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Table of Contents

Introduction
Welcome to Q-View
Terminology
Projects

Quick Start Guide

Q-View Installation
Software Installation
Imager Installation
Q-View Imager Pro Notes

Image Processing
Image Acquisition
Import Images
Manage Images
Align Plate Overlay
Auto-Set Plate Overlay
Auto Spot Finding

Well Assignment
Assigning Wells in Q-View
Assigning Wells in a Spreadsheet
Templates

Data Analysis
Evaluating the Quality of the Curve Fit
Curve Fit Options
Regression Models
Weighting
Negative Well Subtraction
Templates
Viewing and Customizing the Data
Mask Outliers
Statistics
Assay Limits
Error Codes
Export
Q-View Imager Qualification
Settings
Perform Qualification
Reports
Recertification
Build Ranges

Imager & Software IQ/OQ
Settings
Perform Qualification
Reports

Blot Tools
Selection Options
Illuminance Plot
Blot Report

Administrative
Software and Product Updates
Settings
Uninstalling the Software

Support
Technical/Troubleshooting
Contact Quansys

Introduction
Welcome to Q-View Software

Figure 1. Q-View workflow.
Q-View™ Software is a tool for the quantitative analysis of multiplexed chemiluminescent or infrared fluorescent planar-based arrays. Analysis is performed in three easy steps (Figure 1), each covered in detail in this manual:
Terminology
Projects

Figure 2. Multiple projects opened.
Q-View Software stores the images and analyses of related plates together in a file, called a Project, with the "Q-View" extension. As you work, changes to these projects are saved automatically. Multiple projects can be open at the same time (Figure 2).

Before version 3.1, projects were stored as folders. Project folders can still be opened by the software but they will be automatically upgraded to the project file format when opened. Project files are specific to the Q-View Software and cannot be viewed or manipulated by 3rd party software.

Project details can be accessed by opening Project > View Project Details. The project creator, creation date and a list of users that have opened the project along with associated time stamps are recorded.

Project notes can be recorded and viewed by opening Project > Project Notes. These notes allow users to record read-only messages related to the project.

Quick Start Guide
  1. Installation
    1. Install Q-View Software on any number of computers.
    2. Set up your imager and any other equipment to be used for the experiment.
    3. Using a Q-View Imager, ensure the imager has been focused, light leak checks are complete, dark field images have been captured, and set-up is complete before proceeding. All these procedures can be found in the Q-View Imager Pro and LS manuals.
  2. Run and image the Q-Plex Kit
    1. Run the Q-Plex Kit according to the instructions in the kit manual.
    2. Capture the image(s) using a Q-View imager or other compatible, third-party imager.
      With third-party imagers, Only grayscale, TIFF and CR2 image files of at least 12 bits per pixel should be imported for scientific reporting purposes. Images with a white background must be inverted by clicking Image Options > Invert Image.
  3. Analyze images in Q-View Software
    1. Enter the Software Product Code (found on the kit product card) for each plate.
    2. Within Image Processing, set the plate overlay.
    3. Perform Well Assignment.
    4. Within Data Analysis, click Perform Analysis to generate charts and reports. Customize and export the results as desired.
Installation
Software Installation
  1. Ensure the computer meets at least the minimum specification:
    • Windows 7, 8 or 10
      *the Windows version of Q-View is required to operate Q-View imagers
    • Mac OS 10.11 or newer
    • 2 GB RAM
    For optimal performance:
    • 3+ GB RAM
    • 2 or more processing cores
  2. If you do not yet have a Q-View Software installer file, contact a representative from Quansys by email at sales@quansysbio.com or call 1-888-782-6797.
  3. Launch Q-View Software. Click either Evaluate Q-View to start a 60 day trial or enter your assigned company name and license code and click Continue. Follow the prompts on the screen to set up user accounts and Q-View Imagers. You can change these settings later from the Settings menu.
Imager Installation

Figure 3. USB and power sockets.
  1. Carefully unpack the Q-View imager and place it on a clean work area.
  2. Plug the power cord into the back of the imager (Figure 3) and into a surge protector.
  3. Plug the USB cord into the back of the imager (Figure 3) and into the computer. For the Q-View Imager Pro, ensure the power switch is in the on position.
  4. Clean the imager glass with a lint-free cloth and ethanol, rubbing alcohol, or window cleaner.
  5. Launch Q-View Software and open Settings > Administration > Manager Imagers to detect, name and focus the imager and capture a dark field. Dark field capture takes 5 minutes on the Q-View Imager LS and 15 minutes on the Q-View Imager Pro.
Q-View Imager Pro Notes
Image Processing
Image Acquisition

Figure 4. Image acquisition options.
  1. Place the plate in the Q-View imager, close the lid/tray, and click Acquire Image. Select the desired imager, processing method, exposure time(s) and image name(s) (Figure 4). If the desired imager is not listed, click Refresh to scan for imagers.

    Beginning with version 3, Q-View Software utilizes an image processing method called Standard processing. This processing method is recommended for most experiments due to improved curve fitting. Legacy processing is the method used up through Q-View Software 2.17, and should only be used when comparing pixel intensities between projects created with Q-View Software version 2.

    The recommended exposure settings for Q-Plex kits are:
    • Q-View Imager Pro: 300 seconds
    • Q-View Imager LS, Standard Processing: 270 seconds
    • Q-View Imager LS, Legacy Processing: 30, 60, 180 seconds (stacked)
  2. Capture the image(s) by clicking Capture Image. You can continue to Well Assignment while images are being captured.

  3. Figure 5. Plates organized by tabs.
    Add a plate tab for each physical plate to be imaged by clicking the "+" tab (Figure 5). Custom plate names can be set by double clicking the title.
*The 'Enable Internal Illumination' option is available for Q-View Imager Pro models when activated in Settings > Preferences. When enabled, the imager's internal focus lights are turned on during image acquisition. This may be useful for quality control records but should not be enabled when capturing Q-Plex images.

Import Images
Before importing an image, ensure that it is grayscale and has a pixel depth of at least 12 bits. Q-View Software can open TIFF, CR2, BMP, PNG and JPEG files. Low bit depth and lossy images (BMP, PNG and JPEG) are insufficient for analysis and should be imported for display purposes only.

To import an image, click Image Processing > Import Image or drag and drop image files onto the list of images.
Manage Images

Figure 6. Image drop-down menu.
Images can be renamed, deleted or stacked by right-clicking the image name (Figure 6). Image details can be viewed by selecting properties. Click Export to save an image as a lossless TIFF or compressed JPEG. JPEG files are suitable for display purposes only and should not be used for analysis.
Align Plate Overlay
  1. Enter the Software Product Code from the product card into the Product Code field.
  2. Use Image Options to adjust image Gamma, Zoom, and orientation as desired. Images with black spots on a white background need to be inverted by clicking Image Options > Invert Image.

  3. Figure 7. Overlay Options.
    If you are using the Auto-Set Plate Overlay feature (explained below), the initial overlay position will be set automatically. Otherwise, click Overlay Options > Set Plate Overlay, and click and drag the cursor from the center of the top-left well to the center bottom-right well. Use the following Overlay Options (Figure 7), if needed, to manually adjust the overlay:
    • Adjust Plate Overlay: Pivot or expand/contract the entire overlay via clicking and dragging the corner wells highlighted in blue.
    • Adjust Spot and Adjust Well: Click and drag to move either an individual spot in the overlay, or all spots in a well.
    • Auto-Adjust Spots: Q-View Software automatically adjusts all spots to improve the alignment of the overlay.
    • Reset Spots: Reset all spots in the overlay to their default locations.
    • Hide Plate Overlay: View the image without the plate overlay.

Figure 8. Plate overlay.
Auto-Set Plate Overlay
When using a Q-View imager, the Auto-Set Plate Overlay feature, found in Settings > Preferences, can be enabled to have the software provide the initial plate overlay position. This feature is activated after manually setting a plate overlay for the first time. Once activated, the Auto-Set Plate Overlay feature will automatically set the overlay onto all captured images as soon as the product code is entered. Be sure to review the accuracy of the overlay placement for each captured image.
Auto Spot Finding
When using a Q-View Imager Pro or Q-View Imager LS with Q-Plex plates, Auto Spot Finding will be active and will make it easier to accurately set the plate overlay either manually or via the Auto-Set Plate Overlay feature.

Well Assignment

Figure 9. Well assignment.

Assign well types, names, and dilution factors in either Q-View Software or a spreadsheet program such as Microsoft Excel. Use Templates to quickly assign repeat layouts.
Assigning Wells in Q-View
To assign well types and names, select the desired wells by clicking and dragging and then click Groups (Figure 10). Hold the control key to select discontiguous groups of wells. Enter custom names if desired, and then click one of the well types defined below to register the assignment.
Figure 10. Group tools.
Calibrators may be assigned dilution factors or grouped by predefined concentrations for each analyte. Predefined concentrations are created by Quansys and assigned by the user by selecting a well group, clicking the Calibrator button (Figure 10) and then choosing from a list of group names in a subsequent pop-up window.

Use Sequential Naming to automatically name groups of wells with sequentially increasing suffixes.


Figure 11. Dilution tools.
To assign dilution factors (Figure 11), select the desired wells and click the Dilutions button. Enter the dilution factor of the initial dilution in the Initial Dilution field. For example, enter the number "2" for a 1:2 (50%) dilution. If a dilution series was continued from that initial dilution, enter the series dilution factor. For example, to indicate a 1:10 dilution series from the 1:2 (e.g. 1:20, 1:200, 1:2000), enter "10" in the Dilution Factor field. Press any of the directional buttons, e.g. Top to Bottom, to register the assignment.
Assigning Wells in a Spreadsheet
To assign well types, names, and dilutions in spreadsheet software such as Microsoft Excel, click Templates > New Template, then name and save the newly created template file. Once saved, a blank well assignment template will open in the default spreadsheet software. Fill this out and save your changes. Apply the template in Q-View Software as described below.
Templates
To save your current well assignment as a template, click Templates > Save Template. To apply a previously saved template to a plate, either click Templates > Open Template, or drag and drop the template file onto the plate diagram.

Data Analysis
Once you have completed Image Processing and Well Assignment, select Data Analysis. Click Perform Analysis to generate charts, concentrations, and statistics based on optimal regression settings provided by the product definition. Click the Curve Fit Options button to customize regression models, weighting, and negative well subtraction settings.
Evaluating the Quality of the Curve Fit
Backfitting of the calibrator is a practical method for evaluating the quality of the curve fit. This is done by comparing the calculated concentration of each calibrator to its known (theoretical) concentration using the equation: calculated concentration / theoretical concentration x 100%. The Q-View Software displays this result in the report as Percent Backfit. The preferred result for each calibrator point is backfit of 80%-120%, although 70%-130% may be acceptable.
Curve Fit Options

Figure 12. Curve fit regression options.
The default 4PL or 5PL regression models are generally recommended for normal Q-Plex data, as these are widely considered to provide the best overall fit for immunoassay curves. If your data is abnormal or you are trying to optimize the fit for a particular range, one of the other regression models, along with masking, may better fit your needs. You can choose from the following Curve Fit Options (Figure 12):

Regression Models In products with spot replicates, replicates within each well are averaged and treated as single data points. Calibrator curves for assays with spot replicates are fit to the replicate average using the specified regression model.

Calibrator curves for our High Dynamic Range Singleplex assays are automatically fit using the HDR algorithm irrespective of the chosen curve fit option.


Figure 14. Curve fit weight options.
Weighting

Weighting is used to reduce the impact of outliers and offset heteroscedasticity. In ELISA calibrator curves, the residuals tend to increase as the concentration of the calibrator (Y) increases. General weighting (GW) can be applied to normalize the residuals across the curve. Weighting based on residuals (Res), reduces the impact of outliers on the curve fit. The larger the number associated with either weighting method, the more variation there will be between the impact of the calibrator points. The available weighting options are shown in Figure 14.

Negative Well Subtraction


Figure 15. Curve fit templates.
Negative Well Subtraction automatically deducts the average pixel intensity of the negative wells from other wells. The default state, on or off, can be changed in Settings > Preferences. Turning off this setting can be useful when comparing absolute pixel intensities.

Templates

Custom curve fit settings can be saved in the Templates section (Figure 15) of the Curve Fit Options window. To save your current settings as a template, click Save Template. To apply a saved template to a plate, click Open Template or drag and drop a template file onto the Curve Fit Options table. The Reset Optimal button will reset to the optimal settings as defined by the product code.
Viewing and Customizing the Data
Concentrations, pixel intensities, and statistics are displayed based on the selected Data Analysis tab (Figure 16), spot masking, and settings in the Statistics and Assay Limits menus.

Results are organized into three tabs for each plate:
Figure 16. Data Analysis tabs.
Other values that may appear in the Report and Data Tabs are:
Mask Outliers

Figure 17. Masking spots.
Select and right-click (Figure 17) on data points within the Chart, Report or Data tab to Mask (exclude from all calculations) or Unmask spots and wells.

Masking outliers in the calibrator curve is recommended because the accuracy of the regression model affects all calculations. Consider masking a calibrator curve point if the %CV between replicates is greater than 30%, or if the average %Backfit is outside 80% – 120% (these are viewable in the Report tab when selected in the Statistics menu).

Q-View can automatically identify and mask outliers in calibrator curve data. To enable this feature, open Settings > Preferences and select Mask calibrator outliers by default. When you unmasked an auto-masked outlier, Q-View will not auto-mask the point again when fitting curves until you right click a chart or report and select Mask Outliers (Figure 17) from the dropdown menu.
Statistics

Figure 18. Statistics and Assay Limits.
Click Statistics (Figure 18) to select which calculations are shown.

If multiple dilutions of a sample were present, select Auto-Select Dilution to view only the data for the dilution that fell on the most linear part of the calibrator curve, instead of data for every dilution of the sample.
Assay Limits
Click Assay Limits (Figure 18) to modify how extrapolated data is displayed:
Error Codes
The error code column in the report alerts you to potential problems with your assay. Error codes are defined at the bottom of the report and include: Assay Controls (AC), Process Controls (PC), Spot Overlay Out of Bounds (SB) and Masked Outliers (MO).
Export
Click Export to save a chart as an image, or the currently displayed values in the Report or Data tabs as a spreadsheet (CSV).

Q-View Imager Qualification
The Q-View Qualification Plate produces a constant amount of light at intensities across the dynamic range of the Q-View Imager. Qualification captures images of the reference plate and evaluates performance based on defined specifications. Performing qualification on a regular basis ensures that the Q-View Imager consistently meets performance specifications.
Settings

Figure 19. Q-View Imager Qualification menu.
Q-View Imager Qualification settings (Figure 20) can be accessed from the Qualification > Q-View Imager Qualification > Settings menu (Figure 19). The settings are accessible to only admin users.
Perform Qualification

Figure 21. Image capture is restricted.
Power on the Q-View Qualification Plate and place it in the imager face down in the lower right corner of plate deck. In the Q-View Software, select Qualification > Q-View Imager Qualification > Perform Qualification from the menu (Figure 19) or click Q-View Imager Qualification has expired (Figure 21) from the Image Acquisition screen. Confirm that the serial number on the Q-View Qualification Plate matches the serial number in the dialog box. The process takes about 5 minutes to complete.
Reports
To view and export qualification reports, select Qualification > Q-View Imager Qualification > Reports (Figure 19) from the menu.
Recertification
Qualification plate certification expires annually. The expiration date is shown on the back of the plate. Once certification has expired, please contact Quansys Biosciences to arrange to have your plate recertified. Once your plate has been recertified and returned to you, it is necessary to rebuild the qualification ranges.
Build Ranges
To build qualification ranges, open Qualification > Q-View Imager Qualification > Settings > Build Ranges and follow the instructions provided by the software.

Imager & Software IQ/OQ
IQ/OQ testing ensures that the installation and operational functions of the imager and software are performing correctly. These tests can be used upon initial evaluation and subsequent evaluations to ensure that the imager and software continue to operate as expected. Following the IQ/OQ testing, a report is generated that may be used for regulatory compliance and equipment control.
Settings

Figure 22. Imager & Software IQ/OQ menu.
Imager & Software IQ/OQ settings (Figure 23) can be accessed from the Qualification > Imager & Software IQ/OQ > Settings menu (Figure 22). The settings are accessible to only admin users.
Perform Qualification
In the Q-View Software, select Qualification > Imager & Software IQ/OQ > Perform Qualification from the menu (Figure 22) or click Imager & Software IQ/OQ has expired (Figure 24) from the Image Acquisition screen. The process takes about 15 minutes to complete.
Figure 24. Image capture is restricted.
Reports
To view and export qualification reports, select Qualification > Imager & Software IQ/OQ > Reports (Figure 22) from the menu.

Blot Tools
Click Support > Legacy Blot Tools to make custom selections for Western blots, Northern blots, Southern blots, dot blots, and other chemiluminescent electrophoresis gel images. Click Close to return to the previous Image Processing screen.
Selection Options
Click Selection Options and choose one of the following tools to make a custom selection (Figure 25).


Figure 25. Blot tools.
Auto Select (Figure 26): Automatically select a group of similar, contiguous pixels by clicking on the center of the grouping. The number of shades included in the selection correlates to the sensitivity value (0-100) entered in the Sensitivity field.

Rectangle and Ellipse: Create a rectangular or circular selection by clicking and dragging the location where the selection is to be placed.
Figure 26. Auto select tool. On the top, only the outer edge is selected, whereas on the bottom, the entire spot is selected. The light red fill indicates the selection boundaries.


Polygon: Draw a custom, straight-edged selection by clicking on the image where each vertex is desired. Right-click to close the selection boundaries.

The selection border is indicated by a bright red border and filled with translucent red while unselected areas are indicated by a translucent gray fill. Make sure the intended pixel grouping is selected, rather than just a ring around the grouping (Figure 26). Selections can be moved by dragging, cleared with Clear Selection, and exported as a JPEG by clicking Export As Image.

Manipulate the selection boundaries by clicking and dragging, or use the following keyboard commands.

Tool Arrow keys move selection Alt + arrow keys to lengthen/shorten shape +/- keys to resize
Auto Select
Rectangle
Ellipse
Polygon
*Hold shift to amplify actions.
Illuminance Plot

Figure 27. 3D illuminance plot.
To create a plot of the illuminance of a selected area (Figure 27), click View 3D Plot. The 3D plot will appear in a new window. The plot can be viewed at any angle by clicking and dragging. Hold control while dragging to move the plot. Roll the mouse wheel to zoom in and out. Various plotting options are available on the right side of the window.
Blot Report
The Blot Report is an exportable table that includes custom names, number of pixels selected, mean pixel intensities, and standard deviations. To add a selection to the table, click Add to Report. You can add as many entries as you want. The information for the current selection is shown at the bottom of the drop-down. The table can be viewed and edited at any time by clicking View Report.

To view the pixel coordinates and intensities for a selection, click the blue # Pixels label.

Administrative
Software and Product Updates

Figure 28. Q-View Software update options.
Both software and product updates can be obtained automatically if set to do so in Settings > Administration > Q-View Updates (Figure 28). If updates are not occurring, check that Settings > Administration > Network Settings are correct.

If your computer does not have Internet access, contact Quansys at sales@quansysbio.com or 1-888-782-6797 to arrange for regular software updates to be sent to you.

To apply product updates manually, go to Settings > Administration > Q-View Updates and select Update product definitions manually, then click the Update product definitions from file button. A file selection window will appear. Navigate to the product definitions file you received from Quansys and select Open.
Settings

Figure 29. Administrative options.
Use the options under the Settings menu as follows (Figure 29):
Uninstalling Q-View Software
Q-View Software can be uninstalled through your computer’s control panel or by opening the Q-View Software Installer and clicking Remove.

Support

Figure 30. Software
Help button.
This manual is available in the software by clicking the Help button (Figure 30), under Support > Q-View Software Manual, and online at www.quansysbio.com/manuals.
Technical/Troubleshooting
Tips for many aspects of using multiplex ELISA's are available online at www.quansysbio.com/tech-tips.

Technical support data such as projects and imager databases can be sent to Quansys Biosciences directly from the Q-View Software by opening Support > Technical Support Upload and selecting either Upload Project or Upload Imager Database. If your PC running the Q-View Software is not connected to the Internet, the upload options will prepare the data for manual upload which is accomplished by moving the produced file(s) to a computer with Internet access and visiting support.quansysbio.com.
Contact Quansys
To contact Quansys, please:
Call us at 1-888-QUANSYS (888-782-6797)
Chat with us online at www.quansysbio.com
Email us at sales@quansysbio.com for software or other product requests
Email us at techsupp@quansysbio.com for technical and troubleshooting issues
Email us at info@quansysbio.com for other general inquiries