Page 2 Fax: +1-781-790-5649 The information in this user manual is described as accurately as possible. Firmware and software changes and updates may change without prior consent or notification. Copyright © 2014 by NanoEntek, Inc. All right reserved. Published in Korea Documentation: NESMU-JST-001E Revision history: V.0.0 OCT...
Page 3: Table Of Contents
Contents Safety and compliance ..................... 5 General safety precautions ..................5 Safety symbols......................6 Intended use ....................... 7 Product overview ...................... 8 Scope of delivery ......................9 Description ......................10 Front view ......................... 10 Side view ........................11 Rear view ........................11 Control box .......................
Page 4 Contents 11.2.3 Movie Maker....................78 Movie Maker – Single ................. 79 11.2.4 Movie Maker – Sequence ................84 11.2.5 Movie Maker – Matrix ................. 88 11.2.6 ™ JuLI Stage STAT Software ..................90 12.1 Software overview ..................... 90 12.2 Operation ........................92 12.2.1 Main viewer ....................
Page 5: Safety And Compliance
Safety and compliance Safety and compliance General safety precautions  If water or other material enters the instrument, adaptor, or power inlet, disconnect the power cord and contact a service person. For operating environment, refer to Product Specifications.  Do not touch the main plug or power cord with wet hands. ...
Page 6: Safety Symbols
Safety and compliance Safety symbols Symbol Meaning Caution and Warning Protective earth (Ground) This instrument and consumables conforms to the EC Declaration of Conformity. This equipment has been tested and found to comply with the limits for a Class A digital instrument, pursuant to Part 15 of the FCC Rules.
Page 7: Intended Use
JuLI™ Stage is particularly suitable for cellular applications. Exclusion of Liability for Measurement Results NanoEntek does not assume any liability for the correctness of measurement results obtained with the NanoEntek JuLI™ Stage system nor for conclusions based on these measurement results. JuLI™ Stage - User Manual...
Page 8: Product Overview
Product overview Product overview The JuLI™ Stage is a real-time live cell imaging system that can acquire extended image series directly from various cell culture plates (6 to 384 well), flasks, slides and dishes in an incubator. The JuLI™ Stage supports a brightfield and three fluorescence channels, three objective lenses, and sensitive filter-based optics to optimize for various live cell assays.
Page 9: Scope Of Delivery
The JuLI™ Stage real-time live cell imaging system is shipped with the following components. Upon receiving the instrument, please check that all items listed below are included in the shipment. If any part is missing or damaged, contact your local distributor or sales@nanoentek.com. JuLI™ Stage - User Manual...
Page 10: Description
Description Description Front view Cover Used to protect test samples from external light. Simply put on the cover if you use the JuLI™ Stage on the bench and/or look at light sensitive samples. Main body The main body contains all of the mechanical, electrical, and optical parts. JuLI Stage - User Manual...
Page 11: Side View
Description Side view LED arm The arm above the sample holds the LED for the brightfield illumination. Automatic XY-stage The automatic XY-stage precisely navigates to the intended position. The holder is manufactured after SBS standard so that you can insert all standard microplates (6 to 384 well plates).
Page 12: Control Box
Description Control box Serial and camera USB cables The control box can be connected to the PC by connecting the serial and camera USB cables to the USB repeaters. When properly connected the serial and camera LEDs are on. Connection port (control box) Via the white connection cable the JuLI™...
Page 13: Installation
Installation Installation Unpacking the instrument 1. Open the box and remove the material foam from the box. 2. Carefully lift the instrument out of the box, holding it by the handholds in the bottom of the base. 3. Place the instrument on a flat, levelled surface (table or incubator). IMPORTANT! Do not lift the JuLI™...
Page 14: Installing The Instrument
Installation Installing the instrument Installation: Please make sure to carefully follow the installation instructions in this manual in order to ensure safe operation. The final installation should look as shown in the following connection overview: ► ► ► ► ► JuLI™...
Page 15 Installation Connect the other end of the repeater cables to the USB cables of the control box. The LEDs on the control box should be red. Protect the cable connection with the plastic cover. Plug the white connecting cable into the control box using the end of the cable without the ferrite core.
Page 16 Installation Turn on the JuLI™ Stage using the switch on the control box. Start the JuLI™ Stage control software and follow the warm-up guidelines. First use: Before operating the JuLI™ Stage system for the first time, we strongly recommend allowing the instrument to equilibrate to room temperature for 20 minutes outside the incubator while the device is turned on.
Page 17: Operating Environment
Installation IMPORTANT! Use the cover if the instrument operates on the table to protect test samples from external light. Operating environment  Allow at least 10 cm (4 inches) free space at the back of the instrument to allow for proper ventilation and prevent overheating of electronic components. ...
Page 18 Installation The JuLI™ Stage is optimized for a standard size cell culture incubator. It is recommended that the temperature of the JuLI™ Stage instrument can equilibrate to the temperature of the incubator. Environmental requirements When you use the JuLI™ Stage inside an incubator, make sure that the temperature of the laboratory never exceeds 15℃...
Page 19 Installation Internet Connection and Windows Updates on Control PC ® If the control PC is connected to the internet, the Windows operating system is automatically updated (and rebooted if necessary). In order to avoid errors during operation, we recommend:  Check for Windows Updates regularly and make sure that all available security updates have been downloaded and installed before starting a measurement.
Page 20: User Interface
User interface User interface Preview menu The Preview menu contains all functions to quickly set up the vessel layout, imaging conditions, and acquire snapshots and short movies. Setup menu The Setup menu includes all functions of the Preview menu (except snapshot and short movie functions) and additionally the experiment settings.
Page 21 User interface Data menu Settings menu Within the Settings menu you can revise the device serial, change the objective lense, choose the data path, review the remaining disc space, choose your mouse control configuration, review activated software, update the software, and firmware, and toggle on or off the system time which is displayed on the bottom right of the window (neither can the user enter the admin option, nor are there any options for alternative languages).
Page 22: Basic Operation
Basic operation Basic operation The JuLI™ Stage is controlled by a PC (monitor resolution 1920x1080) via wireless mouse and keyboard. ☞ Note: This manual describes all operations using the default mouse setting (Type 1). If necessary, you can change the mouse setting in the Settings menu. Moving Plate navigation In both the Preview and Setup menu you can move to the center of a well by...
Page 23 Basic operation The selected position is marked by a blue square and is equivalent to the field of view. ☞ Note: The field of view for the 4x, 10x, and 20x objectives is 3.55 mm², 0.58 mm², and 0.14 mm², respectively. Well navigation using the image Clicking the right mouse button and dragging over the image will move the current position towards the direction of the arrow in the Preview and Setup mode.
Page 24: Selection
Basic operation 24 well plate 96 well plate ☞ Note: When moving between wells, a popup message will appear to wait for the stage to finish moving. If you click Cancel, the stage will immediately stop at the current position. Selection Well selection A well can be selected by left click which will highlight the well with a grey color.
Page 25 Basic operation Selecting additional positions In order to add multiple positions to a well left-click into the well-representation. Selected positions are highlighted by a light blue square (double-click to delete). Click Add Position to add these positions to the Position List. Subsequently the squares will appear dark blue.
Page 26: Position Zoom
Basic operation Position zoom You can zoom in or out of the position clicking + or – on the right side of the well- representation. JuLI Stage - User Manual...
Page 27: Mouse Wheel
Basic operation Mouse wheel Zoom-in and out Scroll up or down in the image view of either the Preview or Option tab to increase or decrease the size of the image. The zoom function can also be used in all other software modules.
Page 28 Basic operation Focus adjustment While holding the shift key you can scroll up or down to focus the sample. It is the same function as the fine focus arrow buttons on the screen. Coarse focus Fine focus ☞ Note: The coarse focus is not supported by the mouse wheel. Measure distance and size To measure distances or the size of an object click the mouse wheel at one side of an object and hold the mouse wheel down until you reach the end of this object.
Page 29: Operation
Operation Operation Preview menu The Preview menu allows you to select basic system functions and is the first screen after start-up. It is available for checking the sample, setting the optimized focusing, capturing and merging the images. JuLI™ Stage - User Manual...
Page 30 Operation ① Vessel type / calibration ⑪ XY direction control ② Annotation list ⑫ Image overlay button ③ Map mode button ⑬ Image save button ④ Channel selection ⓐ Well zoom in & out ⑤ Exposure control ⓑ Well navigation ⑥...
Page 31 Operation 3. Click Vessel again to close the window without calibration. ☞ Note: Only vessels belonging to the Well Plate type can be calibrated, other vessel will inactivate the Calibration button. ☞ Note: In order to see the well borders during a vessel calibration make sure to have the BRIGHT light source turned on.
Page 32 Operation 3. Click Save to save the calibration information. ☞ Note: The calibration information is always only saved for the plate type currently in use. It will be applied to the current and all following measurements. However, the vessel calibration needs to be repeated whenever the plate type (number of wells and/or manufacturer) is changed.
Page 33 Operation Map mode To browse big samples, look for imaging positions, or calibrate the stitching you can use the Map Mode. Click Map Mode and then Make Map to open the menu. 1. Select the channel, map range, and check whether or not to use the autofocus or to separate the channels (if Separate Channel is turned off, all selected channels will be overlayed in the map).
Page 34 Operation 6. Click Save and X to finish stitching image calibration. Annotation List The Annotation List serves to mark and annotate exact locations in the well that are of special interest. These positions are then also available in the Setup menu. ☞...
Page 35 Operation 1. Click Guide Line to get an idea where exactly the center of the image is. 2. Navigate the guide lines onto the position and click Add to set a mark and assign a name or description to the position. Channel selection 1.
Page 36 Operation Scale Bar Click Scale Bar to display a predefined scale bar on the image in the bottom left corner. Time Stamp The Time Stamp shows the number of images and the time point of the time series measurement in the bottom right corner of the image. Pseudo color During the adjustment of acquisition conditions, the option Pseudo Color can help to visualize image illumination.
Page 37 Operation 3. Click Overlay and edit your images if necessary 4. Activate any combination of channels by clicking the blue dot on the left side of the small images. 5. Click Save to save the snapshot as jpeg, tiff, bmp, or png. Record a movie Clicking Record opens the live-movie menu.
Page 38 Operation Minimum & Close button Minimize or close the control software. JuLI Stage - User Manual...
Page 39: Setup Menu
Operation Setup menu The Position menu contains all functions to easily set up an experiment. ☞ Note: All sections covered under Preview also apply to the Setup tab if not stated otherwise. Sections previously covered are marked with (P). ☞ Note: The acquisition settings and all corresponding functions can be set up below the image view similar to the acquisitions settings in the Preview tab.
Page 40 Operation ① Annotation list ⑬ Save setup toggle button ② Map mode button (P) ⓐ Vessel calibration (P) ③ Channel selection (P) ⓑ Load experimental setup ④ Stitching mode toggle button ⓒ Well navigation (P) ⑤ Z-Stack toggle button ⓓ Well zoom in & out (P) ⑥...
Page 41 Operation Navigation position set  To select all wells click Select All.  After selecting one or multiple wells, clicking Add Position will add the center position of each selected well to the Position list.  There are two ways to proceed if more than one position inside a well is supposed to be measured in only selected or in all wells of the measurement: Select wells first, then select e.g.
Page 42 Operation Project Title Before monitoring, assign a specific title for the project using the pop-up keyboard. ☞ Note: Time Stamp will add the date and time to the name, which improves chronological sorting of the projects. Load Setup Click Load Setup to import the experimental setup and layout from previous projects.
Page 43 Operation Select Channels Make sure to activate all relevant channels for the experiment. ☞ Note: Light blue means active, dark blue means inactive. Stitching Mode To acquire bigger areas, stitching of multiple field of views can be performed (1x1 - 99x99, 1 - 9801 images).
Page 44 Operation The stack can be acquired downwards, upwards, or up- and downwards from the current position. ☞ Note: Stitching and Z-stack acquisition are mutually exclusive. Manual Focus Using Manual Focus images will be acquired in a Z height defined by the user. Activate the manual focus.
Page 45 Operation ☞ Note: In case both manual focus and auto focus are activated, the Z-height of the manual focus will be used where applied, all other positions will be focused using the auto focus. Auto Focus: You can choose how many steps to make until the next well that is supposed to be focused using the auto focus (1 = next well, 2 = second next well).
Page 46 Operation Custom AF: If the auto focus does not perform well you can use the Custom AF. This allows the user to set a specific range for the auto focus to look at, especially when there are differences between the different channels. 1.
Page 47 Operation Time lapse configuration The time lapse can be configured using an end-point, interval and/or the cycle number.  Total time: Depending on the time settings of the PC, the end point of the experiment can be defined using Total Time. ...
Page 48 Operation ☞ Note: During imaging you can click Pause and modify the cycle number by double clicking either interval or cycle. ☞ Note: In order to prevent phototoxicity an interval time of at least 15 minutes is recommended. However, the optimal time interval depends on cell lines. Start After all parameters for the experiment are configured, click Start.
Page 49 Operation IMPORTANT!  The JuLI Stage needs to be at a stable temperature. Before using the JuLI Stage in an incubator for the first time please follow the warm-up routine (30 minutes outside the incubator and 2 hours in the incubator while the JuLI Stage is turned on).
Page 50 Operation ① Title name ⑨ Cycle numbers ② Selected wells+ ⑩ View all wells ③ Annotation list ⑪ Play button ④ Z-Stack ⓐ Well navigation ⑤ Selected channels ⓑ Scale bar button ⑥ Time lapse options ⓒ Time stamp button ⑦...
Page 51 Operation Time lapse slider While capturing time-lapse data all images already acquired (and stored) can be reviewed using the time lapse slider. All well To review all wells at a glance use the toggle button All Wells. JuLI™ Stage - User Manual...
Page 52: Data Menu
Operation Data menu The data menu provides the function to review and export all datasets. Once you selected the project, all test conditions including vessel type, layout, acquisition and time lapse settings are displayed. JuLI Stage - User Manual...
Page 53 Operation ① Project name ⑧ Acquisition settings ② Project list ⑨ View all wells ③ Annotation list ⑩ Export button ④ Z-Stack ⓐ Scale bar ⑤ Plate layout ⓑ Time stamp button ⑥ Well navigation ⓒ Edit button ⑦ Channel selection ⓓ...
Page 54 Operation Well navigation and channel selection The well navigation shows all captured positions per well using blue squares (the selected positions is highlighted in light blue). All channels used during this experiment are blue (dark blue if deselected, light blue if selected) while channels that were not used are greyed out.
Page 55: Settings Menu
Operation Settings menu The Settings menu contains information about the JuLI Stage as well as several user preferences and other settings. Here you can change the objective, change the mouse button configuration, update the software, and display or hide the system time.
Page 56 Operation Objective Lens Provides the information which lens is currently used. ☞ Note: The field of view for the 4x, 10x, and 20x objectives is 3.55 mm², 0.58 mm², and 0.14 mm², respectively. How to change the objective 1. Select an objective by clicking the blue radio button. This will cause the stage to move to a neutral position.
Page 57 Operation Mouse Control Switches for mouse buttons: configurations 1 and 2. ☞ Note: Type 1 is the factory setting. Activation information JuLI Stage Scratch and JuLI Stage Spheroid are optional modules that offer additional analyses as well as assay possibilities. These modules need to be purchased and activated.
Page 58 Operation System Time On the bottom right corner of the software the date and time is visible which can be hidden by clicking Off. JuLI Stage - User Manual...
Page 59: Quick Workflow (Setting Up An Experiment)
Operation Quick Workflow (setting up an experiment) 1. Before the experiment determine the optimal objective and exchange the objective if necessary. See chapter 7.4 “ Settings menu” (How to change the objective). 2. Place the sample into the plate holder of the XY-stage 3.
Page 60: Cleaning And Maintenance
Unauthorized repairs may damage the JuLI Stage or alter its functionality, which will void your warranty. Contact your local distributor or sales@nanoentek.com to arrange for service. IMPORTANT! The JuLI Stage is optimized for a standard size of cell culture incubator.
Page 61 Cleaning and Maintenance IMPORTANT! The well plate’s cover should be wetted by the cell culture media before monitoring. This step is required to avoid condensation inside the well plate cover for monitoring. IMPORTANT! Avoid exposing the JuLI Stage to UV light. UV light may degrade components, including plastics.
Page 62: Troubleshooting
Troubleshooting Troubleshooting Installation Connect the device to the power outlet. JuLI Stage does not power up Check the on/off switch on the side of the control box. Contact your distributor. Monitor is black Move the mouse. Check if the power supply is connected and the monitor is switched on.
Page 63: Specifications
Specifications Specifications 10.1 Product specifications Items Specification Light source Blue, Green, UV LED (Intensity adjustable) 4X, 10X, 20X + Digital zoom Objective Lens Inter-changeable objective lens 3 fluorescences DAPI: Excitation 378/52, Emission 447/60 Fluorescence GFP: Excitation 466/40, Emission 525/50 RFP: Excitation 543/22, Emission 580LP High-sensitivity monochrome CCD (Sony sensor 2/3”) Camera 1,936 x 1,456 pixels (2.8M), 53 FPS, 14 bit...
Page 64: Ordering Information
Specifications 10.2 Ordering information Cat. No. Product Description JuLI Stage basic set (JS1000), Desktop computer JS1000S JuLI Stage, Stater Pack (JP0200), 3 Objective lenses (4X, 10X & 20X) JuLI Stage, Real-Time JS1000 Main device, power supply, control box live cell imaging system CPU: Intel i5, 9 generation or over spec.
Page 65: Warranty
The following defects, however, are specifically excluded:  Defects caused by improper operation  Repair or modification done by anyone other than NanoEntek or an authorized agent  Damage caused by substituting alternative parts ...
Page 66: Juli ™ Stage Edit Software
JuLI™ Stage EDIT Software ™ JuLI Stage EDIT Software 11.1 Software overview With JuLI Stage EDIT, measurement data sets can be reviewed, brightness and contrast settings of single or all images of a project can be edited, and a variety of movies can be created from time lapse measurements.
Page 67 JuLI™ Stage EDIT Software  The Matrix movie function allows you to combine several movies from one or different projects, or channels, or movie sequences, next to each other in a grid of 1x1 up to 9x9. Here for example a 2x2 grid: JuLI™...
Page 68: Operation
JuLI™ Stage EDIT Software 11.2 Operation Double click JuLI EDIT on the desktop to run the program. Click File and Data Path. Enter a directory or click Browse… to choose a directory. Then press OK. ☞ Note: Selecting a single project folder directly will not work, always select a parent folder.
Page 69: Main Viewer
JuLI™ Stage EDIT Software 11.2.1 Main viewer The main viewer is the starting interface for reviewing and selecting image data sets from the JuLI Stage for further editing and movie creation. ① Menu bar ⑦ Data path ② Image view ⑧...
Page 70 JuLI™ Stage EDIT Software ① Menu bar  File menu Data Path: Click Data Path to change the originally chosen or enter a new data path: remember to always select a parent folder containing the projects / measurements. Close: Click Close to close the JuLI Stage EDIT software.
Page 71 JuLI™ Stage EDIT Software ③ Project list All available measurements / projects belonging to the selected data path are listed and sorted by date. By clicking a line in the table, a measurement is selected and thereby marked dark blue. By clicking the header field “Creation Time”, the order of the list can be reversed.
Page 72 JuLI™ Stage EDIT Software ⑦ Data path Display of the data path in use. Use File > Data Path to change the data path. ⑧ Channel selection and overlay Display of all 4 channels available on the JuLIStage: grey channels were not measured in the selected project while light blue channels were acquired and are displayed in image view automatically as an overlay, e.g.
Page 73 JuLI™ Stage EDIT Software ⑩ Project information Once a measurement / project is selected in the list, acquisition details such as objective lens used, total time, number of cycles and interval time are displayed in this window. Also, channel settings of the measurement (exposure time, brightness and LED power) are detailed: ⑪...
Page 74 JuLI™ Stage EDIT Software ⑫ Edit After a project / Z-plane was selected, clicking Edit will open the next window, where two different editing options are offered. JuLI Stage - User Manual...
Page 75: Image Editor
JuLI™ Stage EDIT Software 11.2.2 Image editor ① Image view ⑥ Time lapse slider ② Edit image ⑦ Scale bar ③ Vessel display ⑧ Time stamp ④ Well navigation ⑨ New project ⑤ Edit panel Vessel display & navigation Select well, channel and position inside the well to review and edit image brightness and contrast.
Page 76 JuLI™ Stage EDIT Software Time lapse slider The time lapse slider can be used to navigate through timepoints (cycles) of the displayed data set using the buttons on the left or by dragging the slider bar. Time-point selection Enables the selection of different timepoints or time windows. ...
Page 77 JuLI™ Stage EDIT Software  Background Correction: Uniformly calibrates the brightness of the background of brightfield images  Auto Adjust: Automatically calibrates brightness and contrast  Default: Reverse all changes Save current image Clicking Save Current Image the image currently shown in the image view can be saved as jpeg, tiff, bmp, or png.
Page 78: Movie Maker
JuLI™ Stage EDIT Software Quick Workflow to export a modified data set 1. Select a well and position 2. Choose either all positions of the data set, the current whole well, or only the current position will receive modifications 3. Select one or all time-points or a time window 4.
Page 79: Movie Maker - Single
JuLI™ Stage EDIT Software Movie Maker – Single 11.2.4 The Movie Maker – Single creates movies from all or selected time-points of the whole or a cropped single position. ① Image view ⑥ Edit panel ② Vessel display ⑦ Label options ③...
Page 80 JuLI™ Stage EDIT Software Channel selection Activate all channels you want to be shown in the movie. Position slider The time lapse slider can be used to navigate through timepoints (cycles) of the displayed data set using the buttons on the left or by dragging the slider bar. Timepoint Selection Enables the selection of different time points or even time windows.
Page 81 JuLI™ Stage EDIT Software Scale Bar Insert a scale bar into the movie  Color: Change the color of the scale bar  Background: Change the background color of the scale bar Time Stamp To display a Time Stamp in the movie: ...
Page 82 JuLI™ Stage EDIT Software Preview View a preview of the edited movie. All Positions Apply all settings to all positions. ☞ Note: If All Positions is ON, movies for all wells and all positions in a well will be created with automatically given, individual names. E.g. a measurement containing 6 wells and 5 positions within a well will create 30 movies named for example B02_00005.avi for well B2 and position 5 in well B2.
Page 83 JuLI™ Stage EDIT Software Quick Workflow to create a Single movie Select a well, position, and channels Add labels, size bar, or time stamp Crop image Choose time window Click Add Decide if all positions or not Click Make Movie Set a path, file name if required, and a frame rate Click Save JuLI™...
Page 84: Movie Maker - Sequence
JuLI™ Stage EDIT Software Movie Maker – Sequence 11.2.5 In order to show a series of movies in one sequence you can concatenate movies using Movie Maker - Sequence. ① Image view ⑥ Time lapse slider ② Project list ⑦ Edit sequence panel ③...
Page 85 JuLI™ Stage EDIT Software Find Projects Here you can add additional data sets to the project list. ① Searching options ⑥ Project select option ② Project list ⑦ Insert ③ Selected project list ⑧ Delete projects ④ Back ⑨ Apply ⑤...
Page 86 JuLI™ Stage EDIT Software Add to Sequence After selecting a project click Add to Sequence to go to the edit movie window. Here you can put together a movie series in order to assign it to the sequence list. ① Image view ⑥...
Page 87 JuLI™ Stage EDIT Software Sequence List The sequence list will display the order and alias names of depicted movies of wells/positions or projects which will be used to assemble the sequence movie.  Preview : Preview the full movie sequence ...
Page 88: Movie Maker - Matrix
JuLI™ Stage EDIT Software Movie Maker – Matrix 11.2.6 The Movie Maker – Matrix can combine different movies in a tiled overview to better compare conditions with each other. Each tile can belong to one or can be a sequence of more than one time lapse measurement. ①...
Page 89 JuLI™ Stage EDIT Software Movie Matrix Choose the number of tiles of the movie matrix from 1x1 and up to 9x9 Auto Dimming If activated, movies that are shorter than others will turn black after they finished to better indicate tiles with movies that finished earlier. Quick Workflow to save a matrix movie 1.
Page 90: Juli ™ Stage Stat Software
JuLI™ Stage STAT Software ™ JuLI Stage STAT Software 12.1 Software overview With JuLI Stage STAT, projects containing measurement data are selected and analyzed with one of four evaluations: Growth Curve, Scratch Basic, Attached Cell Counting and Whole Intensity Level. With Plate Editor, detailed experiment conditions of the selected project can be defined and saved in plate maps: each well can be assigned to specific assay conditions (such as cell type and number, compound concentration etc.) in order to...
Page 91 JuLI™ Stage STAT Software GFP, Mean fluorescence intensity RFP, per well [-] (only for DAPI fluorescence channels) Whole GFP, Mean fluorescence intensity Calcium-flux Intensity RFP, per well [-] (only for Level DAPI fluorescence channels) ☞ Note: Only measurements based on multi-well plates (6, 12, 24, 48, 96 or 384 well plates) can undergo further analysis.
Page 92: Operation
JuLI™ Stage STAT Software 12.2 Operation Double click the ‘JuLI STAT’ icon on the desktop to run the program. Select a data directory path. Click Browse… to select the parent folder (located locally or in the network) containing measurement projects. Then click OK.
Page 93: Main Viewer
JuLI™ Stage STAT Software 12.2.1 Main viewer The main viewer is the starting interface for reviewing and selecting measurement data from the JuLI Stage for further analysis. ① Menu bar ⑦ Data path ② Image view ⑧ Channel selection ③ Project list ⑨...
Page 94 JuLI™ Stage STAT Software ① Menu bar  File menu Data Path: Click Data Path to change the originally chosen data path: remember to always select a parent folder containing the projects / measurements. Close: Click Close to close the JuLI STAT Analysis software.
Page 95 JuLI™ Stage STAT Software ③ Project list All available measurements / projects belonging to the selected data path are listed and sorted by date. By clicking a line in the table, a measurement is selected and thereby marked dark blue. By clicking the header field “Creation Time”, the order of the list can be reversed.
Page 96 JuLI™ Stage STAT Software ⑦ Data path Display of the data path in use. Use File > Data Path to change the data path. ⑧ Channel selection and overlay Display of all 4 channels available on the JuLI Stage: Gray channels were not measured in the selected project while light blue channels were acquired and are displayed in image view automatically as an overlay, e.g.
Page 97 JuLI™ Stage STAT Software The initial image view always displays the Default Z-plane, which is 0,0 µm. In order to pick one Z-plane regarded as optimal, select one channel and scroll through the stack using the arrow buttons.  Checking the circles Every Cycles and All Wells followed by Apply will use only this plane of the selected channel in further analyses.
Page 98 JuLI™ Stage STAT Software ⑪ Display All Wells Switching the toggle switch of All Wells to ON will display all wells of the selected plate at a glance – measured ones as well as not measured ones. In this example, in the upper 3 wells of a 6-well plate images were acquired, while the second row was not part of this measurement: ⑫...
Page 99: Analysis Modules
JuLI™ Stage STAT Software 12.2.2 Analysis modules Clicking Select Analysis will direct you to the next window where four different analyses – Growth Curve, Scratch Basic, Attached Cell Counting and Whole Intensity Level - next to a Plate Editor are available: Clicking brings you back to the main viewer.
Page 100: 12.2.2.1 Growth Curve
JuLI™ Stage STAT Software 12.2.2.1 Growth Curve The Growth Curve analysis is used to quantify the cell confluence in images over time, based on both brightfield or fluorescence images. In addition, the mean fluorescence intensity per well is calculated for fluorescence channels automatically. ①...
Page 101 JuLI™ Stage STAT Software Navigation The default setting of the selected measurement always displays the brightfield image and therefore this channel is presented in light blue. Any other acquired channel of the measurement is shown in dark blue, not measured channels in grey. ☞...
Page 102 JuLI™ Stage STAT Software To view the segmentation and confluence result of an image, click e.g. A and then Apply: The yellow colored area outlines the segmentation result of the latest sensitivity and background settings on the image view, and the table shows the numerical results of the selected well, position in well and time point.
Page 103 JuLI™ Stage STAT Software Example results for preconfigured sensitivity and background level settings A, B and C, with B delivering the best separation of foreground and background: ☞ Note: Quick Mode has no effect on the analysis of brightfield images. If the segmentation results of A, B and C turn out unsatisfactory, Edit Image gives more options to optimize brightness and contrast of the original image: ...
Page 104 JuLI™ Stage STAT Software measurement project in JuLI EDIT and save the original measurement under a new name. For more details, see chapter 11 “JuLI™ EDIT Software” ☞ Note: Parameters to optimize confluence segmentation on fluorescent images (Fluorescent Param) are deactivated for the brightfield channel. ☞...
Page 105 JuLI™ Stage STAT Software ☞ Note: Finding the best combination of settings is an iterative process and should always be tested on representative images (different time points, positive and negative controls, etc.). Continue comparing results with Quick Mode turned off (then Click Apply twice). In Quick mode, pixels of an image are binned, thereby reducing the processing time of large data sets.
Page 106 JuLI™ Stage STAT Software Default Gating: off Default Gating: on Here, in the lower left corner a difference can be seen. Also, the Tolerance Factor can be changed to check its influence on the segmentation: Tolerance Factor: 1 Tolerance Factor: 25 Check the Maximum intensity zero box to exclude areas exceeding the maximum intensity (option when Default Gating is unchecked): JuLI...
Page 107 JuLI™ Stage STAT Software Manual intensity gating Manual intensity gating can be useful to e.g. exclude objects with low fluorescence. The intensity plot shows the intensity distribution of pixels in an image, with fluorescence intensity plotted in x and number of pixels on the y-axis. ☞...
Page 108 JuLI™ Stage STAT Software Click Reset to return to the initial graph. Example for excluding low expressing cells from the confluence analysis: here, only cells with intensities above 50 are included in the segmentation mask: Original Default gating Manual gating: 50 to 255 54 % confluence 28 % confluence JuLI...
Page 109 JuLI™ Stage STAT Software Edit Image The segmentation results for fluorescence images can be further improved using Edit Image:  By moving the Brightness and Contrast sliders followed by clicking Apply the confluence analysis results can be viewed instantly and the best settings can subsequently applied to the entire measurement.
Page 110 JuLI™ Stage STAT Software Confluence result list: Numerical results Whenever the Apply button is clicked, the numerical results of the covered cell area, i.e. cell confluence in % (as well as an extrapolated area in µm²) is shown in a table for the selected channel, well, position in well and time point.
Page 111 JuLI™ Stage STAT Software In this example, the brightfield-based confluence in 8 selected wells will be calculated: Click Apply to start the analysis (and creation of graphs and tables) or click once more on Analyze to go one step back. ☞...
Page 112 JuLI™ Stage STAT Software View Results Click View Results ⑩ to proceed to different display options of result tables and graphs: A new window will be opened, divided into three tabs offering different result presentation options: Plate Graph, Well Graph and Results. JuLI Stage - User Manual...
Page 113 JuLI™ Stage STAT Software Plate Graph tab In the Plate Graph tab, a plate overview for all analyzed wells can be visualized depending on channel and readout parameter. Single or multiple wells can be excluded or included into the final results to be compiled as graphs and tables using Export.
Page 114 JuLI™ Stage STAT Software A direct link to the Plate Editor ⑥ can be used to create a detailed experiment  layout for the selected measurement project: assign names, colors or compound concentrations to each well in order to change the colors of curves in graphs or to calculate mean values of e.g.
Page 115 JuLI™ Stage STAT Software ☞ Note: Depending on which part of a cell is stained by the fluorescence dye, the confluence results might differ from the brightfield values as a smaller area of each cell might be subjected to segmentation. The readout Growth Rate can be changed to (mean) Intensity per well by selecting Intensity in the drop down menu of View Type: To remove outliers or unwanted wells from the final graphs and result tables, they...
Page 116 JuLI™ Stage STAT Software Excluded wells appear in red and all results (in all channels) belonging to these wells won´t be shown in graphs and result tables compiled under Export: This selection can be reversed via Include. Finally, clicking Export will create a graph showing the results of all 21 wells and all time points in individually colored lines: As before, clicking another channel or selecting another readout under View Type will display other available readouts.
Page 117 JuLI™ Stage STAT Software There are plenty options to change the appearance of the graphs, most of which are self-explaining and easy to apply. For example, the Main Title, which is the project name by default, can be changed to a more meaningful one, and also Background Color can be selected. Most changes require Set to be clicked to become active.
Page 118 JuLI™ Stage STAT Software ☞ Note: The Interval of the X-Axis requires the entry of a cycle number, independent of the Type selected. ☞ Note: On the X-Axis only timelapse describing entries are selectable, not compound dilutions or any other entries defined in a plate map. Alternative units to express the confluence are also offered: ☞...
Page 119 JuLI™ Stage STAT Software One condition is when more than one field was acquired in an experiment as shown in this example: 5 positions inside the wells were measured: In the Plate Graph tab automatically only one curve per well is displayed and only the selected numerical readouts (mean or median, together with standard deviation or standard error etc.) are exported to the Results table.
Page 120 JuLI™ Stage STAT Software Instead of the former 21 curves, now only 7 curves are shown displaying e.g. Mean and Standard Deviation of the whole well Intensity of triplicates assigned the names 1 to 7: Once the edits to the graph are finalized they can be either printed directly or Copied to the Clipboard to be pasted in an external program such as Word or Powerpoint: Being transferred as bitmaps, graphs cannot be edited after being pasted into an...
Page 121 JuLI™ Stage STAT Software processed with external software such as Excel or other spreadsheets. In case of mean values and standard deviations being available, both data will be exported. ☞ Note: All available readouts of an analysed measurement (and all available units for all readouts) for each channel need to be exported separately, there is no overall export containing all numerical results of an analysis.
Page 122 JuLI™ Stage STAT Software Well Graph tab This tab is especially good for a more detailed look on measurements which contain more than one position within a well. In Well Graph, analysis results for all positions in a well are presented in separate curves. Also, analysis results for each position within a well can be exported in this tab together with their mean values, their standard errors and standard deviations by clicking Results.
Page 123 JuLI™ Stage STAT Software Results tab The Results tab is designed for a quick overview of all numerical data available for a plate and its channels together with a small and instant view on the segmented image belonging to the selected value in the table. Inspecting segmented images can quickly confirm the validity of a value in the table.
Page 124 JuLI™ Stage STAT Software ☞ Note: The enumeration of images in their folders differs from the cycle enumeration displayed in the analysis result tables: if for example 12 cycles were acquired, image one is stored under number 0, while images belonging to the 12th cycle are stored under number 11.
Page 125: 12.2.2.2 Scratch Basic
JuLI™ Stage STAT Software 12.2.2.2 Scratch Basic The Scratch Basic analysis can be used to quantify the wound confluence in images over time - based on both brightfield or fluorescence images. The main differences to the optionally available software JuLI Stage Scratch, are that there is only one readout –...
Page 126 JuLI™ Stage STAT Software ① Project name ⑧ Back ② Image view ⑨ Time lapse slider ③ Channel selection ⑩ View results ④ Bright chan. param. ⑪ Analyze ⑤ Fluorescence chan. param. ⑫ Clear results list ⑥ Vessel display ⑬ Confluence result list ⑦...
Page 127 JuLI™ Stage STAT Software Well A1, Scratch Basic Well A1, Growth Curve ☞ Note: When configuring the optimal analysis settings in Scratch Basic, the instantly created result list displays Growth Rate values per image (not wound). However, once an analysis is applied via Analyze, wound-related confluence results will be displayed.
Page 128: 12.2.2.3 Attached Cell Counting
JuLI™ Stage STAT Software 12.2.2.3 Attached Cell Counting With the Attached Cell Counting evaluation module, adherent, fluorescence stained cells can be counted. Intensity and cell size thresholds next to other parameters can be changed to improve segmentation results. ☞ Note: Attached Cell Counting is only displayed as an analysis option if a fluorescence channel was acquired during the measurement.
Page 129 JuLI™ Stage STAT Software ① Project / measurement name ⑧ Time lapse slider ② Image view ⑨ View results ③ Channel selection ⑩ Analyze ④ Counting parameter ⑪ Clear ⑤ Vessel display ⑫ Cell count result list ⑥ Well navigation ⑬...
Page 130 JuLI™ Stage STAT Software Scrolling in the results table to the right will display the result of counted cells per mm². The size of the imaged position in a well = one Field of View (FOV) depends on the objective used in the measurement: for ...
Page 131 JuLI™ Stage STAT Software Quick Mode unchecked Turning off Quick mode improved the cell separation, then falsely fused cell were separated (yellow circle). But in the example image, some cells were overseparated. (In Quick mode, pixels of an image are binned, thereby reducing the processing time of large data sets.
Page 132 JuLI™ Stage STAT Software Intensity gating: Minimum intensity Set to 20, maximum intensity set to 100, then Apply In the Cell Size tab, additional options to influence segmentation are available: thresholds to exclude certain cell sizes can be set. There is a mouse hover in image view which displays the cell size of the cell pointed at to support this decision process, next to a histogram display (x-axis: size in [µm²], y-axis: number of objects).
Page 133: 12.2.2.4 Whole Intensity Level
JuLI™ Stage STAT Software 12.2.2.4 Whole Intensity Level With the Whole Intensity Level analysis, the mean intensity of images in the GFP, RFP and DAPI channel can be quantified. The interface of this analysis module differs from the others as it displays results in the starting window and there are no parameters to influence the analysis.
Page 134 JuLI™ Stage STAT Software Start by selecting a project/measurement and click Select Analysis - Whole Intensity Level: in the initial screen, images of all channels acquired (light blue) can be inspected separately or as overlay by selecting rows in the Well list ③ display in the Well-based tab and channels in the ④...
Page 135 JuLI™ Stage STAT Software Well-based result display The analysis results can be viewed instantly by selecting a well in the well list (dark blue) and one or more channels (dark blue). When the time lapse slider is moved, the image of the selected channel(s) at exactly this time point will be displayed.
Page 136 JuLI™ Stage STAT Software the value. By moving the time lapse slider, different time points can be displayed, but only one channel at a time: GFP channel, time point 0 GFP channel, time point 40 [RFP channel, time point 0] JuLI Stage - User Manual...
Page 137 JuLI™ Stage STAT Software The export option to display results in ready-to-use graphs and exportable numerical results are found under View Results. Please refer to chapter 0 “12.2.2.1 Growth Curve” to see details about the View Results functionalities. JuLI™ Stage - User Manual...
Page 138: Plate Editor
JuLI™ Stage STAT Software 12.2.3 Plate Editor With Plate Editor, detailed experiment conditions of the selected measurement project can be defined and saved in plate maps: each well can be assigned to specific assay conditions (such as cell type and number, compound concentration etc.) in order to calculate e.g.
Page 139 JuLI™ Stage STAT Software For example, a 96 well plate was selected: There are 4 tabs available to allocate and describe detailed conditions for each well of a plate: Compounds, Cells, Conditions or Regions. (Headers of these tabs cannot be changed.) ...
Page 140 JuLI™ Stage STAT Software Therefore, we start creating an example plate map with one tab only and then inspect its influence on a graph. Click on New in the Conditions tab and enter the description Negative control into both fields – Display Name (will appear in the wells) and Description (will appear in the list).
Page 141 JuLI™ Stage STAT Software Continue, until 7 conditions are defined using different colors: In order to assign each condition to three selected wells each: 1. Mark the wells belonging to a condition by either clicking on single wells or drawing a rectangle with the mouse to mark multiple wells. Selected wells turn gray.
Page 142 JuLI™ Stage STAT Software Now, we can compare the results of an existing analysis with and without plate map: Via Select Analysis and View Results open a Plate Graph. Graphs of the analysis will be displayed in one color: Click on Export: 21 curves belonging to 21 wells are displayed each in an individual color: In order to apply the created plate map, close the Export window again, and in the Plate Graph tab click Plate Editor –...
Page 143 JuLI™ Stage STAT Software The colors of the displayed curves instantly change according to the position of triplicates. Click Export again: Now, instead of 21 curves only 7 curves are displayed, showing the mean of triplicates and their standard error; also, the legend entries display the compound names from the plate map.
Page 144 JuLI™ Stage STAT Software Entries to the Compounds tab need to follow the same series of clicks as described for Conditions, however, there is an additional sub-menu in a separate window which assists the setup of a compound dilutions: 1. Click New to enter the compound name followed by Apply. 2.
Page 145 JuLI™ Stage STAT Software Another layout option would be to reduce the concentration by a factor of 10, from left to right, combining 3 columns into a group (in groups of 3). (The column- or row-wise grouping can only result in groups with integer numbers.) 5.
Page 146 JuLI™ Stage STAT Software 3. Then click HeLa in the cell list and click Add: this will open a new window - the Cells sub-menu. Here the Passage number, the Seeding Density and it´s unit, as well as a dilution similar to the Compounds tab can be used to enter details of the plate map: 4.
Page 147 JuLI™ Stage STAT Software For example, a 384 well plate contains DMSO and positive controls at defined positions. In order to easily mark them and use them in the other tabs, mark all wells belonging to a group and Save them under a name: Then click on one region in the regions list and wells belonging to the regions will be marked accordingly.
Page 148 JuLI™ Stage STAT Software Once the tab is changed exactly these marked wells can be assigned a different layout information. However, after clicking Add the well marks will disappear unless the box keep current selection after adding was checked: then the formerly marked wells will be available after a further tab change: Button Export As Image: ...
Page 149 JuLI™ Stage STAT Software Right clicking a specific well will open a window which enables the color change of single wells. JuLI™ Stage - User Manual...
Page 150 NanoEntek, Inc. 851-14, Seohae-ro, Paltan-myeon, Hwaseong-si Gyeonggi-do, 18531, Korea Tel: +82-2-6220-7940 Fax: +82-2-6220-7999 NanoEntek America, Inc. 220 Bear Hill Road, Suite 102, Waltham, MA 02451, USA Tel: +1-781-472-2558 Fax: +1-781-790-5649 E-mail sales@nanoentek.com Website www.nanoentek.com...

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