image of the grating (Fig. 11)
Fig. 11 Manual Detector Alignment,
Grating at Zoom 1
When the grating is out of focus, you need
to refocus it for the relevant channel by
performing the steps described in section
3.2.3. To check the focus, zoom into the
image using the Zoom function and look at
the center cross within the square (Fig. 12). If
the white portions within the inner most
circle are recognizable, the grating is in
focus.
To zoom into the image you can use these
buttons:
As a reference, the combination of Emission
Selection filter position 1 and Cam 1 should be
taken (see above). This combination should be the
first live image. Here mark a structure close to the
middle of the circled cross using the Graphics
View control. This cross will be the reference for
the following steps.
Now change the light path to the combination of
Fig. 12 Manual Detector Alignment,
Grating, center part zoomed in
Emission Selection filter and Channel (Cam 1 or
Cam 2) that you wish to adjust. The live image will
now show the grating with the current settings.
18 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Lightsheet Z.1 User Interfaces Carl Zeiss
Use the sliders or the input box with arrows of Cam 1 X and Cam 1 Y (for Channel 1, Cam1) or Cam 2 X
and Cam 2 Y (for Channel 2, Cam 2) to move the previously chosen structure of the grating to overlay
with the cross. Finish this step by pressing the Store Current Settings button.
Repeat the procedure for all desired Emission Selection filters and channels, always using the cross
marker as the reference.
It is helpful, especially after an Automatic Detector Alignment has been performed, to use the Profile
View tab in order to evaluate if the image of Cam 1 and Cam 2 overlay. While the grating is
continuously imaged, press the Profile View tab and draw a line or an arrow poly-line on the grating
(Fig. 13). Use the sliders or the input box with arrows of Cam 1 X and Cam 1 Y (for Channel 1, Cam1) or
Cam 2 X and Cam 2 Y (for Channel 2, Cam 2) to move the lines of the grating to overlay each other.
Fig. 13 Manual Detector Alignment, Grating, Profile view tab
You can leave the Manual Detector Alignment by pressing the Close button at the bottom of the
window.
3.2.3 Adjust the Grating Focus for the Automatic or Manual Detector Alignment Tool
During the Automatic Detector Alignment tool wizard and the Manual Detector Alignment tool a
grating is brought into the light path which can be imaged on each channel. When the grating is out-offocus, you need to refocus it for the relevant channel. For checking the focus, it helps to zoom into the
image using the Zoom function and look at the center cross within the square (Fig. 12) If the white
portions within the inner most circle are recognizable, the grating is in focus.
To adjust the focus of the grating start the Manual Detector Alignment by pressing the button in the
Adjustment tool window. The grating will be brought into the beam path and an image is generated
and displayed in the image container of the main software.
02/2013 000000-1790-528 19
CHAPTER 1 - HARDWARE
Carl Zeiss User Interfaces Lightsheet Z.1
Set focus for Cam 1 (Channel 1)
• Use the Light Path of the Manual Detector Alignment to first activate channel 1 (Cam1) and press
afterwards the Continuous button. The resulting live image shows the grating on channel 1 (Cam1).
• Use the Zoom tools of the Manual Detector Alignment to zoom into the center region of the image.
• With the live image running, open the front system door (Fig. 14/1) to the reflector turret for
emission filter selection. This system door has no safety interlock, and opening it will not terminate
image acquisition.
• Turn the screw which is above the turret (Fig. 14/3) with the long Allen wrench (Fig. 14/2).
1 Front system door
2 Allen wrench
3 Screw
Fig. 14 Position of the screw to adjust the focus of the grating for Cam 1 (channel 1)
• Monitor the live image of the grating while you turn the screw in any direction and find the position
of the screw that results in the sharpest image.
• Stop the continuous scan and close the front system door.
Set focus for Cam 2 (Channel 2)
• Use the Light Path of the Manual Detector Alignment to direct light towards channel 2 and
activate channel 2 (Cam2).
• Press afterwards the Continuous button. The resulting live image shows the grating on channel 2
(Cam2).
• Use the Zoom tools for the Manual Detector Alignment to zoom into the center region of the image.
• While the imaging continues remove the black screw cap (Fig. 15/1) on the back of the main system
module Lightsheet Z.1. This opening gives way to a screw that can be turned with an Allen wrench
(Fig. 15/2).
20 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Lightsheet Z.1 User Interfaces Carl Zeiss
• Monitor the live image of the grating while you turn the screw in any direction and find the position
of the screw that results in the sharpest image.
• Stop the continuous scan and place the black screw cap back on the system.
1 Black screw cap
2 Allen wrench
Fig. 15 Position of the screw to adjust the focus of the grating for Cam 2 (channel 2)
02/2013 000000-1790-528 21
CHAPTER 1 - HARDWARE
Carl Zeiss User Interfaces Lightsheet Z.1
3.2.4 Cable Connections for the Detection Module "PCO.Edge"
The detection module "PCO.Edge" is equipped with a liquid cooling system. Please read the safety notes
of the Lightsheet Z.1 system, as well as the separate safety data sheet for the liquid cooling system. The
power supply unit (PSU) for the cooling is connected to the system's power strip at the rear of the laser
module. The cooling hoses should be as short as possible. The connection creates a closed circuit, starting
from the cooling unit leading to the detection module 1 (approx. 200 cm hose length) reflection port
(Cam1). From the latter a hose (approx. 200 cm) leads either back to the cooling unit or, if a second
detection module is used, first to the transmission port (Cam2) (approx. 100 cm) and then a third hose
(approx. 200 cm) leads back to the cooling unit.
Fig. 16 Cable connections for the detection module "PCO.Edge"
The detection module cables are connected as follows:
− CameraLink cables A and B are each plugged into the PC for system control. The upper slots of the
PC for system control are assigned to the detection module of the reflection port (Cam1), [CamLink
A] right and [CamLink B] left. The lower slots are assigned to the transmission port (Cam2),
[CamLink A[ right and [CamLink B] left.
− The power cable is connected to the power strip of the system on the rear of the LB Rack
Lightsheet.
− The trigger cable is always connected to the detection module with [1/IN] and on the rear side of
the main system module Lightsheet Z.1 to [Camera 1/IN] (refection port, Cam1) or [Camera 2/IN]
(transmission port, Cam2).
− The detection module is switched on with the toggle switch on the module itself.
22 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Lightsheet Z.1 User Interfaces Carl Zeiss
3.2.5 Cable Connections for the Detection Module "Standard"
− The following connections exist for this detection module:
− The firewire cable must be plugged into the PC for system control. There are four firewire sockets
on the PC for system control; the detection module from the reflection port (Cam1) is connected to
position [FW 1], from the transmission port (Cam2) to position [FW 3].
− The trigger cable is plugged into the respective detection module and the three ends on the rear
side of the main system module Lightsheet Z.1 to [Camera 1] or [Camera 2], as well as [Camera
1/IN] and [Camera 1/OUT] (reflection port, Cam1) or [Camera 2/IN] and [Camera 2/OUT]
(transmission port, Cam2).
Fig. 17 Cable connections for the detection module "Standard"
02/2013 000000-1790-528 23
CHAPTER 1 - HARDWARE
Carl Zeiss User Interfaces Lightsheet Z.1
3.3 Assembly of the Sample Chamber
− Wear powder-free gloves to avoid fingerprints on the cover slips as well as to protect your hands
from sharp edges.
3.3.1 Assembly of the Sample Chamber Windows
− Assemble the three sample chamber body windows (for the illumination optics on the sides and
the front chamber window for the overview camera with LED white illumination) using the
following steps:
1 Sample chamber body
2 Cover slip
3 O-ring (18 mm O-ring, black)
4 Illumination adapter ring
5 Sample chamber window tool
Fig. 18 Assembly of the three sample chamber windows for illumination optics and overview
camera
1. Take the sample chamber body (Fig. 18/1) and lay it onto the table so that one of the three smaller
windows are facing you.
2. Using a forceps or gloved hands, remove one clean circular 18 mm cover slip; place cover slip into
the empty sample chamber body window so it fits into the smallest groove (Fig. 18/2) – you will
need a fine forceps.
3. Now place the O-ring (18 mm O-ring, black) into the corresponding groove (Fig. 18/3). Make sure
not to disturb the positioning of the cover slip.
4. Take the silver illumination adapter ring (Fig. 18/4) and lay it into the window making sure the flat
side with the four pinholes is facing you.
5. Using the sample chamber window tool (Fig. 18/5), position the circular pins into the pinholes and
turn clockwise until finger-tight. Adapter should be flush with the sample chamber body.
24 000000-1790-528 02/2013
CHAPTER 1 - HARDWARE
Lightsheet Z.1 User Interfaces Carl Zeiss
6. Repeat step 1 - 6 for each of the three windows.
− Assemble the sample chamber window for the detection optic.
1. Turn the sample chamber body (Fig. 19/1) to the largest opening window for the detection
optics.
2. Using a small spatula or the opposite end of a forceps position the blue O-ring tightly into
the groove to ensure no leaking (Fig. 19/2).
3. Assemble the chamber window for the detection optic according to the following steps.
Depending on the detection optics used, insert the correct detection optic adapter:
4. For the detection optic 5x/0.16:
a) Place the large silver detection optic adapter 5x (Fig. 19/3) onto the table with the
grooves facing you.
b) Remove one clean circular 18 mm glass cover slip; place cover slip (Fig. 19/4) into
the large silver detection optic adapter 5x (Fig. 19/3) making sure it is positioned
into the smallest groove – adjust the coverslip position with the forceps if necessary.
c) Now place the O-ring (18 mm O-ring, black) into the corresponding groove on top
of the cover slip (Fig. 19/5).
d) Take the silver retaining ring (Fig. 19/6) and place it inside the detection optics
adapter 5x with the flat side with two notches facing you. Using the circular pin-side
of the sample chamber window tool (Fig. 19/9), turn clockwise until finger-tight.
e) Placing the assembled detection optic adapter 5x into the sample chamber body
window. Using the squared pins of the sample chamber window tool, turn clockwise until finger-tight.
5. For the detection optic 20x/1.0, 40x/1.0, 63x/1.0:
a) Use the corresponding detection optic adapter (see the detection optic adapter
catalogue number and magnification engraving) (Fig. 19/7).
b) Insert the detection optic adapter into the sample chamber body window for
detection with the flat side facing you.
c) Using the sample chamber window tool (squared pin-side) (Fig. 19/9) turn adapter
clock-wise until finger-tight.
d) Take a 15 mm black O-ring (Fig. 19/8) and position the O-ring into the groove inside
the middle of the detection optic adapter using the blunt end of a forceps if
necessary.
02/2013 000000-1790-528 25
CHAPTER 1 - HARDWARE
Carl Zeiss User Interfaces Lightsheet Z.1
1 Sample chamber body
2 O-ring (blue)
3-6 For the detection optic 5x/0.16
− 3 Detection optic adapter 5x/0.16
− 4 Cover slip
− 5 O-ring (18mm O-ring, black)
− 6 Retaining ring
7-8 For the detection optic 20x/1.0, 40x/1.0, 63x/1.0
− 7 Detection optic adapter (20x/1.0, 40x/1.0, 63x/1.0
− 8 O-ring (15mm O-ring, black)
9 Sample chamber window tool
Fig. 19 Assembly of sample chamber window for the detection optic
26 000000-1790-528 02/2013
Loading...