Automation in Micrscopy

Julien Kissenberger Contact:

Ingénieur d'Applications julien.kissenberger@zeiss.com
2017-01-25 06.38.53.48.93
Agenda

1 Automation in Microscopy
2 AxioScan.Z1
3 Celldiscoverer 7

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 2

Agenda

1 Automation in Microscopy
2 AxioScan.Z1
3 Celldiscoverer 7

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 3

Automation
Generalities

What are the current issues in microscopy?

Resolution

Flexibility Repeatability/Reliability

Sensitivity Speed

User friendly

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 4

Automation
Generalities

What is Automation?

• Automation is defined, by the use of control systems for operating equipment

and driving processes with minimal human interventions.

• Automation is achieved by the combination of many components together :

mechanical parts, electrical and electronic devices, sensors and computers.

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 5

Automation
Generalities

How could Automation help me?

• Repeatability/Reliability:
• Limit human errors (ie: microscope setup, calibration…).
• Focus stability.
• Stable environment (ie: Temp., CO2…).

• Speed:
• Fast acquisition.
• Time in front of the system.

• Ease to use:
• Easy software with few clicks.
• Software with workflow processes.

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 6

Automation
Generalities

From manual… … to fully automated…

… for fixed samples

… for living samples

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 7

Agenda

1 Automation in Microscopy
2 AxioScan.Z1
3 Celldiscoverer 7

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 8

AxioScan
Automation for fixed samples on slides

• Slide scanner launched in 2013

dedicated to research.
• Based on the upright microscope,
AxioImager Z2.

• Widefield acquisitions.

• For Brightfield and Fluorescence.

• ZEISS Objectives.

• Possibility to acquire Zstack.

• The system could handle all slide

formats.

• Flexible configuration to fit to your need.

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 9

AxioScan
Flexible configuration

AxioScan.Z1

Objectives Light Sources Loader Filter sets Cameras

White LED 12 slides 6 positions Hitachi

2,5 x
Transmission (25mmx7mm) Fast filterwheel Tri-CCD

100 slides 10 positions AxioCam

5x Colibri Standard
(25mmx75mm) 506 mono
filterwheel
Metal-Halid Hamamatsu
10 x
Lamp Orca Flash 4

20 x

40 x

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 10

AxioScan
Slides

• All slide formats could be scanned from 25x72mm slides to 100x72mm slides.
• Only the tray is handled by the scanner... not the slides. It limits the risk to break
them during the loading/unloading process.
• Slides move horizontally in the scanner. It limits the risk to take off the coverslip, if
it is not fixed (ie: glycerine medium).

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AxioScan
Software

• The slide scanner is controlled by ZEN Blue.

• All the scanning processes are pre-configured in a workflow (scan profile).

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AxioScan
Scanning process

• Possibility to scan the same batch in Brightfield and Fluorescence.

• Possibility to read bar codes for automatic naming.

• For each slide, the software saves an overview image from the slide and the label.
An automatic threshold detects tissue sections using the overview image.

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AxioScan
Scanning process

• Before scanning, the software will perform an autofocus and it determines the
focus.

• Then, the scanning process will start, using the focus map defined previously. The
scanner will acquire 1cm² in 2 minutes (for brightfield @20x/0.8).

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 14

AxioScan
Virtual slides

• The virtual slide, a czi image, contains all the informations from the acquisition
(scaling, exposure time…).

• The images are stored with a lossless compression to avoid harddisk overflow.

• The pyramidal view is activated for each image.

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Agenda

1 Automation in Microscopy
2 AxioScan.Z1
3 Celldiscoverer 7

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 16

AxioScan
Automation for living samples on multiple supports

• Automated inverted microscope

launched at Neurosciences 2016.
• New opto-mechanical concept with high
light efficiency.

• Widefield acquisitions.

• For Brightfield and Fluorescence.

• ZEISS Objectives with AutoCorr.

• Incubation and humidity control.

• Perfusion and injection during

acquisition.

• The system could handle different

supports.

• A sample loader with 2 racks of 12

positions can be added.

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 17

Celldiscoverer
A new optical concept

Carl Zeiss SAS, Julien Kissenberger 2017-01-25 18

Celldiscoverer
Objectives

5x / 0.35 20x / 0.7 20x / 0.95 50x / 1.2

Working distance : Working distance : Working distance : Working distance :
5.1 mm 2.2 mm 0.76 mm 0.84 mm
AutoCorr objective AutoCorr objective AutoCorr objective

For thin or thick Water Immersion

bottom: Glass/PS

• A magnifier changer with 3 positions (0.5x, 1x, 2x) is included in the Celldiscover 7.

• Range of 8 different magnifications : 2.5x, 5x, 10x, 20x, 25x W, 40x, 50x W, 100x W.
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Celldiscoverer
Phase Gradient Contrast

• New contrasting method for transmitted light acquisition: Phase Gradient Contrast
(PGC)..
• Compatible with all objectives and all supports.
• PGC does not impact fluorescence (sensitivity) like standard phase contrast.

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Celldiscoverer
LEDs and fluorescence

• The Celldiscoverer 7 can be equipped up to 7 LEDs (from 385nm to 625nm), to

increase speed and reliability.
• 2 filterwheels, with a switching time of 80ms, complete the configuration.

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Celldiscoverer
Sample holder

• The Celldiscoverer 7 is not only dedicated to multiwall plates, you can acquire
images on all supports, regardless of the bottom.

Holder for Multiwell

Insert for Insert for Insert for

2x slides Petri dish 2x Lab-Tek™
Ø35/60 mm
Carl Zeiss SAS, Julien Kissenberger 2017-01-25 22
Celldiscoverer
Environmental changes

• Many environmental parameters can be applied or fixed during an experiment with

the Celldiscoverer 7.

Heating & Cooling Adding medium / Pipetting Perfusion with POC-R2

Gas exchanges

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Celldiscoverer
Software

• The Celldiscoverer 7 is controlled by ZEN Blue.

• The software can be controlled like an standard inverted microscope or with saved
workflows for automation applications.

Standard
workspace

Automation
workspace

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Celldiscoverer
Applications

• Scanning multiwell plate for automated cell counting.

384 multiwall plate imaged with 2.5x magnification.

Sample courtesy of German Center of Neurodegenerative Diseases (DZNE) Bonn, Core

Research Facilities

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Celldiscoverer
Applications

• Long-term live imaging on embryos.

A developing
cricket embryo.
During the
development from
egg stage 15 to
egg stage 21.
Scale bar 200µm.

Sample courtesy
of Seth Donoughe,
Department of
Organismic and
Evolutionary,
Harvard University
Cambridge, USA

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Celldiscoverer
Applications

• Cells proliferation assay: Control vs Knockdown

0h 22h 45h

0h 22h 45h

Top row: A series of images showing untreated control cells. Bottom Row: A series of images showing cells treated 24 h before acquisition with a
siRNA against AuroraB Kinase.

Sample courtesy of S. Reither, Advanced Light Microscopy Facility, EMBL, Heidelberg, Germany

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Celldiscoverer
Applications

• Studies on HEK cells expressing eGFP

Sample courtesy of Sanofi-

Aventis Deutschland GmbH
R&D IDD/in vitro Biology
Frankfurt.

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Celldiscoverer
Self-calibrated system

• Start-up • Calibration of RL fieldstop

• Initialization of all moving parts
• Calibrate Shading
• Calibration wizard
• Is used during system integration • DF calibrations
• Is used by service on-site customer can • glass
use it on demand • PS thin
• PS thick
• Stage
• Zero-position of mounting frame • Intensity
• Checks camera rotation

• Parfocality
• All objectives
• All magnification changer positions

• Parcentricity
• All objectives
• All magnification changer positions

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Celldiscoverer
Safety first

• The objectives are protected to avoid broken front lenses.

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Celldiscoverer
Safety first

• The autoimmersion system adjusts the proper volume between the objective and
the samples. All the fluids are removed after the experiment in a specific container.

• The Celldiscoverer 7 is delivered with an extra UV light source (around 250nm). It

can be used to sterilize the incubation chamber.

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 THANK YOU FOR YOUR ATTENTION.

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