Logos Celena X User manual

USER MANUAL
LBSM-MD-ML-CLX-001
VC1812-01
www.logosbio.com

DISCLAIMER
The contents of this document are subject to change without notice.
The CELENA®X High Content Imaging System is a set of electrical laboratory instruments for scientific research use only.
It is not a medical, therapeutic, or in vitro diagnostics device.
Do not disassemble the device on any occasion as this will invalidate your warranty.
TRADEMARKS
The trademarks used in this document are the property of Logos Biosystems, Inc. unless otherwise specified.
LED Filter Cubes are LTC Licensed Products provided under an intellectual property license from Life Technologies Corporation.
CellProfiler is provided under the BSD 3-Clause License.
© 2018 Logos Biosystems, Inc. All rights reserved.

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Table of Contents
1. Getting Started...................................................................................................................................................................................................4
1.1 Product contents 4
1.2 Product description 5
CELENA® X High Content Imaging System 5
CELENA® X Controller 5
Software 5
1.3 Setting up 6
Unpacking 6
Connections 6
Shipping guard/restraints 7
Turn on the CELENA® X 7
Install filter cubes and objectives 7
Shut down the CELENA® X 7
2. CELENA® X Explorer .......................................................................................................................................................................................8
2.1 User interface 8
Channels 9
Vessel 12
Z-stack 13
Time lapse 14
Project 15
Toolbar 16
Messages 16
Settings 17
2.2 Workflow 19
Create a project protocol 19
Save a project protocol 21
Load a project protocol 21
Run a project protocol 21
Pause/stop a project protocol 21
View project results 21
3. CELENA® X Cell Analyzer............................................................................................................................................................................ 22
3.1 Overview 22
Project 22
View 23
Image control 24
Analysis 25
Toolbar 25
Messages 26
3.2 Workflow 27
Set up project analysis 27
Load previously analyzed images 28
Annotate images and make simple measurements 28
Edit images 29
3.3 Pipeline module reference 30
Overview 30
Image processing 30
Object identification 33
Measurements 35

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4. Maintenance .................................................................................................................................................................................................... 37
4.1 General care 37
4.2 Change filter cubes 37
4.3 Change objectives 38
4.4 Adjust objective correction collars 39
Appendix A: Troubleshooting ................................................................................................................................................................................ 40
Image quality 40
Explorer 40
Mechanical 40
Appendix B: Specifications ...................................................................................................................................................................................... 41
CELENA® X High Content Imaging System 41
Appendix C: Safety Information............................................................................................................................................................................. 42
Instrument safety 42
Personal safety 43
Instrument symbols 43
Safety standards 43
Appendix D: Ordering Information...................................................................................................................................................................... 44
Instruments 44
Objectives 44
LED filter cubes 45
Accessories 45
Appendix E: Purchaser Notification..................................................................................................................................................................... 46
Limited use label license 46
LTC licensed products 46
BSD licensed products 46
Instrument warranty 47

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1. Getting Started
1.1 Product contents
Your CELENA® X is shipped with the following components:
CELENA® X High Content Imaging System
Camera module (installed as ordered)
Condenser (installed as ordered)
Laser autofocus module (installed as ordered)
Filter cubes (installed as ordered)
Objectives (installed as ordered)
CELENA® X Controller
PC
CELENA® X Explorer (installed)
CELENA® X Cell Analyzer (installed)
Accessories
Universal Vessel Holder
Microplate Holder
Single Slide Holder
Power Cord
Cable PS-1
Cable PS-2
Cable SIG
Cable USB 2.0
Cable USB 3.0
(Optional) Cable Laser AF (included with the Laser autofocus module)
Flathead Screwdriver
CELENA® X Cell Analyzer Verification Key
USB drive, 64 GB (includes the user manual and installation guide)
Keyboard
Mouse
Inspect the product package upon delivery to ensure that all components have been
included. If anything is missing, contact your local sales representative. Damage that may
occur during shipping and handling is not covered by warranty and must be filed with the
carrier.

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1.2 Product description
CELENA® X High Content Imaging System
Your CELENA® X High Content Imaging System is an integrated imaging system designed for
rapid, high content image acquisition and analysis. Customizable imaging protocols, image-
based and laser autofocusing modules, and a motorized XYZ stage simplify well plate
imaging and slide scanning. The integrated CELENA® X Cell Analyzer software processes
images and data for quantitative analysis. Analysis pipelines can be created and used to
identify cellular or subcellular objects, process images for optimal data collection, and make
various measurements.
①Door
②Condenser
③Motorized X/Y stage
④Power button
CELENA® X High Content Imaging System
CAUTION! This instrument uses Class 3B ultraviolet LEDs that are in accordance with
IEC/EN 60825-1. Make the CELENA® X door is closed when imaging to protect
your eyes. Direct exposure to and diffuse reflections of the laser can be
hazardous to the eye.
CELENA® X Controller
The CELENA® X Controller controls the power supply to and mechanical stages of the
CELENA® X.
Software
CELENA® X Explorer
The CELENA® X is controlled by the integrated CELENA® X Explorer software. CELENA® X
Explorer is pre-installed to the computer supplied with the instrument.
CELENA® X Explorer
CELENA® X Cell Analyzer
CELENA® X Cell Analyzer is used to process and analyze images to quantify numerous
cellular phenotypes simultaneously. CELENA® X Cell Analyzer also provides tools to edit
and annotate images as well as create videos.
CELENA® X Cell Analyzer
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1.3 Setting up
Unpacking
Move the unpacked boxes to the site of operation.
CELENA® X
CAUTION! When moving the CELENA® X, do not attempt to lift or move the instrument
without assistance. It is recommended that two or more people lift the
instrument together while taking the proper safety measures to avoid injury.
IMPORTANT! Do not subject the CELENA® X to sudden impact or excessive vibration.
Handle the instrument with care to prevent damage.
IMPORTANT! Wiping the computer supplied with the CELENA® X (i.e., erasing the hard
drive to remove programs, etc.) voids the product warranty.
Open the CELENA® X box and remove the Styrofoam top and sides. Lift the CELENA® X out
of its box by grasping its base firmly.
Place the CELENA® X on a flat, level surface that is free of vibration. Anti-vibration tables are
recommended for optimal use. Leave sufficient space around the instrument for proper
ventilation and to prevent overheating.
IMPORTANT! Do not expose the instrument to intense ultraviolet light.
CELENA® X Controller
Place the Controller near the CELENA® X. A separate surface is recommended for optimal
use but is not necessary.
Connections
Unpack the cables from the accessories box and attach as specified below:
CELENA® X & Controller
CELENA® X & PC
Cable PS-1 (I10331)
USB 2.0 (I10335)**
Cable PS-2 (I10332)
USB 3.0 (I10336)**
Cable SIG (I10333)
Cable AF (I10334)*
*Included if the laser AF module was purchased and installed
**Make sure to plug into the blue USB 3.0 ports at the back of the PC, not the front
Back of the CELENA® X
The CELENA® X is compatible with 4K Ultra HD (UHD) monitors. Use a DisplayPort (DP)
cable to connect a 4K UHD monitor to the provided PC.
The CELENA® X Cell Analyzer Verification Key is a parallel or USB port hardware dongle
that unlocks Cell Analyzer functionality. Attach to the provided PC.
Controller
PC

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Shipping guard/restraints
Your CELENA® X is shipped with two shipping restraints installed (X/Y stage, LED filter
cube stage) to prevent damage to the instrument from shock and vibration during transport.
IMPORTANT! The shipping restraints must be removed before the CELENA® X is turned on.
Remove shipping restraints
1. Unscrew screw A and pull it up to remove it from the LED filter cube stage cover.
2. Unscrew screw B and pull it up to remove it from the X/Y stage.
Note: Store the screws in the accessories box for future use. Make sure they are accessible in
case you need to pack up for maintenance and servicing purposes.
Turn on the CELENA® X
IMPORTANT! The shipping restraints must be removed before the CELENA® X is turned on.
Turn on in this order:
CELENA® X Controller
CELENA® X
Run CELENA® X Explorer
IMPORTANT! Using both Explorer & Cell Analyzer at the same time can affect both imaging
and analysis time. Use just one program at a time.
Install filter cubes and objectives
IMPORTANT! CELENA® X Explorer must be on to install filter cubes and objectives.
For detailed instructions on how to install filter cubes, go to 4.2 Change filter cubes.
For detailed instructions on how to install objectives, go to 4.3 Change objectives.
Make sure the installed filter cubes and objectives match what is set in the CHANNELS panel.
Shut down the CELENA® X
IMPORTANT! Explorer must be shut down before the instrument to allow the stages to dock
for safety.
Turn off in this order:
CELENA® X Explorer
CELENA® X
CELENA® X Controller

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2. CELENA® X Explorer
2.1 User interface
The CELENA® X Explorer is the graphical user interface for the CELENA® X High Content
Imaging System.
CELENA® X Explorer
①CHANNELS: Gives control over light, camera, and focus settings.
②VESSEL: Allows you to select the appropriate vessel, wells, and fields to capture.
③Z-STACK: Allows you to capture multiple planes along the Z-axis.
④TIME LAPSE: Allows you to set up time lapse sequences.
⑤PROJECT: Allows you to run, load, save, and edit automated imaging projects.
⑥Toolbar: Has tools for capturing and visualizing the current field of view.
⑦Viewing area: Shows the current field of view.
⑧System messages: Displays system messages.
⑨Settings: Allows you to set system options and perform calibration procedures.
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Channels
This panel is used to set light, camera, and focus parameters for a project.
①Add: Adds the CHANNELS settings to the project protocol.
②Objectives: Allows you to select from the currently installed objectives.
③Channels: Allows you to select from the currently installed filter cubes and adjust
light and camera settings.
④Focus: Allows you to find focus and set up autofocusing.
Objectives
This panel is used to select from the currently installed objectives.
The magnification and label on each objective reflects its profile, which can be modified in
Settings > Objectives.
Click the desired objective to select the corresponding magnification. You can select only one
objective at a time. The selected objective is highlighted in blue.
Channels
This panel is used to set the light and camera settings.
①ON/OFF: Use to turn the light source on and off. When the light is on, the viewing
area shows the sample illuminated with the selected light source.
CAUTION! This instrument uses Class 3B ultraviolet LEDs that are in accordance with
IEC/EN 60825-1. Make the CELENA® X door is closed when imaging to protect
your eyes. Direct exposure to and diffuse reflections of the laser can be
hazardous to the eye.
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IMPORTANT! Minimize the time that the sample is being exposed to light to prevent
photobleaching and/or phototoxicity.
②Condenser: Is installed at the time of purchase (BF: brightfield, PH: phase
condenser). Click the condenser (BF or PH) button for transmitted light.
The dropdown menu allows you to control the condenser’s iris diaphragm.
AUTO Automatically adjusts to accommodate the selected objective
100% Used for objectives with high magnification
66% Used for objectives with medium magnification
33% Used for objectives with low magnification
0% Used for fluorescence imaging
③Light: Controls the brightness of the selected channel. To adjust, move the slider in
the desired direction or enter the desired value in the text box. Light intensity is
controlled as a single parameter and expressed as a value between 1-1000.
④Filter cubes: Represents the fluorescence channels available for imaging. Up to
four interchangeable filter cubes may be installed at once for multichannel
fluorescence imaging. Click the desired channel to select the corresponding light
source. The selected filter cube is highlighted in blue. You can select only one
channel at a time. Each filter cube can be renamed and its pseudocolor selected in
Settings 󰵊Filter Cubes.
⑤Gain and exposure: Controls the camera capture settings. To adjust, move the
slider in the desired direction or enter the desired value in the text box. Gain is the
camera’s amplification of the signal.
Gain is the camera’s amplification of the signal.
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8-bit: 0-36 dB
12-bit: 0-24 dB
Exposure is the amount of time that the camera shutter is open to allow
light into the sensor.
Exposure range: 0-10,000 ms
Focus
The focus panel is used to find focus and to set up autofocusing for batch processing in the
currently selected channel.
①Focus slider: Used to adjust focus. The focus slider represents the full focal range.
Adjust focus by moving the slider in the desired direction.
②Z-stage speed: Used to adjust the speed at which the Z-stage moves with each
action. For fine focusing at high magnifications, set the focus speed to Slow. When
Step is selected, the Z-stage moves the distance of the selected objective’s depth of
focus with each click.
③Z-position: Shows the position of the Z-stage and used to adjust focus. The focus
position is expressed in mm along the Z-axis. Adjust focus by entering the desired
value in the text box.
④Find focus: Used for instant autofocusing.
⑤Multiscan AF:Used to set repeated autofocusing during an experiment.
The CELENA® X has two autofocus options: Find Focus for instant autofocusing and
Multiscan AF for repeated autofocusing during an experiment.
Find Focus
Find Focus is used to have the CELENA® X find the optimal focal plane based on the image.
Set the range to scan from the current focal position.
A long search range is useful when finding the focal plane of an unknown object.
A short search range is useful for fine focusing.
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Note: The speed of the image-based autofocus is entirely dependent on the set exposure.
Reducing the exposure will increase focusing speed.
Multiscan AF
Multiscan AF is used to set up autofocusing for demanding batch image acquisitions such as
multi-well plate imaging, slide scanning, and time-lapse imaging.
Prior to setting up Multiscan AF, make sure to bring the current field into sharp focus. The
field must be focused sharply to setup subsequent autofocusing correctly.
IMPORTANT! If using this feature, Multiscan AF must be set up for each channel used.
①Autofocus frequency: Used to set the autofocus frequency to use during an
automated scan.
First field of each selected location
Every field
Every _ fields
Optimal AF frequency and range settings in Multiscan AF mode
Optimal AF frequency
Optimal AF range
Multi-well plates
at least 1 field/well
±100 µm
Slides
every 10-20 fields
±10 µm
*Optimal AF frequency is also affected by objective magnification. Adjust accordingly.
*Optimal AF range is also dependent on vessel bottom flatness. Adjust accordingly.
When setting up Multiscan AF, you can select to use either the image-based or laser
autofocus (optional; installed upon purchase).
A comparison of the CELENA® X autofocusing modes
Image-based AF
Laser AF
Imaging speed
Moderate
Fast
6 minutes
1 color, 10 ms exposure,
96-well plate
2 minutes
1 color, 10 ms exposure,
96-well plate
9.5 minutes
3 colors, 10 ms exposure,
96-well plate
3.5 minutes
3 colors, 10 ms exposure,
96-well plate
Applicable magnifications
All
10X-60X
Photobleaching
Yes
No
Scratches, particles in sample
Affected
Not affected
Scratches, particles, and/or
fingerprints on bottom surface
Not affected
Affected
Cell number,
illumination conditions
Affected
Not affected
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②Image AF: Select to set up image-based autofocusing.
Make sure the current field is focused sharply.
Autofocus range: Use to set the range to scan from the current focal
position.
Focus offset: Choose to turn the focus offset on or off. The CELENA® X
will calculate the difference between what the system defines as the
optimal focal plane and what you define as the focal plane of interest and
automatically calibrate the focus accordingly.
③Laser AF: Select to set up laser-based autofocusing.
Make sure the current field is focused sharply and the correct vessel is selected.
Pre-scan: Use to have the CELENA configure the laser autofocus settings.
Test AF: Use to test the accuracy of the configured laser autofocus.
IMPORTANT! Laser AF is not compatible with the following:
!Objectives with magnifications below 10X.
!PHC phase contrast objectives.
!LED filter cubes with an emission wavelength exceeding 750 nm.
!LED filter cubes with an excitation wavelength less than 350 nm.
Vessel
This section allows you to select the appropriate vessel, area, and fields to image.
①Current vessel: Shows the currently selected vessel.
②Select vessel: Allows you to select a vessel. Use the dropdown menus to select the
vessel category and type. If the vessel you need is not available, go to Settings >
Vessels to create a vessel.
③Vessel map: Represents the currently selected vessel.
④Well map: Represents the currently selected well. The field size within each well
changes with the selected magnification.
⑤Acquisition order: Allows you to specify the order in which selected areas are to
be captured.
IMPORTANT! If using an objective with a correction collar, adjust the correction collar as
necessary according to the bottom thickness of the selected vessel.
View a specific area/well: Double-click the desired area/well in the vessel map to move the
stage to its respective location. The currently displayed area/well is rimmed in blue and
indicated by red crosshairs.
Select a specific area/well for imaging: Click and drag to select multiple areas/wells in the
vessel map. Otherwise, click each area/well. Selected areas/wells are filled in with yellow.
Select a specific field for imaging: Click and drag to select multiple fields in the well map.
Otherwise, click each field. Selected fields are filled in with yellow.
Select the acquisition order: Click one of the following buttons to specify the order in
which selected areas are to be captured.
Horizontal, zigzag
Horizontal, rightward
Vertical, zigzag
Vertical, downward
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Z-stack
This panel allows you to set up Z-stack imaging. These settings apply to each added channel.
①Add: Adds the Z-STACK settings to the project protocol.
②Method: Allows you to select from three Z-stack imaging methods.
③Z-stack settings: Allows you to set the Z-stack imaging parameters.
There are three methods of operation:
Z-stack methods
Start/End: Set the start and end positions of the Z-stack.
oSet start: Use to set the current focal plane as the start position.
oSet end: Use to set the current focal plane as the end position.
oOnce the start and end positions have been set, the Z-stack distance is
automatically calculated.
Range (Current): Set the distance above and below the current Z-position.
oAbove (+): Use to set how far above the current focal plane to capture.
oBelow (-): Use to set how far below the current focal plane to capture.
Range (AF): Set the distance above and below the autofocused position for each
well. This method should be used when well-to-well focal variations are extreme.
Multiscan AF must be set up first.
oAbove (+): Use to set how far above the autofocused position to capture.
oBelow (-): Use to set how far below the autofocused position to capture.
Distance: The total distance between the start and end positions of the Z-stack.
This is automatically calculated.
Steps: The number of planes to capture along the Z-axis.
Interval: The distance in µm between each focal plane captured.
Well 1
AF position
Well 2
Well 3
Range (AF)
Start/End
Well 3
Well 2
Well 1
End
Start
Range (current)
Current position
Well 3
Well 2
Well 1
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Time lapse
This panel allows you to set up time lapse imaging for the project protocol. This applies to
each channel. Selected fields are captured at set intervals over an allotted period of time.
①Add: Adds the TIME LAPSE settings to the project protocol.
②Total time: Allows you to set the total imaging time.
③Interval: Allows you to set the time period that must elapse before a new set of
images are captured.
The interval can be set manually or one of the two options below can be used to capture a
new set of images immediately after capturing the previous set with no delay.
As fast as possible: Can be used when imaging in multiple channels to capture the
maximum number of images possible without stopping.
Maximum frame rate: Can only be used when imaging a single field in a single
focal plane with one channel to capture up to 30 frames per second. This option can
be used for high-speed experiments such as calcium imaging.
Note: The interval will take into account other protocol settings such as the autofocus
settings and exposure time.
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Project
The project control panel is used to:
Create and run a project
Open or save a project protocol
①Create project: Allows you to start a project to image. This creates a project folder
where all generated data will be stored.
Project file (.cxproj): Stores project information, images, and associated
metadata. This file can be opened in Cell Analyzer for analysis.
Captured images
Image thumbnails
Note: Save projects on the computer from which you are running Explorer.
Do not save the project to an external hard drive or a USB drive as this can affect
imaging time.
②Project details: Shows you the file path and name of a created project.
③Open protocol: Allows you to open a previously saved protocol. This opens a
previously saved protocol file (.cxprotocol). When you open a protocol, make the
appropriate adjustments to each parameter as needed.
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④Save protocol as: Allows you to save a protocol. This saves a protocol file
(.cxprotocol) for future use.
⑤Protocol details: Shows you the protocol details.
Toolbar
The toolbar has tools for capturing and visualizing the current field of view.
Capture
Save
Pseudocolor
Highlight saturated pixels
Live histogram
Center lines
Gridlines
①Capture: Click once to capture an image in the viewing area and turn off the light.
Click again to clear the image from the viewing area and turn on the light.
②Save: Saves the captured image in the viewing area.
③Pseudocolor: Shows the sample illuminated with the selected light source in
pseudocolor. Go to Settings > Filter cubes to change the pseudocolor for each
channel.
④Highlight saturated pixels: Displays the pixels in saturated areas on an image. Go
to Settings > Camera to change the color to label saturated pixels.
⑤Live histogram: Shows a graphical representation of tonal values in real time.
⑥Center lines: Shows center lines in the viewing area.
⑦Gridlines: Shows gridlines in the viewing area.
Messages
This panel is used to display system messages. You can resize the message panel by dragging
the top border.
Not all system messages indicate problems with your system.
The message text can be copied for troubleshooting.
To copy all the messages, right-click inside the message panel and click Select All
from the context menu. Right-click the selection and select Copy from the context
menu. The selection is copied and can be pasted as desired.
To copy a specific message, select the desired message and right-click the selection.
Select Copy from the context menu. The selection is copied and can be pasted as
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Settings
Settings allows you to set system options and perform calibration procedures. To access the
Settings window, click the Settings wheel above the tool bar.
Camera
The camera settings allows you to select the camera, bit depth, saturated pixel color, as well
as auto white balance the color camera.
①Camera:
oMono: Selects a monochrome camera.
oColor: Selects a color camera,
②Bit depth: Can select to capture images in 8-bit or 16-bit with the monochrome
camera (the actual bit depth of 16-bit images is 12-bit).
③Saturated pixel color: Can select to color saturated pixels in red, green, or blue.
④Auto white balance: Adjusts color intensities to render colors correctly when
using the color camera.
IMPORTANT! Images captured with the color camera cannot be analyzed with CELENA® X
Cell Analyzer.
Objectives
The objectives settings allows you to change objectives, adjust objective correction collars,
and set the description for each installed objective. See 4.3 Change objectives to learn how to
change objectives.
①Change objectives: Can be used to install and remove objectives.
②Adjust correction collars: Can be used to adjust the correction collar of objectives.
③Objective information: Can be used to set the installed objectives and label them.
Filter cubes
The filter cubes settings allows you to change filter cubes and set the pseudocolor and
description for each installed filter cube. See 4.2 Change filter cubes to learn how to change
filter cubes.
①Change filter cubes: Can be used to install and remove filter cubes.
②Filter cube information: Can be used to set the installed filter cubes, assign their
associated pseudocolors, and label them.

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Vessels
The vessels settings allows you to create and edit custom vessels.
①Vessel details: Can be used to select the vessel type, number of wells, well shape,
and vessel name.
②Vessel description: Shows the associated catalog numbers of the selected vessel.
③Vessel dimensions: Shows the dimensions of the selected vessel.
④Save as a new vessel: Allows you to create a new vessel.
Create a vessel
Select the vessel type.
Select the number of wells.
Select the well shape.
Input the vessel dimensions: plate length (A), plate width (B), A1 row offset (C), A1 column
offset (D), well spacing from center to center (E), plate height (G), well diameter bottom (H),
flange/skirt height (I), well bottom thickness (J).
Name the vessel in Save as a new vessel.
Click Save.
Information
This section contains information about hardware, software, and the end user license
agreement (EULA).
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2.2 Workflow
Create a project protocol
Overview
Upon starting Explorer, you will create a new project protocol to capture images.
Select vessel.
▼
Select objective.
▼
Select a channel.
▼
Set light and camera parameters.
▼
Focus on the sample.
▼
Adjust light.
▼
Set Multiscan AF options.
▼
Click Add [CHANNELS].
▼
Repeat for all necessary channels.
▼
Select areas and fields to be captured.
▼
(Optional) Set up Z-stack imaging.
▼
Click Add [Z-STACK].
▼
(Optional) Set up time lapse imaging.
▼
Click Add [TIME LAPSE].
▼
Create a project.
▼
Run the automated scan.
1. Select a vessel
In the VESSEL panel, click Select to bring up the vessel selection window.
Use the dropdown menus to select the vessel category and type.
Available vessel types are well plates, slides, dishes, and flasks.
It is crucial that you select the correct vessel to ensure proper focusing and vessel
navigation. If the vessel you need is not available, go to Settings > Vessels to create a vessel.
IMPORTANT! Make sure the vessel doesn’t fall into the CELENA® X.
2. Select an objective
In the CHANNELS panel, click the desired objective to select the corresponding
magnification.
You can select only one objective for each project/protocol. If the objective you need is not
available, go to Settings > Objectives to install a different objective.
3. Set up the channels
In the CHANNELS panel, set up channels as needed.
CAUTION! This instrument uses Class 3B ultraviolet LEDs that are in accordance with
IEC/EN 60825-1. Make the CELENA® X door is closed when imaging to protect
your eyes. Direct exposure to and diffuse reflections of the laser can be
hazardous to the eye.
IMPORTANT! Minimize the time that the sample is being exposed to light to prevent
photobleaching and/or phototoxicity.
Select a channel
Click the desired channel and adjust the condenser’s iris diaphragm.
For brightfield imaging, click BF or PH for transmitted light. Use the dropdown
menu to control the condenser’s iris diaphragm as desired.