CURIOSIS Celloger Mini Plus User manual
Automated live cell imaging system
2
Email: info@curiosis.com
Website: www.curiosis.com
4F, 10 Teheran-ro 38-gil, Gangnam-gu, Seoul 06221, South Korea
Tel: +82-2-508-5237
Fax: +82-2-508-5246
Copyright © 2022, by CURIOSIS Inc.
All rights reserved. Published in Korea.
CR-IFU-008(Rev.2)
FOR RESEARCH USE ONLY and not for use in diagnostic procedures. Specifications subject to change without
notice
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Table of Contents
1.Pre-requirements 6
2.Operation overview 6
3.Installation 7
4.Create or open a project 8
5.Connect the device 9
6.Software overview 11
6.1. Celloger Mini Plus Scan App 11
6.2. Celloger Mini Plus Analysis App 12
1.Preparation (cell/device preparation) 13
2.Cell monitoring and image setting [in Scan App] 13
2.1 Location adjustment 13
2.2 Light intensity adjustment 17
2.3 Z stacking 17
2.4 Stitching 18
3.Image analysis [in Scan App] 19
3.1 Capture 19
3.2 Merge image 20
3.3 Image Tile 20
3.4 Image adjustment 21
3.5 Preview record 22
3.6 Intensity 22
3.7 Manual Counting 23
3.8 Ruler 23
4.Time-lapse imaging [in Scan App] 24
5.Process single image or time-lapse images [in Analysis App] 25
5.1 Import the original data taken by Celloger® Mini Plus 25
5.2 Merge FL image 26
5.3 Slide show 26
5.4 Make video 27
5.5 Estimate confluency (coverage) 27
5.6 Create confluency graph 29
5.7 Show Z-stacking image 29
5.8 Stitch images 31
5.9 Separate the image 34
1)Celloger Mini Plus Scan App 35
2)Celloger Mini Plus Anaysis App 50
1.General guidelines 52
2.Operating condition 53
3.Safety standards 53
1.Installation 57
1.1 Device IP setup 57
1.2 Using a router 59
2.Operation 59
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Celloger® Mini Plus Live cell imaging device package includes the following items.
Main power cable
1
PoE
1
LAN Cable (5M, 4.8M)
2
USB/LAN adapter
1
USB memory
1
- Instruction Manual
1
- Celloger Mini Plus Scan App
1
- Celloger Mini Plus Analysis App
1
Vessel holder for well plate (Included)
1
Vessel holder for dish 35mm, dual (Optional)
1
Vessel holder for dish 60mm, dual (Optional)
1
Vessel holder for dish 90/100mm, single (Optional)
1
Vessel holder for T-flask 25cm2, single (Optional)
1
Vessel holder for T-flask 25cm2, dual (Optional)
1
Vessel holder for T-flask 75cm2, single (Optional)
1
• Check that all items listed above are included in your package.
• Examine the device carefully for any damage during shipping.
• Contact your local distributor or inf[email protected] if any items are missing or damaged.
• Any loss or damage claims must be filed with the carrier.
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6
1.
For normal and stable operation of the device, the following environmental conditions should be satisfied.
Install Celloger® Mini Plus on a flat and level surface away from vibrating equipment.
Place at dust- or other airborne particles-free area.
During operation, place around 10 cm away from the surroundings for proper air flow of the cooling fan.
During operation inside incubator, do not place close to the incubator door.
Do not put any heavy materials on top of the Celloger® Mini Plus.
If using multiple equipment, there should be a proper clearance among equipment.
Keep the device at temperature between 10 ~ 40 °C.
Relative humidity range is 20 ~ 95 %.
It is not recommended to operate the device at low temperature (Below 10 °C). In those conditions, warm up the
device for over 10 minutes.
Celloger® Mini Plus software can be used on PCs with Window 10.
Large capacity hard drive is necessary to save images from Timelapse. If the capacity is not enough, scanning
will not function properly.
For the ease of use, it is recommended to use a monitor with resolution of 1920 x 1080.
LAN port is required to connect the equipment to PC using LAN cable.
2.
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3.
1. Lift the Celloger® Mini Plus from the box, remove all the covers, and place it on a clean and level surface.
2. Make sure all accessories are present by checking “Package Contents” (p.4). If any items are missing or
damaged, please contact your distributor or Curiosis at inf[email protected]om.
1. Prepare a computer that satisfies the specifications (refer to p.55 for PC specifications) and place it near the
incubator.
2. Connect two LAN cables and power cable to the POE injector. Connect one LAN cable to the device (yellow)
and another LAN cable to the PC (white).
3. Connect power cable to an outlet.
4. Turn on the power by pressing the power switch located next to the power cable port and check if the light
indicator turns on.
1. Install the vessel holder on Celloger® Mini Plus, then place the device inside the incubator.
*Before using the device, make sure to remove all the covers including the lens cover(Black) and clean the
device with soft dampened with 70% ethanol.
Power cable
LAN cable to PC
LAN cable into device
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2. Place the sample on the stage.
3. Check the connection between the PC and the device. To connect the device and PC,
refer to “” (p.9)
4.
Upon executing the , popup screen for project selection is displayed. With
Celloger Mini Plus, the setting can be managed by the porject file (“.cgnproj” file format). Setting
values(Light intensity, Focus position, Schedule, File storage location, etc.) are saved in the project.
In , the setting is managed by config file instead of project file.
Create a new project or open an existing one. You can start a program with default project by clicking
.
Import the project saved
in the past.
Create new project.
Start the program without
customizing a project. In
this case, a default project
“Experiment” is executed.
STATUS LIGHT
Green
Ready
Blue
Scanning
Yellow PC disconnected
Red
Error
Identify
White
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Upon selecting , popup window that requires the selection of project name and storage
location shows up. You can start the program after entering required information and press . The storage
location of the project is the address of default program file and if you want to change the storage location,
cancel and enter address.
5.
1. Open .
2. When the app is executed, popup appears. Select .
3. popup will show.
4. Click to load the network adapters connected to the PC.
5. Select PC network from then the device icon will appear on the right.
6. Click the device icon.
If the icon is not shown, check the following steps and take appropriate actions.
①
②
③
④
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Set the IP address of PC network manually. If the PC IP, device IP and router IP are the same, connection fails
(refer to Appendix B).
Check all cable connection status and POE injector power (refer to in
p.7).
Press the to select the adaptor to which the device is connected. If the device icon
does not appear even when the network adapter is selected, search for device IP address directly from.
Initially, devices are released with the same IP. When using the device for the first time, it is necessary to
reset the IP address. (refer to p.57 for device IP setting)
7. Click . When the connection has successfully completed, the LED status indicator lights in green (from
yellow)
A. reads the connected network adapter from the PC.
B. Network adapter list. shows the network connection status where means
network is enabled, while means disabled.
C. Change the IP address of the selected network adapter.
D. Manually search for the IP address of the device.
E. The list of connected devices.
A
B
C
D
E
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6.
provides two software: and .
is a program for actual operation of device which includes real-
time cell monitoring and time-lapse imaging function. is a program for analyzing
and post-processing the images obtained from and it can be operated even without the device
connection.
Refer to p.35 for full software descriptions
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①
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1.
The cell preparation method is different depending on the type of assay. The method below is a general process
recommended for the monitoring of cells.
1. Before culturing the cell, place in the incubator for prewarming.
Install the system as far as possible from the incubator door and place the device around 10 cm away
from the surroundings for proper air flow of rear cooling fan.
2. Prepare pre-warmed medium and cells on a plate.
3. Discard the medium on vessel and wash the cells with PBS buffer.
4. Discard PBS buffer and treat trypsin-EDTA solution to detach the cell from vessel.
5. Incubate for a few minutes and check cell detachment by Celloger® Mini Plus or microscopy.
6. Remove cell and Trypsin-EDTA to conical tube and add fresh medium to neutralize the Trypsin reagent.
7. Centrifuge the suspension cells on conical tube.
8. Discard the supernatant and add fresh medium into cells.
9. Gently pipette the cell several times.
10. Transfer the appropriate amount of cells into new vessel and dilute the cell concentration by fresh medium.
11. Cover the plate with a lid and gently shake the plate to spread the cells evenly.
Before covering the plate, warm the lid by slightly passing the lid over fire of alcohol lamp to prevent
condensation. This step is not necessary, but it will prevent trapping the condensation on a lid.
12. Place the plate on Celloger® Mini Plus.
Both plate and device should be pre-warmed for 30 minutes prior to time-lapse imaging to prevent
condensation.T
2.
1)
①Select vessel type and desired scan location on the .
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②Press for streaming while is selected in the .
③Move to the desired scan position using the . If the display is too dark or bright, preventing
the cells to be seen, run in p.17first.
2)
➢
①Select the as “X 1mm” and press to move to the point where cells are visible. Conduct
precise adjustment gradually by reducing the .
Selecting clear focal position is one of the major factors that influence the accuracy of analysis.
Therefore, analysis can be precisely conducted when cells are clearly distinguishable from background
and the shape of cells are clearly visible.
’ Z stage absolute coordinate error is 0.001 mm.
②Upon finding the best focal point for scanning, designate the location by pressing in
.
If you want to change the focus, select the position you wish to change in the scan table, click
under and click on or to apply the change. (refer to p.45 for )
Whenis set to a value other thanautofocusing function is executed
during scanning
➢
Autofocusing function of works by selecting clear focal point after screening the
area up and down the location. Therefore, it is necessary to have the focus location within the
designated range in parameter for a more successful autofocusing. Therefore, set
roughly (manually) and use the autofocusing function.
①Move the focal plane using .
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②Open in the menu.
③Designate parameter () in the and press .
✓: Screening scope for autofocusing
✓: The interval of autofocusing calculation within screening scope (0.010 mm or less
for 4X optic and 0.005 mm or less for 10X optics)
✓: If you want to select the point different from focal plane recognized by
, input the difference between the point and Z position coordinates from autofocusing.
✓: Select whether to focus on the black side when performing autofocus.
Focus is on black side when checked, if not, focus is on the clearer side of either black or white.
. The larger and the smaller , the higher autofocusing accuracy.
Meanwhile, the longer the screening time, the longer autofocusing time.
④Set the value except for in or in to set the
frequency of autofocusing function (refer to p.42 for ). If selecting it from
, click or after selecting.
①
②
②
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3)
Since the focus of the channel may be different from that of the , it is recommended to set the focus
position separately.
①Select the desired scan position on the . It is indicated in green once selected.
②Select the channel in and adjust the focus using the by referring to
the .
③Check that the checkboxunder is checked, and click on to specify the
focus and click . (If the position is specified after checking in the , the
checkbox is checked.)
④Check if the designated value is shown in .
①
②
③
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Light source option for is displayed on the lower tab. Select the desired light source to adjust
and change the parameter. It is automatically saved. Brightness is one of the important factors that has an impact
on analysis accuracy. It is important to set the appropriate brightness to distinguish cells from background.
①Select , press and check in streaming on state.
②Adjust the brightness by changing , , and .
1)
①By using , find the position to start stacking.
②Designate the stacking unit in the .
Ex) Step setting for stacking by 10 𝜇m
③In the , click for each position of the range you want to stack by using
.
2)
①Select the position in the where the z stacking function will be applied.
②Select whether or not to execute the z-stacking function in BF and FL in under .
(refer to p.46 for Z-stacking)
③After specifying the interval (= ) to execute the z-stacking function, adjust the scroll bar under
to specify the range.
<Shows Light Source Control in the App connected to Green FL Celloger®Mini Plus>
Scan table
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④After clicking , check that the set z-stacking value appears in .
①Select the position in the scan table where the stitching function will be applied.
②Select whether or not to execute the stitching function in BF and FL in under .
(refer to p.46 for Stitching)
③Set the overlapping area between the images using
scroll bar and designate the range to be scanned
in .
Properties (Z-Stacking)
Scan table
Properties (Stitching)
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④After clicking , check that the set stitching value appears in .
When stitching is performed under autofocus and fluorescence setup, be reminded that it
takes approximately 2 minutes to scan 3 x 3, and 7 minutes and 30 seconds to scan 7 x 7 with the
light conditions of BF (Lamp: 30, Exposure: 0.06, Gain: 1) FL (Lamp: 30, Exposure: 0.5, Gain:1).
3.
icon in captures the image on . (Use Ctrl + I as a shortcut)
1)
It is possible to generate and manipulate the desired features, show it on and save it as it is. To
obtain without features, press () for deactivation and then press .
2)
Press the desired tab of the channel in to show image on through
and press .
<BF channel image>
<FL channel image>
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Press () for activation , merged image is shown on . Adjusted image
as follows can be obtained upon changing setting ( and ) in
of menu.
conducts thresholding for channel image based on intensity and merge it with
image. There are two methods for intensity thresholding.
: Thresholding is executed based on the overall intensity of the image.
Adjust a parameter of while looking at the mask of an image after unchecking the
box.
: Divide the image into local areas and thresholding is conducted
by comparing relative intensity among areas. Adjust parameters of the and while
looking at the mask of the image after checking the box. ( indicates size
of local and indicates the relative offset of the threshold of local area.)
. When the fluorescence areas are distorted or clustered in one place, function
is recommended; and when the fluorescence areas are evenly distributed,
function is recommended.
In the event of imaging using a well plate, each scan position is combined and displayed at once. An
folder is created automatically in the folder where the images are saved for each cycle.
Image Tile
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