Zeiss Axio Zoom.V16 User manual

Stereo Zoom Light Microscopy - ZEISS Axio Zoom.V16

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Contents
Brief Description............................................................................................................................ 3
Reference ...................................................................................................................................... 3
Location......................................................................................................................................... 3
Suggestion for “Materials and Methods” ..................................................................................... 3
Suggestion for “Acknowledgements” ........................................................................................... 4
People and Contacts...................................................................................................................... 4
Optics ............................................................................................................................................ 4
Features and Specifications .......................................................................................................... 5
Objective ....................................................................................................................................... 5
Filter-Sets ...................................................................................................................................... 6
Additional Equipment ................................................................................................................... 6
System Overview........................................................................................................................... 7
WARNINGS and Advice ................................................................................................................. 7
Potential Techniques..................................................................................................................... 8
Set Up............................................................................................................................................ 9
Sample Preparation................................................................................................................... 9
Initial Set Up.............................................................................................................................. 9
Final Set Up ............................................................................................................................. 12
ZEN 2.1 (blue edition) Guidelines................................................................................................ 13
Single Image Acquisition or Visualization................................................................................ 14
Multidimensional Image Acquisition (Multi-Channel Images)................................................ 18
Channel Combine or Image Overlay ....................................................................................... 26
Extracting Single Fluorescence Images of a Multichannel Image........................................... 30
Closing ZEN 2.1........................................................................................................................ 31
Downstream Image Processing and Analysis.......................................................................... 32
Open.................................................................................................................................... 32
Display Adjustments............................................................................................................ 32
Add Scale Bar or Arrows...................................................................................................... 34
Distance Measurements ..................................................................................................... 35
Cropping Region of Interest (ROI) ....................................................................................... 36
Export .................................................................................................................................. 37
Other Guidelines ......................................................................................................................... 40
Further Protocols, Tutorials and Guidelines ............................................................................... 43

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Brief Description
Axio Zoom.V16 is a stereo microscope that successfully combines a large field of view,
zoom, and working distance. Axio Zoom.V16 combines a 1x zoom with a numerical
aperture of NA 0.25. It achieves a considerable numerical aperture for a medium zoom
range: superior fluorescence brightness in large object fields.
Axio Zoom.V16 is the fluorescence zoom microscope fully automated for large field
visualization having a motorized zoom from 0.7x to 11.2x, motorized Z focus and easy-
to-use system control panel.
Reference
Zeiss Axio Zoom.V16
Location
Room 5.25 (Microscopy Cluster), 5º floor, Instituto de Tecnologia Química e Biológica
António Xavier (ITQB NOVA)
Suggestion for “Materials and Methods”
Images were acquired on a Zeiss Axio Zoom.V16 stereo microscope equipped with a
Zeiss Axiocam 503 mono CCD camera and controlled with the Zeiss Zen 2.1 (blue edition)
software, using the 1x 0.25 NA objective, the fluorescence filter sets GFP + mRFP and
the Bright Field optics.

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Suggestion for “Acknowledgements”
This work was partially supported by PPBI - Portuguese Platform of BioImaging (PPBI-
POCI-01-0145-FEDER-022122) co-funded by national funds from OE - "Orçamento de
Estado" and by european funds from FEDER - "Fundo Europeu de Desenvolvimento
Regional”.
People and Contacts
Adriano O. Henriques, Microbial Development Group ([email protected])
Mónica Serrano, Microbial Development Group ([email protected])
Carolina Cassona, Microbial Development Group ([email protected])
Fernando Cruz, Microbial Development Group ([email protected])
Phone [phone]
Extension 1524
Optics
Transmitted Light:
•Bright Field
•Dark Field
•Oblique Light
Incident Light:
•Fluorescence

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Features and Specifications
•Incident Light Source: HXP 200 C illuminator (w/ integrated electronic shutter)
•Illumination: HXP 200 (200 W mercury short-arc reflector lamp)
•Fluar illuminator Z mot: Motorized reflector turret for up to four Z reflector
modules (filter sets)
•Motorized Stand 300: Focus motor 3 w/ central profile column placed on a
Transillumination base 300 (Transmitted Light Source)
•System Control Panel: SYCOP 3 (joystick, scroll wheels, buttons, and a
touchscreen into a handy, mobile control unit)
•Electronic Module: EMS 3 (for connecting SYCOP3)
•Objective nosepiece Z
•Eyepieces: PL 10x/23 Br. foc.
Objective
•Magnification: 1x
•Zeiss System: PlanNeoFluar Z
•Class: Achromatic objective
•Numerical Aperture (A): 0.25
•Free Working Distance (mm): 56
•Pixel Size for 0.7x (µm): 6.4857
•Pixel Size for 11.2x (µm): 0.4054
Notice that the zoom microscope is equipped with two eyepieces PL 10x/23 Br. foc.
Furthermore, a camera adaptor 60N-C1 1x is also installed. In general, the Axio
Zoom.V16 has a motorized zoom from 0.7x to 11.2x

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Filter-Sets
Filter Cube
Excitation Filter
Dichromatic Mirror
Emission Filter
Emission Colour
GFP1
BP 470/40
495
BP 525/50
Green
YFP1
BP 500/25
515
BP 535/30
Green/Yellow
mRFP1
BP 572/25
590
BP 629/62
Red
1High efficiency (HE) filter set: These filters are distinguished by particularly steep cut-
off edges and extremely high transmittances
BP stands for bandpass filter
Additional Equipment
Camera
•Brand: Zeiss®
•Type: CCD Cameras
•Model: Axiocam 503 mono (D)
•Adaptor: 60N-C1 1x (magnification)
•Features: Sensor CCD; 2.8 megapixels (1936 x 1460 active pixels); 4.54 µm pixel
size; 38 Frame Rate @ 1936 x 1460 (frames per sec)
Computer
•Type: Desktop
•Brand: Asus®
•Model: i9S
•Features: Intel I5 4460 up to 3.2 GHz (quad-core); 8GB RAM; 1TB HDD
•OS: Windows 7 Professional
•Available Software: Zeiss Zen 2.1 (blue edition)
Monitor
•Brand: Samsung
•Model: S24C450B

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System Overview
WARNINGS and Advice
•Before you get started for the first time on this microscope consult the
responsible people for proper training
•ONLY turn ON the lamp if it has been off for more than one hour
•Do not turn OFF the lamp if someone else will be using the microscope in the
next few hours. In order to avoid unnecessary working hours or usage constraints,
please inform the next user about the microscope occupancy
•You must turn ON THE LAMP FIRST and then the remaining devices
•THE LAMP must be the LAST TO TURN OFF
•Never LOOK DIRECTLY into the beam paths as they are capable of permanently
damaging the human eye
•Make sure you have everything set (as for instance, samples, plates, etc) before
starting the observations

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•Automatic movements can be interrupted by pressing the STOP button. Prevent
the objective from colliding with the specimen or transillumination base. The end
positions for the motorized range of movement should be set when appropriate
•When the motorized focusing drive is lowered, there is the risk of pinching
fingers in the working area. Do not reach into the working area or beneath the motorized
focusing drive during lowering
•The diaphragm slide should initially be at the rear stop in order to achieve a large
homogeneous luminous field. Pull it slowly to the front while observing the microscopy
image to achieve the desired effect
•The transmitted light can be completely covered with the diaphragm slide (front
stop) to achieve a dark background, e.g. for fluorescence illumination
•Slight careful deflection of the joystick results in fine focusing, while full
deflection produces faster focusing movements as a course screw would produce
•If you personalized some ZEN 2 pre-setting, reset the software for the default
definitions. Do not save any alteration to prior experiment sets
•Make sure you have the correct tube opening regarding the intended light path
(eyepieces or camera)
•Save all data into your own storage device or cloud as we routinely clean the
computer’s data
•Do not forget to register your utilization in the logbook
•A free software license is available for ZEN lite at ZEISS website. Install and use
such version in your own personal computer for downstream image analysis (see
Downstream Image Processing and Analysis)
•Contact the responsible people in case of any doubt
Potential Techniques
•Large-Field Observations
•Large-Field in vivo Timelapses
•Biofilm Observation

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Set Up
Sample Preparation
Colony and Biofilm Observation
This stereoscope allows visualization of colonies and biofilms previously
grown/developed onto plates supplemented with any media. Simply place the plate in
the centre of the transillumination base. Do not remove the cover.
Initial Set Up
1. Before you start, ensure the lamp has been off for more than one hour
2. Switch on the devices in the following order:
2.1. Incident Light Source HXP 200 C (Lamp)
2.2. Transmitted Light (turn the rotary knob clockwise)
2.3. Electronic Module EMS 3 (turn the device on in the back)

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2.4. Computer
3. All motorized components will run through an initializing phase, including the
SYCOP 3 (System Control Panel)
4. Meanwhile, in the computer’s desktop directory, run ZEN (blue edition)
and select ZEN Pro
5. Place the specimen in the centre of the round insert plate
6. Ensure that the aperture diaphragm is completely open. To do this, turn the
rotary knob up (on the right-hand side of the microscope)

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7. Adjust the tube towards camera image acquisition by pulling out the control bar.
Alternatively, if you only intend to use the eyepieces for visualization, please consult
other guidelines (See Operating Manually)
8. Although you can operate the microscope in manual mode, without using ZEN
2.1 (See Operating Manually), we strongly advise you to use the available software for
observation and image acquisition (Go to Single Image Acquisition or Visualization)

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Final Set Up
1. Before you switch off the devices confirm the next user and if so, ask whether he
or she will be using the microscope in the next hour
2. Remove the specimen from the transillumination base
3. If there is an interval of more than one hour to the next user, switch off the
devices. Press the Shut Down button on the Home page of SYCOP 3 touchscreen
4. When the safety query is answered by pressing the YES button, the
system will be shut down, while the remaining devices will be in the standby mode
5. Then, switch off the devices in the following order:
5.1. Electronic Module EMS 3 (turn the device off in the back)
5.2. Transmitted Light
5.3. Incident Light Source HXP 200 C (lamp)
6. Save all data into your own personal storage device or cloud and switch off the
computer (see FIRST Closing ZEN 2.1)
7. Sign your registration in the logbook
8. Take everything you brought and keep the room and desk clean for the next user

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ZEN 2.1 (blue edition) Guidelines
The ZEN 2.1 (blue edition) can be equipped with several modules, applications and
licenses. This guideline is strictly specific for the ZEN 2.1 (blue edition) version available
on the microscope’s computer. In other words, only for ZEN 2 pro on the computer.
The ZEN 2.1 user interface is divided into three main areas. Via the tabs in the Left
Tool Area (4) 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 Centre Screen Area (5) is used to display your images, while the Right
Tool Area (6) provides you with an overview of all open documents and is used for
advanced file management.

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Single Image Acquisition or Visualization
1. Go to the Locate tab
2. In the Favorites section, select the contrast method or filter set you would like
to acquire or visualize
The FL off mode activates the transmitted light and closes the shutter of incident
light (switching off fluorescence optics). To fully activate the transmitted light, you
should also open the diaphragm slide until the rear stop (in the Transillumination base
300), so that a large homogeneous luminous field can be obtained.

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You can now pull the diaphragm slide slowly to the front for adjusting the light
intensity. Notice that the bright field contrast method is regularly set as default (See
Other Guidelines -> Contrast Methods and Filter Sets).
Finally, once you finished the visualization in the FL off mode (transmitted light
illumination), do not forget to close the transmitted light by moving the diaphragm slide
until the front stop.
3. Click in the Live button. Once the Live mode is activated the camera live image
will be displayed in the Centre Screen Area

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4. Click in the blue header of the Camera tool. Then, in the Settings section, select
the adequate exposure time or Auto Exposure option.
Although, it is possible to increase or decrease the Exposure Time during visualization
as function of the fluorophores’ signal. To get back to the default settings, click in the
Default button.
5. Start focusing your sample using the SYCOP 3. You should move the specimen
carefully to find interesting and prominent detail.
With the minimum magnification, use the joystick and side wheels to swipe the
motorized focusing down or up. Repeat the process for consecutive magnifications
Joystick
Robust Forward Tilt
Coarse Focusing
Moving upwards
Joystick
Robust Backward Tilt
Coarse Focusing
Moving downwards
Joystick
Soft Forward Tilt
Fine Focusing
Moving slightly upwards
Joystick
Soft Backward Tilt
Fine Focusing
Moving slightly downwards
Wheels
Swipe Forward
Fine Focusing
Moving extremely slightly upwards
Wheels
Swipe Backward
Fine Focusing
Moving extremely slightly downwards

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To increase or decrease the objective magnification in the SYCOP 3, go to the
Microscope tab and select the magnification in the Function sub-tab
1 –Joystick
2 –Side Wheels
Hint: When focusing on the Bright Field contrast method try to adjust slightly the
diaphragm slide in order to obtain greater detail and contrast
Hint: If you are getting problems focusing the sample, use the Min/Max
button in the Histogram (Centre Screen Area -> Display -> Histogram). This will adjust
the gray levels which may be responsible for darker, dim or overexposed images (See
Downstream Image Processing and Analysis -> Display Adjustments)
6. After focusing the sample, click in the Snap button to acquire the first
image
2
2
1

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Multidimensional Image Acquisition (Multi-Channel Images)
1. Go to the Acquisition tab
You can create a new blank experiment. Alternatively, you can use one of the pre-
set experiments. Try to follow the protocol until the end and then look at the pre-set
experiments that may be useful for your experiment. In this way, we can keep the
workspace organized and without redundancy.
You can use and alter the available pre-set experiments as long as those alterations
are not saved at the end! We advise this option for short-term experiments
2. To create a new, "empty" experiment, click in the Options button of the
Experiment Manager section. The Options dropdown list will open.
3. Click on the New entry.
4. Enter a name for the experiment, e.g. "experiment_3_channel"

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5. To create the experiment, click in the Save button
You have created a new blank experiment. For any change in the experiment, an
asterisk (*) will appear after the file name. This means that the experiment was modified
and not saved. Save your experiments from time to time to ensure that your settings are
not lost.
6. Start focusing the sample (See above ZEN 2.1 (blue edition) Guidelines -> Single
Image Acquisition or Visualization)
7. Now in the Multidimensional Acquisition group, click in the blue header of the
Experiment Designer tool
8. Make sure to box the Enable Multi Block Execution option

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9. In the Acquisition Block section, click in the Add New button.
a) Add as many blocks as contrast methods/filter sets you would like to
acquire. For instance, to acquire images of Bright Field, GFP, mRFP and YFP, four blocks
must be added. This approach will generate one image for each contrast method and
filter set.
Later, you will be able to combine two images at a time into one using ZEN 2.1
(See ZEN 2.1 (blue edition) Guidelines -> Channel Combine or Image Overlay)
b) Alternatively, you can add only one block. This approach will generate a
multichannel image of all selected filter sets. Combine only filter sets into a single block
though, as it is required one block for each single contrast method.
In the end, you can extract individual fluorescence images of a multichannel
image (See ZEN 2.1 (blue edition) Guidelines -> Extracting Single Fluorescence Images of
a Multichannel Image)