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Prism6 & Crystal Reader software User Manual

47

VII. Troubleshooting

Display Screen Error Messages

Prism6 and Crystal Reader software:

Description

error message

Recommendations

A red light appears in the third position at
the front of the Prism6 instrument

Reboot  the  Prism6  instrument  (press  the  power
button  in  the  front  for  a  few  seconds  until  the
Prism6  instrument  shuts  down,  then  press  again
to start it). Restart Crystal Reader software.

The Crystal Reader software application
stops responding

Go to “Control Panel” > “Task Manager” and stop
the Crystal Reader process, then start it again.

Image acquisition issue (Black or non-
existent flag)

Re-scan the concerned chamber

Non-optimal image assembly in a
chamber

Re-scan the concerned chamber

Error

message:

Unable

to

scan

chamber(s)

Re-scan the non-scanned chamber(s)

Error message: It is not possible to
deactivate the blue channel, it is used for
droplet detection.

Ensure to select at least 3 detection channels,
including the Blue channel.

Error: it is necessary to scan three
channels, including the blue one, to
perform a scan

"Error message: Prism6 instrument
configuration

not

applicable

with

detection channel selection."

Depending

on

the

Prism6

instrument

configuration,  up  to  6  detection  channels  are
available.

H24000.3:  3-color  Prism6  configuration  with  the
detection in Blue, Green, and Red channels.
H24000.6:  6-color  Prism6  configuration  with
detection  in  Blue,  teal,  Green,  Yellow,  Red  and
Infra-red  channels.  Please  contact  the  Technical
Support  to  enable  detection  channels,  if
applicable.

Error:  the  current  Image  Analysis
Configuration File (.yaml) is not designed
to  be  used  on  this  scanner  type.  Please
ensure  the  consistency  between  the
“.yaml” file and the experiment settings.

Please use the scanning templates provided by
Stilla Technologies accessible at

https://www.stillatechnologies.com/technical-
resources/naica-system-prism6/

. Please ensure

the consistency between the

“.yaml” file and the

experiment settings when editing

“.yaml” files

.

Error: the selected Crystal Miner Plot
configuration file (

“.ncp”) uses different

channels than the ones activated.

Please use the scanning templates provided by
Stilla Technologies accessible at

https://www.stillatechnologies.com/technical-
resources/naica-system-prism6/

. Please ensure

the consistency between the “.ncp” file and the
experiment settings

.

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Содержание Prism6 H24000.3

Страница 1: ...STILLA Prism6 Crystal Reader software for the naica system User Manual Prism6 H24000 3 H24000 6 Crystal Reader software v3 0 7 3 ...

Страница 2: ...e resulting from the use of this information Elements of the naica system and its uses are covered by US and corresponding non US patent claims owned by or under license to Stilla Technologies Details available at www stillatechnologies com patents Purchase of this product includes a limited non transferable right under such intellectual property for use of the product for internal research only N...

Страница 3: ... the experiment context 20 Name the experiment 24 Edit experiment details 25 Check the scanning parameters 27 2 Step 2 and 3 Select the chambers to scan Define chamber context 28 3 Step 4 Launch the scan 30 Check chamber image quality 31 4 Step 5 Check image quality 33 V ADVANCED FUNCTIONALITIES 36 1 How to rescan existing chambers How to scan new ones 36 How to rescan all the chambers of the expe...

Страница 4: ...Prism6 Crystal Reader software User Manual 3 3 Third party licenses 59 ...

Страница 5: ...s and 10 000 to 20 000 droplets Opal chips also called a droplet crystal PCR is then performed within the chips and the crystal is imaged to reveal the droplets that contain amplified targets The last step consists of counting the number of these positive droplets to precisely extract the absolute quantity of nucleic acids With Crystal Digital PCR the combination of powerful image analysis and int...

Страница 6: ...ftware is pre installed on the Prism6 delivered as part of the naica system The naica system performs Crystal Digital PCR within microfluidic chips Sapphire chips Opal chips The naica PCR MIX reagents are recommended for use to achieve optimal Crystal Digital PCR performance on the naica system For detailed instructions for the Geode and the Crystal Miner software please refer to the respective Us...

Страница 7: ... Power cords Monitor Monitor USB cable Mini display port cable USB mouse USB keyboard naica system accessory box Blue envelope naica system mouse pad Prism6 safety Information The following warning labels are located on the Prism6 instrument Caution the possibility of pinch risk Please ensure to correctly insert the tray holders onto the movable tray of the Prism6 instrument to avoid pinch risk Av...

Страница 8: ...r Manuals are subjected to changes The latest version can be accessed at the Technical Resources webpage o Stilla Technologies cannot be held responsible for any damages or injuries arising from improper operation o Do not use the Prism6 instrument if any parts are broken chipped rusty or if the power cables are damaged in any way o Do not open the housing of the Prism6 instrument if not explicitl...

Страница 9: ...ocedures provided by Stilla Technologies Do only package and ship the instrument when instructed by a Stilla Technologies representative o Regular instrument service maintenance is recommended to ensure optimal system performance at all times Please contact Stilla Technologies for the respective service offers Labeling symbols present on the Prism6 instrument Manufacturer Product reference part nu...

Страница 10: ...e disposed of in an appropriate recovery and recycling structure CE marking manufacturer s declaration that the product meets the requirements of the applicable EC directives I Power on o Power off Protective conductor terminal Labels present on the Prism6 instrument ...

Страница 11: ...ilure mode 2 USB 3 0 ports for data transfer Note Differences in USB key compatibility with the Prism6 front panel USB ports can occur Stilla Technologies recommends to use a USB key with a maximum capacity of 8Gb The compatibility of individual USB keys needs to be validated by the end user for functional data transfer 1 soft power button to turn on the instrument if it has been powered up before...

Страница 12: ... Clean indoor laboratory Temperature 15 C to 30 C Relative humidity 20 to 90 non condensing Altitude Operating at max 1000m above sea level Barometric pressure 795 hPa to 1060 hPa Noise 45 dB at a distance of 1 meter Electrical AC input 100 240V 50 60Hz Installation category II Input mains fluctuation Max 10 Input power Max 400W Additional fluorophores within the specified wavelengths can be used ...

Страница 13: ...ility Contact the Technical Support for any questions The original configuration and calibration files for the Crystal Miner software are located on the Prism6 instrument by default at C Program Files Stilla CrystalReader config Stilla Technologies does not recommend reading modifying or deleting any of the following files AnalysisConfiguration_GenericTemplate yaml AnalysisConfiguration_GenericTem...

Страница 14: ... a new experiment o Embedded drop down menu for chip specific experimental templates New Sapphire chip experiment New Opal chip experiment OPEN EXPERIMENT open a previously saved experiment file to use identical experimental settings OPEN TRAY CLOSE TRAY engagement button to open close the Prism6 instrument flap to eject retract the movable tray o If the movable tray is fully retracted this button...

Страница 15: ... Prism6 instrument flap Do not manually engage with the movable tray Do not manually pull out the moveable tray any further c Instructions for chip positioning Note Prior to chip positioning for scanning read the respective Sapphire chip and Opal chip Instructions for Use for detailed instructions on how to clean the bottom surface of each chip and for automatic chip ID barcode reading using a han...

Страница 16: ...solution rinse with water using a lint free wipe Position one of the chips Sapphire chips or Opal chips in the individual slots of the corresponding tray holder Sapphire tray holder or Opal tray holder Check for correct positioning The respective chips should fit perfectly into the individual slots Instructions to position the chips in the tray holder Example provided for Sapphire chips and Sapphi...

Страница 17: ...ip in the tray holder Each type of chip has a poka yoke corner matching the respective tray holder Note Sapphire tray holder top left corner Opal tray holder bottom right corner Ensure the respective chip type is pushed against the top right corner of the slot ...

Страница 18: ... to be used Note Make sure to use the specific Sapphire tray holder or Opal tray holder with the respective chip types only Do not operate Sapphire chips on Opal tray holder Do not operate Opal chips on Sapphire tray holder Do not mix chip types on one tray holder The tray holder is clearly identifiable by the tray holder label Instructions for loading the tray holder in the movable tray of the Pr...

Страница 19: ...n the movable tray Gently push the tray holder towards the back of the moveable tray The tray holder is correctly positioned after the spring loaded ball bearing blue circle is set in position with a distinct click Ensure the tray holder is lying flat on the movable tray ...

Страница 20: ...o set up the experimental conditions for image acquisition e Definition of a Crystal Digital PCR experiment An experiment is a set of samples processed during an individual Crystal Digital PCR workflow on the naica system o The samples of an individual experiment were all processed on the same respective chip type Sapphire chips or Opal chips Chip types cannot be mixed in a single experiment for i...

Страница 21: ...ment screen The example visualizes the experimental set up for a Sapphire chip experiment The categories on the left panel can be edited for each experiment 1 Step 1 Create a new experiment define the experiment context Each experiment starts with the selection of the scanning template ncx format for an experiment The scanning template contains all the information and components required to set up...

Страница 22: ...CR Mix Name_AssayIndicator_version ncx Examples For an experiment using the naica multiplex PCR MIX compatible with TaqMan probe and the Sapphire chip select ScanningTemplate_Prism6_SapphireChip_naica multiplex PCR MIX_Taqman_v1 0 ncx For an experiment using the naica PCR MIX compatible with EvaGreen fluorescent intercalating dye and the Opal chip select ScanningTemplate_Prism6_OpalChip_naica PCR ...

Страница 23: ... the experiment Recently used scanning templates are provided for direct selection from the displayed menu Recent files To select a different scanning template proceed to BROWSE and select the respective scanning template from the set of default scanning templates provided at the location C Program Files Stilla CrystalReader templates The user can use the chamber details default settings or load t...

Страница 24: ...e the user a faster way to set up an experiment context that will be run several times Figure 7 Crystal Reader software pop up window prompt the chamber details configuration that the user can load Figure 8 The Crystal Reader software home page for the Sapphire chips ...

Страница 25: ...fault but can be applied for the Opal chips o Enter the experiment name in the New Experiment field Note Stilla Technologies recommends establishing traceability rule for experiment naming e g date prefix chip IDs user initials objective of the experiment 20210507_04527983 04568924 04698435_ABC_valid_6plex Figure 10 Enter Experimental Details in the New Experiment interface Name the experiment ...

Страница 26: ... unselect the checkbox to include exclude a detection channel from scanning Selecting a minimum of three detection channels including blue are mandatory it is the main detection channel Note Ensure to select the appropriate detection channel for the experiment to avoid any false positive results In case of selection of a wrong detection channel re scan the chips o Go to Embedded Files selection me...

Страница 27: ...omatically use the default image analysis configuration file that is optimal for most experiments prepared by the user The specific files for different applications are available on the Monitor at this location by default C Program Files Stilla CrystalMiner config Spillover Compensation Matrix If the Spillover Compensation Matrix field is empty it is possible to load an already created spillover c...

Страница 28: ...s the threshold or polygons settings and the population configuration Figure 13 The Scanning Parameters menu o Click on the Scanning Parameters drop down to edit the exposure time of each excitation LED in ms Focus value The focus value representing the optimal z distance of the imaging plane in mm The focus value is a set value based on the original instrument calibration that do not need to be m...

Страница 29: ...the user wishes to proceed and erase the images of the already scanned chambers 2 Step 2 and 3 Select the chambers to scan Define chamber context o Click on the pencil icon located below the chip holder display to modify the selection of the chips chambers to be scanned by default the 12 chambers for the Sapphire chips and 48 chambers for Opal chips are selected Clicking on the cross icon will dea...

Страница 30: ...p code to gain time Chamber Context optional to enter additional information to make the scan data unique useful if scanning the chamber multiple times For each detection channel Blue Teal Green Yellow Red and Infra Red LED Sample Type see details below Reference Concentration if the Sample Type is S meaning that the target concentration is known the expected concentration in copies µL Dilution Fa...

Страница 31: ...mber details are displayed on the chamber button of the Tray Holder The sample ID and sample types per channel are displayed on the chamber button Note Before the scan is done the flag button is grayed in deactivated The user will be able to click on this button once the chamber has been scanned and analyzed 3 Step 4 Launch the scan o Click on the SCAN button then validate the path to save scanned...

Страница 32: ...an Already scanned chambers are displayed on the Crystal Reader software scan page interface for a visual pre analysis of chamber images while the scanning is still in progress A quality flag is added in the chamber rectangle as soon as it is ready to be explored for pre analysis Green quality flag indicates scanning finished without remarks of quality parameters Yellow and Green quality flag indi...

Страница 33: ...Note Quality flags only apply to the Blue LED channel Stilla technologies recommends to visually inspect all the other the LED channels Stilla Technologies recommends to first continue to check the scanning performance by looking at the image quality Image quality can either be inspected directly in Crystal Reader software or after opening the Crystal Miner software application Following the butto...

Страница 34: ... adjust the image contrast Click on the Reset button to reset the image contrast It is possible to navigate across the chambers using o the chip holder layout on the top right click on the rectangle of a chamber to explore it o The left right arrow buttons on the top to explore the previous next chamber o The left right top left arrows of the keyboard to explore the chamber in the specified direct...

Страница 35: ...tal Reader software o The Grayscale Histogram tool bottom right might help you to appreciate the distribution of pixel intensities expressed in RFU Relative Fluorescence Unit where 0 RFU indicates black and 216 1 65535 RFU indicates saturation Figure 23 Step 5 Use the histogram to place intensity cursors and reveals the pixels corresponding to the RFU interval in the Crystal Reader software ...

Страница 36: ...Figure 24 Overlay of the saturation map in the Blue detection channel in the Crystal Reader software saturated pixels are displayed in orange color In this case it is a dust particle CAUTION Ensure that all the pixels located in the droplets are not saturated in any channel otherwise the saturated droplets will be considered as not analyzable by the software and will be rejected from the analysis ...

Страница 37: ...tionalities for more details about chamber rescan In case of artifacts such as dust yellow flag for the number of saturated objects not due to saturation of droplets clean the back surface of the chip by slowly sliding it 1 time on the length directly on a cleaning wipe delivered with the respective chips and rescan The saved experiment can be now analyzed with Crystal Miner software please refer ...

Страница 38: ...re highlighted in blue color For this scenario click on the validation icon and the scan will start For this scenario unselect the previously scanned chambers and activate the new ones to be scanned For this scenario unselect the previously scanned chambers to be kept and click on the new ones desired to be added to the experiment 2 How to edit the Settings for different user profiles o From the H...

Страница 39: ...n in the Experiment Details form Output Storage Path default storage path to save the ncx experiment files Username chosen username Password chosen password Prism6 Settings Focus Value mm z distance to the imaging plane used for image acquisition set by the user Prism6 Serial Number SN unique serial number of the Prism6 set by Stilla Technologies Focus Calibration button advanced functionality to ...

Страница 40: ... logged user has the same rights as a non logged one except that his her user preferences for the input output storage directories will be saved even after quitting Crystal Reader software He she is also allowed to modify his her password Unless modified after software installation a default Manager account is created with the login manager and the password manager it is recommended to change this...

Страница 41: ...uorophore names Modify the default min max and step values for the z values to be tested during the Focus Calibration for all chip models o In the path fields USERPROFILE can be used this is a Windows environment variable that redirects to the path of the current Windows session s user For example if the user is logged in Windows with the account John USERPROFILE has the following value C Users Jo...

Страница 42: ...crystal and to place this chip on the middle slot of the chip holder i e slot 2 For optimal focus calibration on the Opal chip it is recommended to use three chips including chambers with good quality droplet crystal in positions A D and H Select the chamber s of interest i e the chamber for which the optimal acquisition focus needs to be calibrated by clicking on the chamber rectangle in the chip...

Страница 43: ...ers as well as the chamber image of the first selected chamber at this optimal focus A table with all the computed sharpness scores for each selected chamber and each tested z value is also provided together with a graph showing the sharpness score for each chamber as a function of the tested z value o The user may select the other tested focus values by clicking on them in the table to check the ...

Страница 44: ...eader software User Manual 43 Figure 31 Example of the result of focus calibration for the Opal chip in the Crystal Reader software 4 How to Pool Unpool Chambers Figure 32 Pooling feature of the Crystal Reader software ...

Страница 45: ... select these chambers in the chip layout using Ctrl Click or Shift Click and then click on the POOL CHAMBERS button resp UNPOOL CHAMBERS button in the Chamber Details tab Fig 14 All chambers pooled together will automatically share the same Pool ID displayed as an incremented number in the chip layout as well as the same sample name chamber context and for each detection channel the same sample t...

Страница 46: ...swer as promptly as possible If applicable Stilla Technologies offers to engage for remote troubleshooting by using the shared desktop application Team Viewer which is installed on the Prism6 monitor To enable remote software maintenance on the Prism6 monitor provided by Stilla Technologies check that the Prism6 monitor is turned on and connected to the Internet Then double click on the Team Viewe...

Страница 47: ...re for decontamination of the Geode please view the Decontamination Protocol Disposal of naica system equipment The disposal of the Prism6 at the end of the product s life should comply with the current legislation in force in the country of use regarding electrical and electronic waste The Prism6 instrument should be packed and shipped back to Stilla only after a Stilla Technologies Service Speci...

Страница 48: ...form a scan Error message Prism6 instrument configuration not applicable with detection channel selection Depending on the Prism6 instrument configuration up to 6 detection channels are available H24000 3 3 color Prism6 configuration with the detection in Blue Green and Red channels H24000 6 6 color Prism6 configuration with detection in Blue teal Green Yellow Red and Infra red channels Please con...

Страница 49: ...ferent number of channels than the current experiment Please check the consistency of these elements Please use the scanning templates provided by Stilla Technologies accessible at https www stillatechnologies com technical resources naica system prism6 If these observations persist please contact Technical Support ...

Страница 50: ...ht corner of the chamber There are too many droplets within the chamber Stacked droplets are considered artifacts and thus excluded from the analysis To relax the crystal bring back the chips to the Geode In the menu select Template Protocols and launch the program named Sapphire Protocol Unpacking Droplets After the reading step the neural network of Crystal Miner should recognize more droplets D...

Страница 51: ...roplets There are two or more droplets crosses instead of one oversegmentation or one droplet instead of two or more undersegmentation which may lead to false positive or false negative results Manually inspect the droplets in case of red crosses the software automatically rejests the droplets no action is required However in case the crosses are green then manual exclusion of the crosses is neces...

Страница 52: ... the exposure time usually by a factor 2 set for the detection channel where saturation is observed and re scan the experiment Shiny aggregates within droplets The PCR mix can aggregate when it has been heated up for too long Optimize the PCR conditions decreasing denaturation times and or the PCR master mix concentration with a scan rescan It is also possible to use the Template PCR program FAST ...

Страница 53: ...top of the inlet port or between oil and PCR mix during the pipetting step Check the expiry date of the used chips listed on the box Make sure the white PCR caps are on the inlet port during PCR Image stitching event The Crystal Reader software stitches several images of the chamber taken subsequently into one final image Droplets along the stitching lines are occasionally excluded to avoid over u...

Страница 54: ...chip holder is positioned flat A foreign particle might be present between the chip and the holder forcing the chip to be tilted Please clean the foil of the chip and the holder with a lint free wipe and re scan the concerned chamber The focus value is not well calibrated Refer to the How to perform focus calibration section and re scan the concerned chamber ...

Страница 55: ... if the number of analyzable droplets is sufficient Presence of different sizes of droplets in the chamber polydispersity The procedure using the antistatic spray was omitted or not properly performed The chamber is still analyzable as the partitioning step was successful Depending on the assay it is the user discretion if the number of analyzable droplets is sufficient Please refer to the IFU for...

Страница 56: ...ps The Geode is not tilted enough front back axis or is tilted in the wrong direction Contact Technical Support if unsure about the correct positioning of the Geode The small and defined population of droplets with different sizes The population is often positioned close to the chip injectors and it is composed of droplets often smaller than the normal ones The normal cycling of temperature and pr...

Страница 57: ... properly place the Opal chip PCR caps During the loading of the chips potential multiple droplets could have generated Those droplets were probably not merged properly using the electrostatic pen Make sure that the electrostatic pen is working For more information about how to use the electrostatic pen with the opal chips refer to the IFU for Opal chips The partitioning step did not occur Check t...

Страница 58: ...en the inflated pouch upon reception If the inflated pouch containing the chips was not opened upon reception and the chips are still under the expiry date it is recommended to open the inflated pouch and to keep the chips at atmospheric pressure for at least 2 weeks before using them Presence of a large droplet coalescence area in the chamber The procedure using the antistatic spray was omitted o...

Страница 59: ...ible traces on the chamber The traces could be due to the partial drying of the antistatic spray Repeat the procedure of cleaning the Opal chip foil with a lint free tissue sprayed with the antistatic spray Ensure to completely dry the chip and rescan ...

Страница 60: ...he software version o From the HOME page of the Crystal Reader software click on the ABOUT button The software version as well as the third party license information will be displayed Figure 34 The About page of the Crystal Reader software 3 Third party licenses The Crystal Reader software uses the following third party software components Boost Catch CMake CNTK cuDNN Docopt cpp Eigen ETL GLC Lib ...

Страница 61: ...rty websites or check the Crystal Reader software and Crystal Miner software installation directory by default in C Program Files Stilla CrystalMiner licenses Note The license information of all the third party software components is also accessible in the Crystal Reader software by clicking on the About button on the Home page ...

Страница 62: ...s Mathé 94800 Villejuif FRANCE Phone 33 09 82 29 50 50 North American Office Stilla Technologies Inc 66 Cherry Hill Drive Suite 110 Beverly MA 01915 USA Tel 1 833 888 0150 For sales related queries please contact our local office in your region or refer to www stillatechnologies com Pat www stillatechnologies com patents MKT 00089 Rev A ...

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