LEAP™ System - Core Facilitiescorefacilities.isbscience.org/wp-content/uploads/sites/5/2015/07/LEA… · procedures. The LEAP System is designed to perform both user-interactive

LEAP™ System User Manual

LEAP Software Version 1.3 Doc. No. 17-LEAPSYSUMB1.3-R05

Published By Cyntellect® 6620 Mesa Ridge Road, Suite 100 San Diego, CA 92121 Telephone: (858) 450-7079 Fax: (858) 550-1774 http://www.cyntellect.com

Copyright and Patents

Copyright © 2002-2008 by Cyntellect. All rights reserved. The LEAP System, Software, and portions of this document are protected by one or more patents, including U.S. Patent Numbers 5,874,266; 6,143,535; 6,514,722; 6,534,308; 6,642,018; 6,753,161; 6,804,385; 7,092,557; 7,129,070; 7,300,795, Australia Patent Number 743,239, BREVET NO. 1011697 (France); D.B.P. 98913223.8 (Germany); UK Patent No. GB 1011697 and/or other pending patent applications. Cyntellect reserves the right to make modifications and additions to the information in this document without notice. No part of this document may be reproduced or transmitted in any form or means, electronic, photographic, mechanical, or otherwise, for any purpose without the express written permission of Cyntellect.

Trademarks Cyntellect is a registered trademark. LEAP™ is a trademark of Cyntellect. Other products or company names mentioned in this document might be trademarks or registered trademarks of their respective owners, and are treated as such.

Appropriate Use

For research and development uses, only. Not for use in diagnostic or therapeutic procedures. The LEAP System is designed to perform both user-interactive and automated functions for the measurement, analysis, and induction of events in cell specimens by imaging cells in microplates, analyzing images, and inducing changes in selected cells via laser irradiation. The LEAP System is designed to be used in a temperature and humidity controlled laboratory environment that is free from aerosols, liquid sprays or spills, and excessive airborne dust.

WARNING! Use this product only in the manner described in this document, and while observing all specified safety precautions. When used other than as specified, the safety features may be impaired or defeated. Failure to adhere to the safety precautions and/or procedures outlined in this document may result in system failure, personal injury, or death, for which Cyntellect shall not be held liable under any circumstances.

WARNING! Risk of electric shock! The LEAP™ System contains voltages and electric currents that are potentially hazardous. To reduce the risk of electric shock, do not remove instrument covers without proper training. Qualified service personnel should perform all repairs.

WARNING! The LEAP™ System can emit visible and/or invisible Class IV laser radiation when access doors or exterior panels are open and the safety interlocks are failed or defeated. Avoid eye or skin exposure to direct or scattered radiation. Skin exposure might result in a burn injury. Eye exposure might result in serious injury, possibly including blindness.

LEAP System User’s Manual for v1.3 Page 2 of 51

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Contents

1. Introduction to LEAP ............................................................................................ 444566689

10101013141415

38383943444550

2. About This Guide ..................................................................................................

2.1 Documentation and Labeling Conventions ...................................................

2.2 Technical Assistance....................................................................................

3. Safety and Limitations..........................................................................................

3.1 Laser Safety .................................................................................................

3.2 High Intensity Lamp Safety...........................................................................

3.3 Electrical Safety............................................................................................

3.4 Moving Part Hazards ....................................................................................

4. LEAP System Overview......................................................................................

4.1 Introduction.................................................................................................

4.2 Hardware and Optics..................................................................................

4.3 User Interface and Software.......................................................................

5. LEAP Operation...................................................................................................

5.1 System Startup and Shutdown ...................................................................

5.2 Instrument Operation..................................................................................

5.3 Calibrations ...................................................... Error! Bookmark not defined. 6. LEAP Processing ................................................................................................

6.1 Introduction.................................................................................................

Appendix A: Software License Agreement ...............................................................

Appendix B: Filter Configuration...............................................................................

Appendix C: Consumables List .................................................................................

Appendix D: Error Event Reporting...........................................................................

Appendix E: Example Power & Spot Size Data ........................................................

1. Introduction to LEAP

The LEAP (Laser-Enabled Analysis and Processing) System combines high-throughput cell imaging and laser-mediated cell manipulation. LEAP is based on robust semiconductor manufacturing technologies, providing users with significant speed advantages and unrivaled versatility. Laser-mediated cell manipulation capabilities validated on LEAP include cell enrichment and cloning, OptoClean™ (cell purification), LaserFect™ (optoinjection of ions, nucleic acids, proteins, etc.), and CALI (chromophore-assisted laser-inactivation for functional proteomics). The integrated cell imaging and manipulation capabilities on LEAP represent an enabling technology for new avenues of life science research and drug discovery. LEAP is protected by the following patents and others pending: U.S. Patent Numbers 5,874,266, 6,143,535, 6,514,722, 6,534,308, 6,642,018, 6,753,161, and 6,804,385; Australia Patent Number 743,239.

2. About This Guide

2.1 Documentation and Labeling Conventions

This manual and the System’s labeling utilize conventions to communicate safety hazards via symbols, and their associated severity levels via signal words. The hazard symbols, summarized in Table 2-1, allow for easy and rapid recognition of the hazard type. The signal word definitions, summarized in Table 2-2, are in compliance with the ANSI Z535.4 standard for product safety signs and labels. Table 2-1 Hazard symbols

Symbol Hazard

Voltage or electrical current

Intense visible or invisible electromagnetic radiation from lasers or other sources

Fire or heat

General


Table 2-2 Hazard severities.

Signal Word Severity

DANGER Indicates an imminently hazardous situation that will result in severe injury or death if it is not avoided.

WARNING Indicates a potentially hazardous situation that could result in severe injury or death if it is not avoided.

CAUTION Indicates a potentially hazardous situation that may result in minor or moderate injury. It may also alert against unsafe practices.

NOTICE Indicates a hazard or unsafe practice that could result in damage to material or property, or could result in the loss or corruption of data or information.

2.2 Technical Assistance

Cyntellect 6620 Mesa Ridge Road, Suite 100 San Diego, CA 92121 Telephone: (858) 450-7079 Fax: (858) 550-1774 E-mail: [email protected] Website: http://www.cyntellect.com

If you have a technical question that you are unable to answer after consulting the documentation provided with the LEAP System, please contact Cyntellect for assistance. So that we may assist you in the most expeditious manner possible, please be prepared to answer the following questions.

♦ What were you doing when the problem occurred? Try to recall the sequence of actions that

you took just prior to the problem’s occurrence. ♦ If any error messages appeared, what did they say? What error codes were displayed, if any? ♦ Is the problem reproducible? ♦ If you have attempted to solve the problem yourself, what actions did you take and what were

the results?


mailto:[email protected]


3. Safety and Limitations Read and understand the safety material in this manual before attempting to use or train on the LEAP System. If you are uncertain about any of the safety issues highlighted in this manual or have additional safety concerns, do not hesitate to contact Cyntellect directly, with your questions or concerns, before attempting to use the LEAP System.

3.1 Laser Safety

The LEAP System is a Class 1 laser device in normal operation. The LEAP System, however, contains two Class 3B lasers that emit at two laser wavelengths. One laser emits invisible ultraviolet (355 nm) radiation while the other emits visible green (532 nm) radiation.

Laser emissions @ 1000 Hz: Wavelength: 532 nm 355 nm Ave Power: 27.7 mW 19.4 mW Pulse Width: 0.5 nsec 0.5 nsec In normal operation the laser energy is contained within the instrument by the covers. To insure safe operation, the covers require tools for removal and are interlocked. However, if the safety interlocks have failed or are defeated, intentionally or otherwise, and the covers removed, it is possible for laser radiation to be emitted outside the instrument. By design, the laser path is contained within the interior frame of the instrument and beam blocks have been strategically located to keep the beam from exiting the instrument even when the front, back and end covers have been removed for servicing. Nonetheless, caution must be exercised and proper safety equipment used any time the instrument covers have been removed.

WARNING! The LEAP™ System can emit visible and/or invisible Class 3B laser radiation when access doors or exterior panels are open and the safety interlocks are failed or defeated. Avoid eye or skin exposure to direct or scattered radiation. Skin exposure might result in a burn injury. Eye exposure might result in serious injury, possibly including blindness.

Therefore, it is mandatory for all persons in the room surrounding the LEAP System to wear safety goggles that are rated for filtering Class 3B radiation levels for each of the two wavelengths of laser emission (355 nm and 532 nm) whenever the laser is powered on and any of the following circumstances occur: • the exterior cover is removed from the instrument. • either of the lasers have been removed from the instrument without disconnecting the power

from that laser.

3.2 High Intensity Lamp Safety

The LEAP System contains a high-intensity lamp used for the excitation light source. This lamp emits across a broad spectrum of electromagnetic radiation, including ultraviolet, visible, and infrared wavelengths. This lamp is not on the safety-interlocked circuit, so it can continue to


operate with the exterior cover removed or the access door open. There are three general hazards associated with this lamp.

3.2.1 Intense Light Hazard

One hazard is that the light emits a very high irradiance level of visible and invisible radiation, which can be damaging to skin and eye tissues when the light is tightly focused onto a small area of tissue. To mitigate this hazard, the user must observe the following precautions: • Do not look into the beam of intense light, or into a transmission or reflection of any

component of the intense light. • Wear safety goggles for intense light when operating the lamp with the exterior panels

removed. • Do not pass your skin into the path of the beam of intense light, or into a transmission or

reflection of any component of the intense light.

WARNING! The LEAP™ System’s high-intensity lamp can emit harmful visible and/or invisible electromagnetic radiation when access doors or exterior covers are open. Avoid eye or skin exposure to direct or scattered radiation. Skin exposure might result in a burn injury. Eye exposure might result in serious injury, possibly including blindness.

3.2.2 High Operating Temperature

The second hazard is that the lamp subsystem has a high operating temperature, which: (a) might cause a burn to the skin in the event of direct contact; (b) might ignite materials (such as paper), liquids (such as alcohol or acetone) or vapors (such as from alcohol or acetone) that have a low flashpoint; and, (c) might cause the System to overheat if the function of the air-flow cooling system were impeded in any way. To mitigate this hazard, the user must observe the following precautions: • When the exterior cover shielding of the high-intensity lamp is removed, be aware that the

exposed surfaces can be hot enough to burn your skin. Therefore, do not touch any component of the high-intensity lamp subsystem until it has completely cooled to a safe handling temperature.

• Do not impede the performance of the lamp’s cooling system. Keep the System’s exhaust vents

and air intake vents free of obstruction. Keep the System’s air intake filters in a clean state by cleaning them periodically.


CAUTION! The LEAP™ System’s high-intensity lamp has a high operating temperature. Exposed surfaces of this lamp and its associated subsystem might be hot enough to cause burns to the skin, and might ignite materials, liquids or gases that have a low flashpoint. Do not touch any component of the high-intensity lamp subsystem until it has completely cooled to a safe handling temperature. Keep flammable liquids and materials away from the lamp subsystem.

3.2.3 Explosion Hazard

The third hazard is that the lamp’s bulb can explode when it experiences a severe thermal stress (such as cooling too rapidly) or when it reaches the end of its service life. The explosion will generally be contained within the confines of the lamp housing, but there exists the chance that some debris, consisting of hot glass and metal fragments, might escape to the vicinity surrounding the lamp housing. To mitigate this hazard, the user must observe the following precautions: • When the exterior panel covering of the high-intensity lamp is removed, be aware that the bulb

might explode without warning, at any time, even when power is removed and the lamp is cooling down.

• When the exterior panel covering of the high-intensity lamp is removed, wear laser safety

goggles whenever operating the lamp or waiting for it to cool down to a safe handling temperature.

CAUTION! The LEAP System’s high-intensity lamp can explode when it experiences a severe thermal stress or when it reaches the end of its service life. The bulb might explode without warning, at any time. Therefore, wear laser safety goggles when operating the lamp with the exterior panels removed.

3.3 Electrical Safety

The LEAP System contains voltages and electric currents that are potentially hazardous. Under normal circumstances, the user and other persons in the vicinity of the System are protected from accidental contact with these electrical hazards by physical barriers (exterior panels and access doors) and by electrical grounding of the instrument. To reduce the risk of electric shock: • Do not remove instrument covers without proper training. • Ensure that all three-pronged power cords from the System (total of 4) are plugged only into

properly grounded 110 VAC receptacles. • In the event of a spill of an aqueous or other conductive solution within the instrument, power

down the system and unplug the power cords before attempting to clean up the spill. • In the event of a foreign object falling into the instrument through an opening (such as the

access door or one of the air vents), power down the system and unplug the power cords before attempting to retrieve the foreign object.


• Only qualified service personnel should perform repairs on or within the instrument.

WARNING! Risk of electric shock! The LEAP™ System contains voltages and electric currents that are potentially hazardous. To reduce the risk of electric shock, do not remove instrument covers without proper training. Qualified service personnel should perform all repairs.

3.4 Moving Part Hazards

The LEAP System contains mechanical components that move within the instrument. Some of these components move in a linear fashion (for example, the x-axis and y-axis movements of the specimen stage), and some of these components have a rotational motion (for example, the galvanometer motors and the filter wheels). The LEAP System’s moving components can pose risks of pinching, crushing, cutting, twisting or entrapping body parts, particularly hands and fingers. To avoid injury by the System’s moving components, you must observe the following precautions. • While a protocol is executing, keep the access door in the closed position. • While a protocol is executing, the various mechanical components of the LEAP System can

move at any time, without warning. Do not reach into the instrument while a protocol is executing, unless the protocol specifically requests that a new specimen plate be loaded onto the stage, at a particular time.

• Do not reach into the instrument to remove or load a specimen plate while the stage is still

moving. Wait until the stage has come to a complete stop, before reaching into the instrument. • Keep clothing, jewelry, hair, and other loose materials clear of the System’s mechanical

components. Moving components can catch hold of such loose materials, thereby forcing a body part into a dangerous position.

CAUTION! The LEAP System contains moving mechanical components that are capable of causing bodily harm. Do not reach into instrument while parts are moving. Keep clothing, jewelry, hair, and other loose materials clear of mechanical components.


4. LEAP System Overview 4.1 Introduction

LEAP™ is a technology platform that combines the power of laser-based semiconductor manufacturing equipment with medical knowledge regarding laser-based manipulation of living tissues. The result is a high-tech cell imaging and laser-based cell processing platform that images up to 100,000 cells per second, and targets up to 1,000 cells per second with a laser to achieve various types of laser-mediated cell manipulations. These unique capabilities enable a number of important applications such as: (i) in situ purification of fragile, adherent and/or rare cells; (ii) delivery of a wide range of macromolecules into cell types that are often refractory to traditional methods; and (iii) ultra high-throughput imaging. Ultra high-throughput processing on LEAP is achieved by using a large field-of-regard (FOR) F-theta lens with high-speed galvanometer mirrors that scan a large surface area. These scanning mirrors can obtain a series of images (“fields-of-view” or FOV) as well as steer the laser beam to hit target cells. The use of automated and robust components from the semiconductor manufacturing industry in LEAP provides a level of technological sophistication that is unique in the life sciences arena.

4.2 Hardware and Optics

The LEAP instrument contains several hardware and optical subsystems:

• multi-color fluorescence excitation and imaging optics (three 8-position filter wheels, for excitation filters, dichroic filters, and emission filters)

• LED-based Brightfield illumination

• image magnifier (3X, 5X, 10X, and 20X)

• intensified mega-pixel CCD camera

• rapid auto-focus

• lasers and laser beam delivery optics (with spot size and intensity controls)

• F-theta lens, which defines the FOR, with scanning galvanometer mirrors

• x/y/z stage system for plate loading and accurate positioning

The LEAP possesses a unique optical system that enables it to image and manipulate cells at unprecedented speeds. The large FOR lens views a significantly larger area than conventional microscope imaging systems. The FOR allows the direct viewing of 4 complete wells of a 384-well plate and 1 complete well of a 96-well plate (Fig. 4-1). Within the FOR, multiple images are collected to provide full coverage of the well at any specified magnification (Fig. 4-1).



A

B

Figure 4-1. Field of Regard vs. well coverage in LEAP. (A) The FOR covers 4 wells of a 384-well plate. (B) The FOR covers 1 well of a 96-well plate. The dark circles represent the wells of the plate. The translucent circles correspond to the FOR in each layout. The green box represents the image at 3X magnification.

Magnification 384-Well Plate 96-Well Plate

3X 1 image - 384-well

16 images - 96-well

5X 4 images - 384-well 32 images - 96-well

10X 16 images - 384-well

120 images - 96 - well

20X 45 images - 384-well 408 images - 96-well

Figure 4-2. Multiple images (squares) are acquired within optical field in order to provide the ability to image the entire well (circle). A physical stage movement and focus occurs only when moving the optical view from well to well.


4.3 User Interface and Software

The software interface is designed around the workflow that the user needs to execute. The optical components are controlled through the graphical user interface, allowing easy visualization and changing of instrument settings by simply clicking on each component in the interface diagram.

NOTICE! The LEAP System computer should not be used for applications other than those provided with the LEAP. Doing so may cause damage to the hardware or system settings, leaving the instrument in a compromised or totally inoperable state that can only be corrected by trained service personnel.

The system is organized in two screens on separate monitors (Fig. 4-3). The left side of the left screen provides the workflow as a series of collapsible menus that start from loading a sample into the system to viewing the results. The right screen generally contains configuration settings for the workflow.

Figure 4-3. LEAP User Interface. The user interface is generally split between workflow (left) and configuration settings (right).



5. LEAP Operation 5.1 System Startup and Shutdown

System Startup

• Turn on the LEAP instrument by switching on the main power button.

• Turn on the LEAP computer.

• Login to the LEAP Application computer e.g. User Name: leap and Password: leap (remember that passwords are case sensitive).

• Launch the LEAP Application using the “LEAP” icon,

located on the desktop (Fig. 5-1). The program will open and the “System Status” window will appear on the left hand monitor (Fig. 5-2).

• Select the check box to turn on the Excitation Lamp if you are going to use it during

instrument operation (Fig. 5-2). When you check the box, a cooling fan will become audible. You may uncheck the box to turn off the lamp between procedures if you are not going to shut down the instrument.

Warm-Up

• Warm-up periods for optimal subsystem performance are communicated in the Initial Startup

Screen (Fig. 5-2). Note that for LEAP System Version 1.2, the Application will not safeguard against premature utilization of these subsystems; therefore, it is the User’s responsibility to ensure that the appropriate warm-up period has elapsed before utilizing these subsystems.

Figure 5-2. Initial Startup Screen.

Figure 5-1. LEAP Application Icon.


A. Laser: 10 minutes. B. Excitation Lamp: 5 minutes. C. Camera: 10 minutes.

System Shutdown

• Be sure you have removed any plate that is loaded in the instrument before shutting the System down. Remove the plate by clicking the “Remove Plate” button (Fig. 5-3) in the Protocol menu.

• Exit program by clicking on “File” and

choosing “Exit”.

• Shut down the computer using the Windows Start/Shutdown menu.

• Switch off the Instrument power button.

5.2 Instrument Operation

5.2.1 Load a Plate Map

• The plate is loaded into and removed from the Instrument through the sliding access door on the top panel.

• Select the ‘Load Plate Map” icon in the Protocol Menu (Fig. 5-4A). A new window will

appear (Fig. 5-4B) where you will locate the previously made plate map file. Once a plate map is loaded, the user can then select where the data will be saved by clicking on “Set Results Path…” . Click “OK”.

Figure 5-3. Removing a plate. “Remove plate” will position the plate at the load/unload position, and open

BA

Figure 5-4. Selecting “Load Plate Map” (A) will bring up a window (B) Click on the “…” button to locate the Plate Map file.

• The access door on the top panel of the LEAP will open with the stage in the load position

Do not reach into the Instrument while the stage is in motion!


• Place the plate carefully onto the stage. Ensure that the plate is seated flat in the stage before attempting to process the plate. If the plate is not seated securely in its proper position, the instrument will be unable to reliably focus on the entire plate. Make sure the metal clamp on the bottom right of the stage is open when the user loads the plate and it’s seated securely against the corner of the plate when the user is finished.

o The proper orientation of the well plate will be achieved when well position A-1 is in

the upper-left hand corner in relation to an operator in the front of the instrument.

• The “Insert Plate” popup window asks for confirmation that the plate was inserted and that all hands are away from the instrument (Fig. 5-5).

Figure 5-5. Select “OK” once the plate has been placed inside the instrument.

• Selecting “OK” closes the access door and aligns the plate (Fig. 5-6).

Figure 5-6. Upon loading a plate, an automated alignment procedure will be executed that adapts to the plate geometry.

5.2.2 Load a Plate • When the Plate Map feature is not utilized, the user can load a plate by clicking on the “Load

Plate” icon in the Protocol Menu (Fig. 5-7A). A new window will appear (Fig. 5-4B) where you enter the Plate “Label” (e.g. the Plate ID) and select the type of plate you have placed into the instrument. Click “OK”.


A BFigure 5-7. Selecting “Load plate…” (A) will bring up a window (B) to enter the Plate “Label” and select the Plate Type.

Do not reach into the Instrument while the stage is in motion!


• The access door on the top of the LEAP will open with the stage in the load position.

• Place the plate carefully onto the stage. Ensure that the plate is seated flat in the stage before

attempting to process the plate. If the plate is not seated securely in its proper position, the instrument will be unable to reliably focus on the entire plate. Make sure the metal clamp on the bottom right of the stage is open when you load the plate and seated securely against the corner of the plate when you are finished.

o The proper orientation of the well plate will be achieved when well position A-1 is in

the upper-left hand corner in relation to an operator in the front of the instrument.

• The “Insert Plate” popup window asks for confirmation that the plate was inserted and that all hands are away from the instrument (Fig. 5-8).

Figure 5-8. Select “OK” once the plate has been placed inside the instrument.

• Selecting “OK” closes the access door and aligns the plate (Fig. 5-9).

Figure 5-9. Upon loading a plate, an automated alignment procedure will be executed that adapts to the plate geometry.


5.2.3 Plate Navigation and Well Selection

• Navigation from well to well is performed manually by selecting the appropriate well for viewing from the “Plate” tab (Fig. 5-10). After loading a plate, click on any well in the map to move to that well.

• The User is able to zoom in on the interactive plate icon by selecting the interactive plate icon

and using the roller ball on the mouse.

• The blue box will be located over the selected position.

Figure 5-10. Interactive plate icon used for navigating to particular wells within a plate. (Left): C-lect™ 96-well plate, (Right): C-lect™ Mirror. Blue box indicates the position of the stage over the F-theta lens.

• To select wells for processing, the user can either right click on the mouse and choose ‘Well

Selection’ or click on ‘Well Selection’ button below the plate icon (Fig. 5-11).

• Selecting more than one well allows the protocol to process all wells within the selection. Note that when using a Plate Map, the user does not need to select wells before processing.


Figure 5-11. Selecting rows, columns, rectangular regions, or the whole plate can be done using logical spreadsheet-type selection methods.

• During processing there is direct feedback on the position of the data acquisition. On each

well, progress in indicated by a colored square indicating the channel being acquired and the position within the well where the image is being recorded (Fig 5-12).

Figure 5-12. Colored squares indicate both the channel being acquired as well as the position within the well the image is being recorded. Green squares indicate processing of the default green channel and red squares indicate processing of the default red channel.

Column Whole Rectangular Selection Plate

Row



Saving and Restoring LEAP configuration states

n can be saved and recoveredsingle file.

ion conditions (Fig. 5-13) via Load settings…

• Configuration conditions including Protocol,

Optical Path, Image Pre-processing, and Image Segmentatio from a

• After loading a plate, it is possible to load a prior

set of configurat.

can be saved for rapid reuse (Fig. 5-14, 5-15).

• Once a set of conditions is established that will

be either routine or provide a good starting point for processing, it

• All configuration settings are also saved with the

data from an experiment. It is possible to use settings from a prior experiment by importing the configuration settings file from the data directory of that experiment using the File menu (Fig. 5-14). From Import Settings from Results Folder… a dialog will allow selection of the appropriate file.

Figure 5-14. Configuration settingcan be loade

s d and saved from the

File menu.

Figure 5-13. Load configuration settings that were established and saved prior to run.

Figure 5-15. Save/Load common configurations from a single source

5.2.4 Machine Control The Machine Control tab (Fig. 5-16) allows you to view and change the hardware settings when using the LEAP.

Optical Path Controls • A schematic representation of the System hardware contains interactive menus which allow

you to control some of the current hardware settings of the System. A summary of the settings you are currently using is displayed on the bottom left of the diagram. A popup with the filter wavelength or magnification setting will appear as you move the mouse over the filter diagram.

• Settings for the laser wavelength, laser attenuator and beam expander can be saved and loaded. If loaded, the name of the file will appear in the Laser Path Configuration box. (Fig. 5-16).

Figure 5-16. Machine Control Interactive Diagram.


Beam Expander / Laser Attenuator (See Calibration - Section 5.3) • This is the menu for settings controlling the operation of the laser, including selecting which

laser to use (UV (355 nm) or Green (532 nm)), varying the energy of the laser pulse (Laser Attenuator) and the position of the focus of the laser (Beam Expander).

• The Beam Expander adjusts the focus of the laser. In some cases, the goal is to hit the cell with a focused beam (B.E. position that creates the smallest spot size). In other cases, the goal is to hit the cell with a de-focused beam (B.E. position that creates a larger spot size, which is less (in mm) than the position that created the smallest spot size). See Appendix A for Beam Expander position (mm) vs. spot size radius (μ).

• The Laser Attenuator adjusts the amount of energy that is transmitted from the laser to the sample. For example, the laser energy is decreased during Optoinjection procedures to prevent cell death whereas higher energies of the Green (532 nm) laser may be used for killing cells.

X, Y, Z Position

• The X, Y, Z motors are the motors that control the movement of the stage/plate in relation to the F-theta lens. They are useful for manual scanning around a plate or inside a well.

• The X Position will shift the well along the x axis. The Y Position will shift the well along

the y axis. The Z Position will shift the cell into and out of focus.

• Absolute position can be entered via the "Move" buttons which will popup for entering the position.

• The arrow buttons will move the object (well or cells) in the direction of the arrow. The slide

controls adjust the step size of the movement.

Magnification The magnification wheel contains options for 3x, 5x, 10x or 20x viewing. At 3x magnification, an entire 384 well plate sized well is visible in one FOV. Table 5-1 indicates the pixel sizes at each magnification.

Table 5-1. Pixel sizes at each magnification. Magnification Pixel Size 3X 1.957 μm 5X 1.177 μm 10X 0.582 μm 20X 0.292 μm


5.2.5 Image Acquisition Settings • Select the tab (Fig. 5-17A) for the image channel you wish to set up on the right-hand

monitor. • Select the “Optical Path” menu (Fig. 5-17B) which provides the ability to setup the required

illumination source, filter settings, camera settings, and focusing capabilities.

• All conditions set from this menu can be saved into a file and later retrieved (Fig. 5-17C). If you have prior settings to restore select the “Load” button and select the file. After you have made changes select the “Save As…” button to save those settings with a descriptive name.

A

C

B

Figure 5-17. Optical Path Menu with File Load window.

Optical Path • Setup the filter combinations for the imaging conditions (e.g. fluorescence with a particular

set of filters or bright field) for that image channel.


o Table A1 (Appendix A) provides a recommended list of available filter combinations for particular dyes. Any combination of filters can be selected, and it is encouraged that the user verifies that the combination of filters provides the appropriate image.

NOTE! If you cannot see any wells, verify that the excitation lamp is turned on. If the lamp is on, then check your camera settings or change to the Brightfield Illumination Source and the Dichroic to 409LP. Some combinations of filters do not allow any light to enter the camera. Other combinations will let too much light through and the camera will automatically shutdown to avoid damage. See Optical Path/Machine Control section.

Figure 5-18. Wavelength Drop-down Menus

• Current filter settings are displayed. You can use the drop-down menus to change your

Illumination Source and Excitation, Emission and Dichroic filters (Fig. 5-18).

• Illumination Source can be either Brightfield or Fluorescence. Brightfield is white-light illumination. Fluorescence is controlled by the selected filters.

Camera Settings

Figure 5-16. Camera Settings Box

Figure 5-19. Camera Settings Box

o Frame Exposure Time (ms)

The slide bar controls the length of time over which the camera integrates the signal to define a frame. Thus, in Fig. 5-19, a “frame” is defined as an exposure of 151.26 ms. If for example, the “Number of Frames to Average” is set to 2, then 2 frames of 151.26 ms each are averaged together to achieve the Total Capture Time (ms) of 302.52.


o Gain (volts)

The camera includes an electron multiplying (EM) register at the end of the serial register. The gain setting controls the amount of ion impact amplification within this register which allows the viewing of extremely low light level images, such as from fluorescently labeled cells.

o Number of Frames to Average (1-8)

Frame averaging is used to minimize camera noise that may be due to a high gain value.

“Number of Frames to Average” determines the number of frames that are averaged

together to provide the final image. Frame averaging will improve the signal-to-noise ratio of the images by decreasing the random noise. The image of the cells should improve with the averaging of larger numbers of frames. The trade-off for a better image is the greater amounts of time necessary to acquire large numbers of frames.

o Binning (1, 2 or 4)

This feature will reduce resolution but increase sensitivity and speed. Default setting is 1, which will not change the image.

When set to 2, the software will add the intensity of a 2x2 pixel area and put it into 1

pixel, and then stretch the image back to the original size. Therefore a 1000x1000 pixel image will still be a 1000x1000 pixel image, but each 2x2 pixel square will have the same value.

When set to 4, the software will use a 4x4 pixel area instead of a 2x2 pixel area.

Adjust and find optimal Camera Settings:

Figure 5-20. “Camera Live” button is circled in red.

• Select the “Camera Live” Button (Fig. 5-20) located on the top of the right hand monitor to see a live, real-time image.

• To minimize camera noise, adjust first the Frame Exposure Time between 0 to 32 ms,

keeping the Gain slider at 0. If the signal is still not visible at 32 ms, increase the Gain until the signal is visible or the camera noise becomes apparent. If the signal is still not visible, then increase the Frame Exposure Time until the signal is visible.

• Slide the Exposure and Gain settings until you can see all of the cells in the image. Then

minimize the Exposure and Gain settings so that you can still see all the cells without overexposing the image (Fig. 5-21). The best exposure and gain settings allow imaging of both the brightest and the dimmest objects simultaneously.


If no cells are visible, they may be out of focus. Use the focus panel and adjust focus if necessary (see next section).

Good Exposure Over Exposure Under Exposure Figure 5-21. Exposure examples.

• Select the “Snap Image” button to take a picture and turn off the live camera (Fig. 5-22). • The user may also snap an image, without using the live video, to help prevent photo

bleaching of a fluorescent sample because the sample is being exposed to excitation light for the duration needed to take a picture (Total Capture Time (ms)).

Figure 5-22. “Snap Image” (circled)


Focusing the sample

• Focusing (Fig. 5-23) on the sample can be achieved by either moving to an absolute position, interactively via the arrow buttons or by clicking on the Auto Focus or Refocus button.

• An absolute position can be entered via the “Move” button (red circle in Fig. 5-23A). The

“Move” button brings up a popup window (Fig 5-23B) for entering the value. • Interactive adjustment of focus occurs by selecting the “Up” and “Down” arrow buttons to

move the stage. The sliding bar (Fig. 5-23A) to the left of these buttons controls how far each click on “Up” or “Down” will move the stage. Use the minimum (left-most) job speed for a fine focus and the maximum (right-most) speed for a rough focus.

• Clicking on the Auto Focus or Refocus buttons will tell the instrument to find the ideal focus

for the sample, based on the image with the maximum contrast (ie the image with the sharpest edges). The difference between the two buttons is that the range scanned (on the Z axis) is larger for the Auto Focus than the Refocus. Therefore use the Refocus button only when the sample is slightly out of focus.

Figure 5-23. Z Position (Focus) Menu. Use up and down arrow buttons to interactively move the stage. To enter an absolute focus position, select “Move”, the “Change Position…” pop-up window will appear (B), enter the number and select “OK”.

A

B


5.2.6 Image Preprocessing Preprocessing options allow the user to perform image processing before the data is analyzed. The preprocessing options must be selected independently for each fluorescence channel. • On the right hand monitor, click the “Image Preprocessing” tab. The Image Preprocessing

window will open (Fig. 5-24).

Well Masking

• The well masking operations delineate the working area of the image by masking any regions that fall outside the well. With the Mask Well operation selected, all subsequent processing is calculated and performed within the masked region (e.g. background correction) including object finding.

• Mask Well: Execute well masking

operations. o Search (%): Percent of area from the

center to evaluate for masking well. o Adjust (Pixels): Number of pixels to

expand or shrink well mask.

• Attempt to Find Well Mask: This setting attempts to match the well mask to the well.

• To view the well mask outline selected the option from the Overlay pull down menu (Fig. 5-30A)

Background Correction

Background Correction executes a correction for background variations within an image. The ability to adapt to a wide variety of background conditions is a common challenge in biological samples. • Correct Background: Process each image

to correct for background variations (Fig. 5-25). Figure 5-24. Image Preprocessing

Window. Click on the check box(es) circled here in red to apply the feature.


• Additive: Algorithm samples the image over a grid of sampling regions establishing a value based on the Point Method chosen. For example, if Minimum is chosen, then the minimum of the sample region is recorded. After processing all sample regions, a surface is fit to the data creating the ‘background’ image for subtracting from the image. These sampled points may represent either true background or actual object values. The ‘Trim Outliers’ value is used to trim the higher pixel values from the distribution. The ‘Visible Background’ and ‘Coverage’ parameters determine the sampling grid and area of image covered.

B A

Figure 5-25. Sample images without (image A) and with (image B) default settings for background correction.

• Additive Options

o Point Method [Minimum / Average / Maximum]: calculation performed within a region of the sample grid. • Minimum: minimum of the pixels • Average: average of the pixels • Maximum: maximum of the pixels

o Trim Outliers: Using a histogram of the values for each sampling region, it is possible to remove outlying object intensities from the background intensities.

o StdDevs: Standard deviation about the mean of the values to trim from the distribution.

• Additive & Multiplicative: Use both the Additive correction (background subtraction) as

well as a Multiplicative algorithm. The Multiplicative algorithm corrects for non-uniform illumination across an image. The algorithm samples the image over a grid of sampling regions establishing a value based on the Point Method chosen. For example, if Average is chosen, then the average of the sample region is recorded. After processing all sample regions, a surface is fit to the data creating the multiplicative correction image for multiplying with the image. The ‘Trim Outliers’ value is used to trim the extreme pixel values from the distribution. The ‘Visible Background’ and ‘Coverage’ parameters determine the sampling grid and area of image covered.

• Multiplicative Options

o Point Method: [Minimum / Average / Maximum]: calculation performed within a region of the sample grid.

o Trim Outliers: Using a histogram of the values for each sampling region, it is possible to remove outlying object intensities from the background intensities

o StdDevs: Standard deviation about the mean of the values to trim from the distribution.


• Fit Precision Defines the overall precision of the surface that is fit to the data. Too low of a precision can lead to inadequate compensation for the variances within the image.

• Visible Background [Normal / Low]: In general, this variable determines the number of

points the software will look at to determine the background correction. The selection of this variable is based on how much area in the well is a non-signal. A setting of ‘Normal’ uses a 20 x 20 grid and a setting of ‘Low’ uses a 10 x 10 grid. Therefore, a high density culture would have a lower area of background and thus a larger region should be used to evaluate the background pixels, use the setting of ‘Low’. For lower density cultures, where more area of the well would be background, a setting of ‘Normal’ would be best.

• Coverage (%): Percent area of image from the center over which to place the sampling grid. • Suppress Tails Outside of Mask: Option is used in conjunction with well masking. When

background correction is applied to a masked area it is possible the regions outside the mask will have wildly random patterns. This option sets all values outside the mask to zero.

• Click the checkbox to turn on and off background correction. The default settings in “Background

Correction” are usually sufficient. Remove Lines

• Remove Lines removes ALL lines, including really short ones - the ones that look like cells. These lines are usually the result of scratches or stray fiber-type objects that can complicate interpretation of the image.

• Remove

o Applies the remove line filter when checked.

• Horizontal o Removes linear objects that are mainly horizontal.

• Vertical o Removes linear objects that are mainly vertical.

• Both o Removes linear objects that fall both along the vertical and the horizontal directions.

• Variance

o Removes linear objects that are not 100% vertical or horizontal. The user determines the width of the lines to filter.

• Cutoff

o Cutoff is an option to keep short lines in the image. The Cutoff number is in pixels and will not affect (Mostly) objects smaller than the setting.

o The default setting, “ON”, is usually sufficient for normal operations.

Conditional Smooth


• This is a noise removing filter tuned to remove the relatively low frequency speckle noise generated by the camera.

• Each pixel value in the image is replaced by the mean value of it neighbors, determined by

the radius, IF the value is less than the mean plus the threshold.

• For real pixel edges (like those on the edge of a cell) the pixel value is higher than the condition and therefore left alone. This filter is also called an “Edge Preserving Smooth” filter.

• Processing time increases exponentially with the increase in the “Radius”.

• The default setting, “off”, is usually sufficient for operation.

Edge Enhancement • Clicking on ‘Enhance’ will apply the feature which is based on a LaPlacian of Gaussian

equation. • Smoothness determines the amount of the Gaussian blur • Weight applies the amount, or strength, of the effect.

Bright Field Enhancement • Clicking on ‘Enhance’ will apply Cyntellect defined kernel operations for enhancement of the

LEAP brightfield signature. • Weight applies the amount, or strength, of the effect


5.2.7 Image Segmentation

Image segmentation is the process of separating objects from the background and separating them into individual entities. Often these objects are cells. These settings are critical for accurate cell identification within images.

• Select the “Image Segmentation” tab (Fig. 5-

26) on the right hand monitor below the Image Preprocessing tab.

Global Threshold (intensity)

The global threshold settings (Fig. 5-26) apply equally across the entire image. It works best when the background is uniform and even across the entire image. A minimum grey level can be set as well as a maximum grey level. The resulting black and white binary image will define the areas that possess intensity values above the "Min Grey" and below the "Max Grey" levels (white). The black regions represent the areas where the pixel intensities are below the minimum and above the maximum. The "Min Grey" level is used to separate objects from the background signals within the images. The "Max Grey" level is typically used to prevent very high intensity objects (i.e. saturated) from being further processed.

• The Auto button will automatically adjust the Min Grey levels for each image.

Figure 5-26. Image Segmentation Window.

B

A

Figure 5-27. Examples of Min. Grey too low (A) and too high (B).


Local Threshold (intensity)

Local Threshold provides a means of adjusting the targeting when there is variation in the background across the image. This is the most commonly used segmentation method.

• The "Radius" defines the extent of the region across the image that is considered in the algorithm. Usually the “Radius” should be set to the pixel diameter of the objects in the image.

• Within the area set by the “Radius” slider, any pixel intensity below the “Threshold” setting will

result in a zero level (black area when looking at the ‘Preview Segmented Image’). This threshold is similar to the "Min Grey" of the Global Threshold.

• This processing can be applied to either bright or dark objects by selecting "Bright Objects" or

"Dark Objects". • By clicking on “Auto”, the software will automatically determine the best “Threshold” setting at

the set “Radius” for each image. • Note: Processing time will increase with an increase in the radius slider.

Radius Too Low Radius Too High Good Image Segmentation Figure 5-28. Local Threshold: changing the size of the radius.

Morphology

• Provides a means to find objects based on shape (e.g., circularity). This more complex algorithm generates a topographical map of the targets. This is useful for bright field imaging of suspension cells.

o Radius - approximate radius of object in pixels o Threshold - intensity threshold o Bright Objects: For processing lighter objects on a darker background. o Dark Objects: For processing darker objects on a lighter background. o By clicking on “Auto”, the software will automatically determine the best

“Threshold” setting at the set “Radius” for each image.

• Note: Processing time will increase with an increase in the radius slider. Separate Touching Objects


• Check box to apply a watershed algorithm to separate touching objects.

Object Size Range

• It is possible to select to filter the objects found, after the previous threshold operations, based on size. Only objects (e.g. cells) that are greater than the "Min Area (Pixels)" and less than the "Max Area (Pixels)" will be left within the segmented image.

Object Elongation Range

• This filter may be suitable when a mixture of adherent and semi adherent cells are present because it is a calculation of each object of the major axis over the minor axis.

• A value of 1 is equal to a perfect circle and as the value increases the object is more oval

shaped. Object Compactness Range

• This filter is a ratio of the derived perimeter (based on the radius of the object) and the actual perimeter.

• A value of 1 is equal to a perfect circle and as the value increases the object contains multiple ‘fingers’. For example a lymphocyte would have a small value and a dendrite would have a large value.

Objects Found

♦ Number of objects found using the current image segmentation settings.

Preview Segmented Image

• This will change the currently displayed image to a binary (black and white) image applying all the chosen settings from above. Grey pixel areas will be changed to either black or white. Black is ignored as background and white pixel areas can be subsequently gated out by using the ‘Noise Gates’ filters.

Auto Update Objects

• Checking this box will immediately update the target display upon changing any image

segmentation setting.

Clear Objects

• Clicking on this button will remove the green labeled objects from the displayed image. Once cleared, the laser will have no targets to shoot.

Find Objects


• By clicking on this button, the computer will update the target display with the current image segmentation setting. This button is useful when “Auto Update Objects” is not checked. (Fig. 5-26)

Find Objects Report

• Provides detailed information on the objects found by the segmentation algorithms. The user

can then copy and paste the data into an Microsoft Excel workbook (Fig. 5-29)

Figure 5-29. Object (Blob) Report.

5.2.8 Object Indicator

• When selected, this will provide a display of a green crosshair, dot or outline (Fig. 5-30) on all found objects.


A

Crosshair Dot Outline

C DB

Figure 5-30. Identified objects can be identified by different symbols, including crosshairs (B), dots (C) and outlines (D). These symbols are changed by the target indicator icon drop down menu, located in the upper menu bar on the right hand monitor (A). Current selection is the Outline symbol.


5.2.9 Focus Map

• This feature fits a 3D curve in the Z axis to the plate based on the focus of points (wells) chosen by the user.

• To utilize this feature the user will go to a well, autofocus and then right click over that well and

select ‘Set Focus Point’ (Fig. 5-31). Once a point is set, a black ring will outline the well.

• The user can clear a Focus Point by right clicking on the mouse and selecting ‘Clear Focus Point’. If the user wants to delete all points, then select ‘Clear Focus Map’.

• It is recommended that the user uses at least five points for the Focus Map. In Fig. 5-31, six

points are used at C3, C22, H12, I13, N3 and N22.

Figure 5-31. A 384 well plate will all wells selected for processing (orange ring) and six wells selected for the Focus Map (black ring).

• The user can choose to use the Focus Map instead of Autofocus during processing. (See individual Application Guides.)

• If both Autofocus and the Focus Map are chosen when the plate is processed, then the software

will used the Focus Map as a starting point for the focus and then refocus on each well.


6. LEAP Processing 6.1 Introduction

Protocols are used to encapsulate the various operations of data acquisition and laser targeting parameters for specific applications. Common components, such as segmentation, camera settings, etc. are constant across all of the protocols. Components specific to each protocol are available only when that specific protocol is selected. There are a variety of applications available for the LEAP™ and new ones continue to be developed. Each application is described in a separate Application Guide. New protocols can be added by installing the application via a CDROM installation disk.

The LEAP™ comes with the following protocols pre-installed: o Cell Purification o Automated Laser Targeting o Manual Laser Targeting o Cell Counting o Cell Viability o Cell Optoinjection o Grid Optoinjection

Optional protocols can be installed to supplement the functionality of LEAP.

See the specific Application Guides for details on each protocol. Data from each of these protocols is available to the Results Viewer application. The Results Viewer allows the viewing and exporting of data collected on LEAP. See the Results Viewer Application Guide.


Appendix A: Software License Agreement

CYNTELLECT INC. SOFTWARE END USER LICENSE AGREEMENT

IMPORTANT: THIS SOFTWARE LICENSE AGREEMENT IS A LEGAL AGREEMENT BETWEEN YOU (EITHER AN INDIVIDUAL OR, IF PURCHASED OR OTHERWISE ACQUIRED BY OR FOR AN ENTITY, AN ENTITY) AND CYNTELLECT INC. READ IT CAREFULLY BEFORE COMPLETING THE INSTALLATION PROCESS AND USING THE SOFTWARE. IT PROVIDES A LICENSE TO USE THE SOFTWARE AND CONTAINS WARRANTY INFORMATION AND LIABILITY DISCLAIMERS. BY INSTALLING OR USING THE SOFTWARE, YOU ARE CONFIRMING YOUR ACCEPTANCE OF THE SOFTWARE AND AGREEING TO BECOME BOUND BY THE TERMS OF THIS AGREEMENT. THIS AGREEMENT IS ENFORCEABLE AGAINST YOU AND ANY LEGAL ENTITY THAT OBTAINED THE SOFTWARE AND ON WHOSE BEHALF IT IS USED. IF YOU OR YOUR EMPLOYER OR COMPANY DO NOT AGREE TO THE TERMS OF THIS AGREEMENT, THEN CYNTELLECT INC. IS UNWILLING TO GRANT YOU THIS LICENSE, YOU MUST NOT INSTALL OR USE THE SOFTWARE.

THIS AGREEMENT SHALL APPLY ONLY TO THE SOFTWARE SUPPLIED BY CYNTELLECT INC. HEREWITH REGARDLESS OF WHETHER OTHER SOFTWARE IS REFERRED TO OR DESCRIBED HEREIN.

THE SOFTWARE MAY INCLUDE PRODUCT ACTIVATION AND OTHER TECHNOLOGY DESIGNED TO PREVENT THE UNAUTHORIZED COPYING AND USE. THE TECHNOLOGY MAY PREVENT YOUR USE OF THE SOFTWARE IF YOU DO NOT FOLLOW THE ACTIVATION PROCESS DESCRIBED IN THE SOFTWARE AND DOCUMENTATION. YOU ACKNOWLEDGE AND AGREE THAT THE SOFTWARE WILL CEASE TO FUNCTION UNLESS AND UNTIL YOU ACTIVATE THE SOFTWARE.

1. Definitions

a) “Cyntellect” means Cyntellect Inc. and its licensors, if any.

b) “Instrument” means Cyntellect’s LEAP™ (Laser Enabled Analysis and Processing) System

c) “Software” means all the information with which this agreement is provided, including, but not limited to, the Cyntellect software program(s) and third party software programs related to the operation of one or more Instruments, in each case, supplied by Cyntellect herewith, and corresponding documentation, associated media, printed materials, and online or electronic documentation (“Documentation”).

2. License Grants. As long as you obtained the Software from Cyntellect or one of its authorized licensees, and as long as you comply with the terms of this agreement, Cyntellect grants you the non-exclusive license to use the Software in connection with the operation of the Instrument in the manner and for the purposes described below and in the Documentation. The licenses granted in this Section 2 are subject to the terms and conditions set forth in this agreement:

a) You may use the Software on the computer on which it is pre-installed by Cyntellect or its licensee. If the Software is not pre-installed, you may install and use the Software on a single computer; OR install and store the Software on a storage device, such as a network server, used only to install the Software on your other computers over an internal network, provided you have a license for each separate computer on which the Software is installed and run. A license for the Software may not be shared, installed or used concurrently on different computers.

b) In the event the Software is distributed along with other Cyntellect software products as part of a suite of products (collectively, the “Suite”), the license of the Suite is licensed as a single product and none of the products in the Suite, including the Software, may be separated for installation or use on more than one computer.

c) You may make one copy of the Software in machine-readable form solely for backup purposes. You must reproduce on any such copy all copyright notices and any other proprietary legends on the original copy of the Software. You may not sell or transfer any copy of the Software made for backup purposes.

d) You agree that Cyntellect may audit your use of the Software for compliance with these terms at any time, upon reasonable notice. In the event that such audit reveals any use of the Software by you other than in full compliance with the terms of this Agreement, you shall reimburse Cyntellect for all reasonable expenses related to such audit in addition to any other liabilities you may incur as a result of such non-compliance.

e) Distribution of any Software code is specifically prohibited.

f) The Software is licensed as a single product. You may not separate the Software's component parts for use on more than one computer.


3. License Restrictions

a) Other than as set forth in Section 2, you may not make or distribute copies of the Software, or electronically transfer the Software from one computer to another or over a network.

b) You may not alter, merge, modify, adapt or translate the Software, or decompile, reverse engineer, disassemble or otherwise attempt to discover the source code of the Software or otherwise reduce the Software to a human-perceivable form.

c) Unless otherwise provided herein, you may not rent, lease, or sublicense the Software.

d) You may permanently transfer all of your rights under this agreement only as part of a sale or transfer of the Instrument, provided you retain no copies, you transfer all of the Software (including all component parts, the media and printed materials, any upgrades, this agreement, the serial numbers, and, if applicable, all other software products provided together with the Software), the recipient agrees to the terms of this agreement, and you notify Cyntellect in writing of the transfer (and the transferee’s agreement to be bound) within 60 days of the transfer. If the Software is an upgrade, any transfer must include all prior versions of the Software from which you are upgrading. If the copy of the Software is licensed as part of the whole Suite (as defined above), the Software shall be transferred only with and as part of the sale or transfer of the whole Suite, and not separately. You may retain no copies of the Software. You may not sell or transfer any Software purchased under a volume discount.

e) Unless otherwise provided herein, you may not modify the Software or create derivative works based upon the Software.

f) You may not export the Software into any country prohibited by the United States Export Administration Act and the regulations thereunder.

g) You may receive the Software in more than one medium but you shall only install or use one medium. Regardless of the number of media you receive, you may use only the medium that is appropriate for the server or computer on which the Software is to be installed.

h) You may receive the Software in more than one platform but you shall only install or use one platform. If the Software is delivered in multiple versions or languages, you may only run one version or language of the Software, and you may not run the additional versions in any other language on any other computer.

i) You shall not use the Software to develop any application having the same primary function as the Software.

j) In the event that you fail to comply with this agreement, Cyntellect may terminate the license and you must destroy all copies of the Software (with all other rights of both parties and all other provisions of this agreement surviving any such termination).

k) NOTE – THE USE OF THE INSTRUMENT OR SOFTWARE FOR ANY OF (i) PURIFICATION OF CELLS BASED ON IN SITU MEASUREMENT OF INDIVIDUAL CELL PROTEIN SECRETION, FOR THE PURPOSE OF THERAPEUTIC PROTEIN PRODUCTION, (ii) DIAGNOSIS OF DISEASE IN HUMANS OR ANIMALS, OR (iii) THERAPEUTIC TREATMENT OF HUMANS OR ANIMALS, REQUIRES A SEPARATE LICENSE NOT GRANTED HEREIN. A SEPARATE SIGNED LICENSE AGREEMENT MUST BE ENTERED INTO WITH CYNTELLECT.

4. Upgrades. If the Software is an upgrade or update to a previous version of the Software, you must possess a valid license to such previous version in order to use such upgrade or update. If this copy of the Software is an upgrade from an earlier version of the Software, it is provided to you on a license exchange basis. You agree by your installation and use of such copy of the Software to voluntarily terminate your earlier agreement and that you will not continue to use the earlier version of the Software or transfer it to another person or entity unless such transfer is pursuant to Section 3.

5. Prior Same Version License. If this copy of the Software is licensed as part of the Suite (as defined above), and you have a prior license to the same version of the Software, and the Suite was licensed to you with a discount based, in whole or in part, on your prior license to the same version, the Software is provided to you on a license exchange basis. You agree by your installation and use of this copy of the Software to voluntarily terminate your agreement with respect to such prior license and that you will not continue to install or use such prior license of the Software or transfer it to another person or entity.

6. Ownership. The foregoing license gives you limited license to use the Software. Cyntellect and its suppliers retain all right, title and interest, including all copyright and intellectual property rights, in and to, the Software and all copies thereof. The structure, organization and code of the Software are valuable trade secrets and confidential information of Cyntellect. All rights not specifically granted in this agreement, including Federal and international Copyrights, are reserved by Cyntellect and its suppliers.

7. Limited Warranty And Disclaimer

a) Cyntellect Inc. warrants that, for a period of one (1) year from the date of delivery of the Instrument: (i) when used with a recommended hardware configuration, the Software will perform in substantial conformance with the documentation


supplied with the Software; and (ii) the physical media on which the Software is furnished, if provided by Cyntellect, will be free from defects in materials and workmanship under normal use.

b) EXCEPT AS SET FORTH IN THE FOREGOING LIMITED WARRANTY, CYNTELLECT AND ITS SUPPLIERS DISCLAIM ALL OTHER WARRANTIES AND REPRESENTATIONS, WHETHER EXPRESS, IMPLIED, OR OTHERWISE, INCLUDING THE WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. ALSO, THERE IS NO WARRANTY OF NON-INFRINGEMENT AND TITLE OR QUIET ENJOYMENT. CYNTELLECT DOES NOT WARRANT THAT THE SOFTWARE IS ERROR-FREE OR WILL OPERATE WITHOUT INTERRUPTION. NO RIGHTS OR REMEDIES REFERRED TO IN ARTICLE 2A OF THE UCC WILL BE CONFERRED ON YOU UNLESS EXPRESSLY GRANTED HEREIN. THE SOFTWARE IS NOT DESIGNED, INTENDED OR LICENSED FOR USE IN HAZARDOUS ENVIRONMENTS REQUIRING FAIL-SAFE CONTROLS, INCLUDING WITHOUT LIMITATION, THE DESIGN, CONSTRUCTION, MAINTENANCE OR OPERATION OF NUCLEAR FACILITIES, AIRCRAFT NAVIGATION OR COMMUNICATION SYSTEMS, AIR TRAFFIC CONTROL, AND LIFE SUPPORT OR WEAPONS SYSTEMS. CYNTELLECT INC. SPECIFICALLY DISCLAIMS ANY EXPRESS OR IMPLIED WARRANTY OF FITNESS FOR SUCH PURPOSES.

c) IF APPLICABLE LAW REQUIRES ANY WARRANTIES WITH RESPECT TO THE SOFTWARE, ALL SUCH WARRANTIES ARE LIMITED IN DURATION TO NINETY (90) DAYS FROM THE DATE OF DELIVERY.

d) NO ORAL OR WRITTEN INFORMATION OR ADVICE GIVEN BY CYNTELLECT, ITS DEALERS, DISTRIBUTORS, AGENTS OR EMPLOYEES SHALL CREATE A WARRANTY OR IN ANY WAY INCREASE THE SCOPE OF ANY WARRANTY PROVIDED HEREIN.

e) (USA only) SOME STATES IN THE UNITED STATES DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTIES, SO THE ABOVE EXCLUSION MAY NOT APPLY TO YOU. THIS WARRANTY GIVES YOU SPECIFIC LEGAL RIGHTS AND YOU MAY ALSO HAVE OTHER LEGAL RIGHTS THAT VARY FROM STATE TO STATE.

8. Exclusive Remedy. Your exclusive remedy under the preceding is to notify Cyntellect with a description of the problem. Provided that any non-compliance with the above warranty is reported in writing to Cyntellect no more than one (1) year following delivery to you, Cyntellect will use reasonable commercial efforts to supply you with a replacement copy of the Software that substantially conforms to the Documentation, provide a replacement for defective media, or refund to you your purchase price for the Software and Instrument at its option. Cyntellect shall have no responsibility if the Software has been altered in any way, if the media has been damaged by misuse, accident, abuse, modification or misapplication, or if the failure arises out of use of the Software with other than a recommended hardware configuration. Any such misuse, accident, abuse, modification or misapplication of the Software will void the warranty above. THIS REMEDY IS THE SOLE AND EXCLUSIVE REMEDY AVAILABLE TO YOU FOR BREACH OF EXPRESS OR IMPLIED WARRANTIES WITH RESPECT TO THE SOFTWARE AND RELATED DOCUMENTATION.

9. Limitation Of Liability

a) NEITHER CYNTELLECT NOR ITS SUPPLIERS SHALL BE LIABLE TO YOU OR ANY THIRD PARTY FOR ANY INDIRECT, SPECIAL, INCIDENTAL, PUNITIVE, COVER OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, DAMAGES FOR THE INABILITY TO USE EQUIPMENT OR ACCESS DATA, LOSS OF BUSINESS, LOSS OF PROFITS, BUSINESS INTERRUPTION OR THE LIKE), ARISING OUT OF THE USE OF, OR INABILITY TO USE, THE SOFTWARE AND BASED ON ANY THEORY OF LIABILITY INCLUDING BREACH OF CONTRACT, BREACH OF WARRANTY, TORT (INCLUDING NEGLIGENCE), PRODUCT LIABILITY OR OTHERWISE, EVEN IF CYNTELLECT OR ITS REPRESENTATIVES HAVE BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES AND EVEN IF A REMEDY SET FORTH HEREIN IS FOUND TO HAVE FAILED OF ITS ESSENTIAL PURPOSE.

b) (USA only) SOME STATES IN THE UNITED STATES DO NOT ALLOW THE LIMITATION OR EXCLUSION OF LIABILITY FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES, SO THE ABOVE LIMITATION OR EXCLUSION MAY NOT APPLY TO YOU AND YOU MAY ALSO HAVE OTHER LEGAL RIGHTS THAT VARY FROM STATE TO STATE.

c) THE FOREGOING LIMITATIONS ON LIABILITY ARE INTENDED TO APPLY TO THE WARRANTIES AND DISCLAIMERS ABOVE AND ALL OTHER ASPECTS OF THIS AGREEMENT.

10. Basis of Bargain. The Limited Warranty and Disclaimer, Exclusive Remedies and Limited Liability set forth above are fundamental elements of the basis of the agreement between Cyntellect and you. Cyntellect would not be able to provide the Software on an economic basis without such limitations. Such Limited Warranty and Disclaimer, Exclusive Remedies and Limited Liability inure to the benefit of Cyntellect’s licensors.

11. U.S. Government Restricted Rights Legend. This Software and the documentation are provided with “RESTRICTED RIGHTS” applicable to private and public licenses alike. The Software is a “commercial item,” as that term is defined at 48 C.F.R. 2.101 (Oct 1995), consisting of “commercial computer software” and “commercial computer software


documentation,” as such terms are used in 48 C.F.R. 12.212 (Sep 1995) and is provided to the U.S. Government only as a commercial end item. Any technical data provided with such Software is commercial technical data as defined in 48 C.F.R. 12.211 (Sep 1995). Consistent with 48 C.F.R. 12.211 through 12.212, 48 C.F.R. 227.7202-1 through 227.7202-4 (Jun 1995), and 48 C.F.R. 252.227-7015 (Nov 1995), all U.S. Government End Users acquire the Software with only those rights expressly set forth in this agreement.

Without limiting the foregoing, use, duplication, or disclosure by the U.S. Government is subject to restrictions as set forth in this agreement and as provided in DFARS 227.7202-1(a) and 227.7202-3(a) (1995), DFARS 252.227-7013 (c)(1)(ii) (Oct 1988), FAR 12.212(a) (1995), FAR 52.227-19, or FAR 52.227-14, as applicable. For purposes of these regulations the Manufacturer of the Software is Cyntellect Inc.

12. Third Party Software. The Software may contain third party software which requires notices and/or additional terms and conditions. Such required third party software notices and/or additional terms and conditions, if any, are located at Cyntellect’s web site at http://www.cyntellect.com and are made a part of and incorporated by reference into this agreement. By accepting this agreement, you are also accepting the additional terms and conditions, if any, set forth therein.

13. General

a) This agreement shall be governed by the internal laws of the State of California, without giving effect to principles of conflict of laws. You hereby consent to the exclusive jurisdiction and venue of the state courts sitting in San Diego County, California or the federal courts in the Southern District of California to resolve any disputes arising under this agreement. In each case this agreement shall be construed and enforced without regard to the United Nations Convention on the International Sale of Goods.

b) This agreement contains the complete agreement between the parties with respect to the subject matter hereof, and supersedes all prior or contemporaneous agreements or understandings, whether oral or written. You agree that any varying or additional terms contained in any purchase order or other written notification or document issued by you in relation to the Software licensed hereunder shall be of no effect. The failure or delay of Cyntellect to exercise any of its rights under this agreement or upon any breach of this agreement shall not be deemed a waiver of those rights or of the breach.

c) No Cyntellect dealer, agent or employee is authorized to make any amendment to this agreement.

d) If any provision of this agreement shall be held by a court of competent jurisdiction to be contrary to law, that provision will be enforced to the maximum extent permissible, and the remaining provisions of this agreement will remain in full force and effect.

e) All questions concerning this agreement shall be directed to: Cyntellect at 6620 Mesa Ridge Road; Suite 100, San Diego, CA 92121, Attention: President.

f) “Cyntellect” and other trademarks contained in the Software are trademarks or registered trademarks of Cyntellect in the United States and/or other countries. Third party trademarks, trade names, product names and logos may be the trademarks or registered trademarks of their respective owners. You may not remove or alter any trademark, trade names, product names, logo, copyright or other proprietary notices, legends, symbols or labels in the Software. This agreement does not authorize you to use Cyntellect’s or its licensors’ names or any of their respective trademarks.

g) If you are a business, company or organization, you agree that upon request from Cyntellect or its authorized representative, you will within thirty (30) days fully document and certify that use of any and all Cyntellect software at the time of request is in conformity with your valid licenses from Cyntellect.



Appendix B: Filter Configuration

Table A-1. Standard filters on LEAP Excitation

(nm) Dichroic

(nm) Emission

(nm) Common Dyes

377/50 409 LP 438/24 DAPI, Hoechst, Alexa Fluor 350, Aminomethylcoumarin, Lysosensor Blue, Marina Blue, Pacific Blue, BFP, EBFP

438/24 458 LP 483/32 AmCyan, CFP, ECFP, BOBO-1, SYTOX Blue 472/30 495 LP 520/35 Cy2, CyQuant, DiO, YOYO-1, GFP, EGFP,

482/35 506 LP 536/40 Carboxyfluorescein. FITC, Alexa Fluor 488, Calcein, Fluo-4, Mitotracker Green, Oregon Green, Rhodamine 110, rsGFP

525/40 555 LP 585/40 Rhodamine, TMR, TRITC, Alexa Fluor 532, 5-TAMRA, Dil, POPO-3, PO-PRO-3,

531/40 562 LP 593/40 Alexa Fluor 546, Alexa Fluor 555, Calcium Orange, Cy3, LysoTracker Yellow, MitoTracker Orange, Phycoerythrin (PE), dsRed

562/40 593 LP 624/40 Propidium Iodide (PI), Cy3.5, Alexa Fluor 568, Alexa Fluor 594, Calcium Crimson, MitoTracker Red, Texas Red, HcRed

628/40 660 LP 692/25 Cy5, Alexa Fluor 647, Alexa Fluor 660, APC (Allophycocyanin), BODIPY 650/665, DiD, SYTO Red 60, 62, 63, TOTO-3

Optional filters available for installation by Cyntellect 500/24 520 LP 542/27 Calcium Green, Fluo-3, Rhodamine 123, YFP


Appendix C: Consumables List

• The list of LEAP consumables and contact information is available from the Help menu.


Appendix D: Error Event Reporting Introduction When encountering problems or questions while using the LEAP software, please follow the steps described below to gather information that will help Cyntellect address the problems or questions. Future software will address automatic gathering of this information. All screen captures should be performed using the “Print Screen” button, a paste into Microsoft Paint, and save file as a JPG file. 1. Create a zip file with the name of the LEAP + Date + Time, e.g. CYNT-102206-1545

or CompanyName-102306-1620, etc. You can create multiple zip files with different time stamps during the day if multiple issues occur. All files will be saved within this zip file before emailing it to Cyntellect. For example, to create a CYNT1-102006-1534.zip file in the Desktop:

a. Right-click on the Desktop and select New Compressed (zipped) folder;

b. Name the zip file/folder appropriately, e.g. CYNT1-102006-1534.zip.

2. Perform a screen capture of the Protocol Options with the Machine Control tab

and save into the zip file created in #1, e.g. Protocol.jpg


e.g., Cell Purification protocol

Machine Control

Protocol Options

3. Perform a screen capture of the Protocol Gating screen with gates, if gating is

involved in protocol and save into the zip file created in #1, e.g. Gating.jpg


4. Perform a screen capture of the LEAP Image Display screen and save into the zip

file created in #1, e.g. LEAPImage.jpg

5. In the LEAP Image Display, select Channel 1, select Save As…, enter a channel name with date, e.g. Channel1 091106, and save into the zip file created in #1.

6. Select Channel 2, select Save As…, enter a channel name + date, e.g. Channel2

091106, and save into the zip file created in #1.


7. Save LEAP Computer event log:

1. Right-click My Computer, select Manage:

2. In Computer Management,

select System Tools Event Viewer Application. Right-click on Application and select Save Log File As…

3. Navigate to zip file created in

#1, enter a filename, e.g. ApplicationLog091106, and Save


8. Copy the following files into the zip file. Right-Click and drag to zip file created in

#1, select Copy Here. C:\Program Files\Cyntellect\LEAP Version 1\NavigationConfig.xml C:\Program Files\Cyntellect\LEAP Version 1\Hardware Control\leaphardware.config C:\Program Files\Cyntellect\LEAP Version 1\Hardware Control\ leaphardwarecalibration.config And the folder:

C:\Documents and Settings\All Users\Application Data\Cyntellect\LEAP\ChannelConfigs\ 9. Email the zip file to Cyntellect:

Summary Files to be collected and emailed to Cyntellect:

Description Number of files

JPG files saved in Steps 2,3,4 3 C:\Documents and Settings\All Users\Application Data\Cyntellect\LEAP\ChannelConfigs\

folder

Event log 1 C:\Program Files\Cyntellect\LEAP Version 1\NavigationConfig.xml C:\Program Files\Cyntellect\LEAP Version 1\Hardware Control\leaphardware.config C:\Program Files\Cyntellect\LEAP Version 1\Hardware Control\ leaphardwarecalibration.config

3


Appendix E: Example Power & Spot Size Data Green Laser Example Laser Energy Measurement vs. Attenuator Position

Green (532 nm) LaserPower vs. Attenuator Position

0

2

4

6

8

10

12

14

0 20 40 60 80 100 120 140 160 180 200

Attenuator Position

Lase

r Pow

er (µ

J)

Example Laser Spot Size Calibration


UV Laser Example Laser Energy Measurement vs. Attenuator Position

UV (355 nm) LaserPower vs. Attenuator Position

0

0.5

1

1.5

2

2.5

0 50 100 150 200 250 300

Attenuator Position

Lase

r Pow

er (µ

J)

Example Laser Spot Size Calibration
