I.DOT User Manual

FOR RESEARCH USE ONLY

Not for use in diagnostic procedures.

Intellectual Property Rights

This I.DOT One System manual and its contents are the property of Dispendix GmbH ("Dispendix"). It is intended solely for the use of Dispendix customers for operating the I.DOT One System and its consumables. The I.DOT System, including its software and hardware components, and this user manual are subject to patents, copyright, trade secret rights, and other intellectual property rights owned by or licensed to Dispendix. No part of the software or hardware may be reproduced, transmitted, transcribed, stored in a retrieval system, or translated into other languages without prior written consent from Dispendix.

Limited License

Subject to the terms and conditions of the I.DOT One System, Dispendix grants a limited, nonexclusive, non-transferable, non-sublicensable, research use only license to use the proprietary I.DOT One System in accordance with the manual and other written instructions provided by Dispendix.

Table of Contents

• 1. Preface
• 2. Setting Up Dispensing Runs
  • 2.1. Protocol Settings Setup
  • 2.2. Target Layout Setup
    • 2.2.1. Dispensing Options
    • 2.2.2. Edit Well groups
    • 2.2.3. Copy Well groups
  • 2.3. Source Plate Setup
    • 2.3.1. Enable source liquid duplicate popup
  • 2.4. Overview
  • 2.5. Dispense
  • 2.6. Re-dispense Missed Droplets
  • 2.7. Results
• 3. PrintLog
• 4. Comma-Separated Values (CSV) File
  • 4.1. Header Structure
  • 4.2. Task Section
• 5. Advanced User Settings
  • 5.1. Liquid and Liquid Class Library
    • 5.1.1. Liquid Library
    • 5.1.2. Liquid Class Library
  • 5.2. Creating a New Liquid Class
    • 5.2.1. Automated Creation of a new Liquid Class
    • 5.2.2. Manual Creation of a Liquid Class
  • 5.3. About Plate Format Library
  • 5.4. About Advanced Device Settings
    • 5.4.1. Reset Liquid Classes

1. Preface

Purpose

This user manual details specific information pertaining to the safe and proper handling and operation of the I.DOT low volume non-contact dispensing system and I.DOT consumables.

Conventions Used

• ⚠️ Hazard Note: Indicates a potential hazard to personal safety or potential for equipment damage.
• ℹ️ Important Note: Presents essential content indicating a potential for assay failure, diminished data quality, and/or a loss of data if the information is ignored.
• ? Tip: Information contained in a tip may offer helpful suggestions, alternative procedures, methods, or shortcuts.
• Bold text: When appearing in text or a procedure, bold text highlights a specific button, key stroke, or menu option.
• Italic text: Used to emphasize an important word or expression within the text, or to indicate the title of a book, journal, or other documentation, or the special or unusual meaning of a word or phrase.

2. Setting Up Dispensing Runs

This chapter describes how to set up the target and I.DOT source plates, prepare the instrument, start, and analyze a dispense run.

When opening the I.DOT Assay Studio software, it prompts to open a saved protocol or create a new protocol.

[Figure 1: Starting screen of I.DOT Assay Studio software, showing options to open or create a protocol.]

The top bar of the software screen guides through each single step of a dispense run, highlighting the current step in blue color.

2.1. Protocol Settings Setup

Choose "Create New Protocol" to access the Protocol Settings screen (as shown in Figure 2).

[Figure 2: Protocol Setup screen displaying various settings.]

Editable Items:

• Enable/disable the deionization function.
• Enable/disable the "Dispense to waste" function (used for priming source wells to ensure no air bubbles). This includes:
  • Adjusting the volumes for priming of each source well.
  • Adjusting the number of priming cycles for each source well.
• Edit the error handling behavior if no droplets are detected during priming (e.g., Ask, Abort, Continue).
• Edit the optimization level to reduce dispensing time (e.g., by reordering steps and using parallel dispensing).
• Edit the "Save liquids in local liquid library" setting.

Press "Ok" to proceed to the Target Layout setup.

Protocol settings can be modified at any time by navigating to "Menu > Settings > Protocol settings".

2.2. Target Layout Setup

This section covers setting up the target plate layout.

[Figure 3: Target Layout Setup screen showing a plate grid and options.]

Editable Items:

• Optional: Enter a Plate Identifier.
• Optional: Enter Plate Barcode ID.
• Select the target plate format (e.g., MWP 384).
• Optional: Select a Waste Well position if waste dispense is enabled.

Press "Ok" to confirm.

Target plate properties can be edited at any time by clicking the ⚙️ button in the top-left corner of the Target Layout window.

The barcode reader is available and can be enabled in "Advanced Device settings" (see chapter 5.4). The barcode of the source and target plate can be scanned if it is on the left side of the plate. Click the ?️ button to scan.

Target Well Selection

Select wells for liquid dispensing by clicking on desired positions in the Target Layout tab. Selected wells are highlighted in blue and form a "Wellgroup".

[Figure 4: Target layout screen with selected wells highlighted in blue, forming a well group.]

Adding Additional Target Plates:

• Click the ➕ button to add a new, empty target plate layout.
• Click the ? button to duplicate an existing target plate layout. A popup allows selection of which plate to duplicate and where (New target plate, New protocol, or Existing protocol), as shown in Figure 5.

[Figure 5: Duplicate Target Plate popup window allowing selection of plate and destination.]

Arrow buttons allow switching between different target plate layouts.

2.2.1. Dispensing Options

The I.DOT Assay Studio software offers four dispensing options, indicated by icons:

• Constant: A simple liquid transfer for dispensing a specific volume or concentration.
• Dilution: Creates direct dilutions within a selected well group.
• Normalization: Equalizes filling levels of target wells to the same volume.
• Array: Creates arrays of spots within one or multiple wells.

[Figure 6: Dispensing option "Constant" selected, showing parameters like volume and concentration.]

When dispensing by concentration, the software calculates the required volume based on total assay volume, liquid stock concentration, and desired target concentration. Total assay volume can be set for the entire plate or for individual well groups.

The "Dilution" option allows setting volume/concentration start and end points for linear or logarithmic distribution, adding "0" steps, choosing distribution types, adding replicates, and changing dilution patterns.

[Figure 7: Dispensing option "Dilution" selected, showing parameters for dilution series.]

The "Normalization" option equalizes filling levels. Users can choose to normalize to the highest fluid volume, a specific volume, or a specific percentage of the assay volume.

[Figure 8: Dispensing option "Normalization" selected, showing normalization choices.]

The "Array" option allows creating arrays of spots within wells by defining ID, size (X/Y), pitch (X/Y), and location.

[Figure 9: Dispensing option "Array" setup screen, showing parameters for array size and position.]

[Figure 10: Defining dispensing options within an array, showing the selected array pattern.]

[Figure 11: Final array displayed in the target layout.]

Note: Mixing, Dilution, Normalization, and Array operations affect all wells within the selected area of a well group.

2.2.2. Edit Well groups

To edit a well group, select it from the list on the left. Click within the well group on the target plate layout; blue squares will appear at the corners.

• Adjust size: Hover over blue squares until they turn red, then click and drag.
• Adjust position: Click and drag the middle of the well group.
• Remove wells: Drag them to the bin or press the "Del" key.

[Figure 12: Assigning target wells, showing selection and modification of well groups.]

2.2.3. Copy Well groups

Copy entire well groups (including dispensing option, volume, and liquid) by clicking the ?️ button for the respective well group. Select the desired liquid and location for the new well group.

[Figure 13: Copy well group setup screen, showing options to select liquid and destination.]

2.3. Source Plate Setup

After defining the target layout, navigate to "Source Layout" in the top bar to proceed.

[Figure 14: Source Layout-Setup screen, showing plate configuration and liquid assignment.]

Editable Items:

• Optional: Enter a Plate Identifier.
• Optional: Add the Plate Barcode ID.
• Select the source Plate Type (details in chapter 5.1.2).

Press "Ok" to confirm.

Source plate properties can be edited via the ⚙️ button. Additional source plates can be added via the ➕ button.

Unassigned liquids required for the protocol are listed on the left side of the screen.

[Figure 15: Source Layout-Setup screen focusing on Unassigned Liquids.]

Liquids are assigned by manually dragging them to the desired position on the source plate or automatically using "Auto Assign All". Assigned liquids move to the "Assigned liquids" section on the right.

[Figure 16: Source Layout-Setup screen showing Assigned liquids.]

Liquids can be assigned individually by dragging or by selecting multiple liquids and dragging them together.

[Figure 17: Multi-select feature for assigning liquids, showing selection and assignment actions.]

The initial fill volume of each well can be adjusted manually or by percentage to account for dead volume or prevent wells from running empty. Changes applied to selected wells will be applied to all selected wells.

[Figure 18: Source plate setup screen showing fill volume adjustment.]

[Figure 19: Source plate setup screen showing confirmation prompt for fill volume changes.]

After defining the source plate layout, click "Overview" in the top bar.

2.3.1. Enable source liquid duplicate popup

Enabling the "source liquid duplicate popup" (highlighted in Figure 20) allows defining the number of source wells for a liquid during source layout setup.

[Figure 20: User interface settings screen, with "Enable source liquid duplicate popup" option highlighted.]

When liquids are in the "Unassigned liquids" section, clicking the ?️ button next to a liquid opens a popup (Figure 21). This popup shows the number of source wells for which the liquid volume will be averaged.

[Figure 21: Source liquid duplicate popup window.]

2.4. Overview

The "Overview" section displays a simulation mode to review protocols. It shows the total dispensing time and number of dispensing steps.

Clicking "Start Simulation" allows monitoring all dispensing steps. Simulation speed can be adjusted to speed up or slow down the process. Adjusting the dispensing step allows observation of specific protocol steps.

[Figure 22: Overview screen in simulation mode, showing source and target plates, simulation controls, and progress.]

Click "Dispense" in the top bar to proceed.

2.5. Dispense

Once the dispensing protocol is set up, prepare the I.DOT source plate with the respective liquids.

Click "Start Dispensing". The software will prompt to insert the source plate into the upper tray and the target plate into the lower tray of the device.

[Figure 23: Dispense screen showing the protocol setup and prompts to insert plates.]

Dispensing runs can be paused (⏸️) at any time.

ℹ️ Note: Dispensing runs can be aborted by clicking "Abort Dispensing" ❌. Source wells may need refilling, and the target plate may need exchanging for repetitions.

Upon completion, a "Dispensing completed" popup appears. Clicking "Ok" automatically opens the results tab.

2.6. Re-dispense Missed Droplets

If drop detection identifies missing droplets during a dispense run, a "Missed Droplets" window appears immediately after the run. This window lists steps where droplets were missing.

By selecting wells and clicking "Re-dispense Missed Droplets", the I.DOT system will fill these target wells according to the counted missed droplets.

[Figure 24: Missed Droplets window, listing dispensing steps with missing droplets.]

2.7. Results

The "Results" tab displays the performance of the dispensing run. Green wells indicate that the expected volume was dispensed (based on droplet count), while red wells indicate missing liquids.

Used liquids and their positions on the source plate are shown on the left. The software provides performance information for each source well.

Clicking on any target well shows the liquids and volumes dispensed into that specific well on the right side.

[Figure 25: Results of a dispense run, showing target plate wells colored green or red based on performance.]

A threshold can be adjusted in the results tab to tolerate a percentage of less volume. Wells with droplet counts above the threshold are shown in green; below the threshold are red; on the limit are yellow.

For questions, contact support@dispendix.com.

Dispensing run results are automatically saved as "PrintLog.txt". Printlogs can be reviewed in the I.DOT Assay Studio software.

3. PrintLog

The printlog summarizes the results of a dispensing run in a text file.

[Figure 26: Example content of a PrintLog.txt file, showing dispensing step details.]

Description of items saved in the printlog:

All PrintLogs are automatically labelled with date and time and saved in the "PrintLogs" subfolder of the I.DOT Assay Studio directory.

4. Comma-Separated Values (CSV) File

The CSV function allows importing and exporting I.DOT dispensing protocols. Protocols can be created using the I.DOT Assay Studio software or a CSV file. When opening a CSV file, choose between "Read-only Mode" (protocol not editable) or "Edit Mode".

[Figure 27: Prompt to open a CSV file in Read-only Mode or Edit Mode.]

The CSV file for the I.DOT device consists of a header and a task section.

[Figure 28: CSV format of an I.DOT protocol, showing header and task sections.]

4.1. Header Structure

The structure and content of the header section are declared as follows:

Header Content (Part 1 of 2):

Header Content (Part 2 of 2):

Important: The structure and content of the header section are crucial for proper import of .csv files into the I.DOT Assay Studio software. The 4th line of the Header should never be changed!

4.2. Task Section

All dispensing steps are defined within the task section. Each step includes source well position, target well position, desired volume, and the exact name of the liquid from the liquid library. Steps should be added row-wise to the CSV list.

5. Advanced User Settings

These settings are optional for operating the I.DOT but useful for adjusting dispensing runs for individual needs.

5.1. Liquid and Liquid Class Library

Data on different liquids and their dispensing parameters are saved in libraries.

5.1.1. Liquid Library

The Liquid Library displays all liquids used in protocols, showing their stock concentration and chosen liquid class. Liquids can be deleted or added.

[Figure 29: Liquid list displayed in the Liquid Library.]

Clicking on a liquid and selecting the ?️ button enables editing (Figure 30). Information on liquid and I.DOT source plate compatibility is saved for each liquid.

[Figure 30: Define Liquid window, showing liquid properties and compatibility.]

Assign favorite liquids using the blue star ⭐ icon. Favorite liquids appear in the dropdown menu when selecting a liquid for a protocol.

5.1.2. Liquid Class Library

The Liquid Class Library contains dispensing parameters for different liquid types (aqueous, DMSO, glycerol, solvents) for I.DOT source plate types:

• S.60 Plate
• S.100 Plate
• S.200 Plate

The number indicates the well orifice size (60, 100, or 200 µm).

New liquid classes can be added by importing provided I.DOT XML Liquid Class files (*.libx) or by creating new ones.

ℹ️ Note: Liquid classes cannot be deleted without enabling advanced settings (see chapter 5.4).

5.2. Creating a New Liquid Class

Select "Add New Liquid Class", enter a name, choose the desired source well type, and select "Define".

[Figure 31: Liquid Class Library, showing existing liquid classes and options to add/import.]

Choose one of the following options to define a new liquid class:

• Copy from existing: Dispensing parameters are copied from an existing liquid class and can be edited.
• Measure: Automated creation of parameters (detailed in section 5.2.1).
• Manually: Manual entry of parameters (detailed in section 5.2.2).

[Figure 32: Adding a new liquid class, showing the choice between Copy, Measure, and Manually.]

5.2.1. Automated Creation of a new Liquid Class

A Liquid Class Wizard guides through the steps to measure and set up individual liquid classes.

Steps to measure a liquid class:

1. Define the source well position for liquid class calibration.
2. Select the target plate format and position for waste disposal.
3. Select the liquid volume to be filled into the source well.
4. (Optional) Edit the minimum and maximum dosing energy for the liquid class.
5. Define pressure settings for calibration and the number of measurement points per setting. Calibration can be performed at multiple pressure settings.

[Figure 33: Liquid Class Wizard, illustrating the steps for calibration.]

To start measurement, select a row and click "Measure selected row", then follow software instructions.

After measurement, the software calculates the liquid class. A graph of the results can be displayed by clicking the arrow next to the table. The graph can be intersected with [0,0] by enabling a checkbox (Figure 34).

[Figure 34: Measured values and plotted graph of the liquid class in the Liquid Class Wizard.]

Select "Save" and "Finish" to save the liquid class.

For adjustments, contact support@dispendix.com.

ℹ️ Recommendation: Using a calibrated pipette for calibration is recommended. Higher filling volumes in step 3 yield more precise results. Measuring triplicates confirms stable droplet volumes.

ℹ️ Note: Once saved, a liquid class cannot be edited.

5.2.2. Manual Creation of a Liquid Class

A liquid class can be defined as a polynomial equation based on the correlation between dosing energy and generated droplet volume. Parameters can be adjusted manually.

Adjust parameter values for "M", "N", "Min", and "Max" (Figure 35).

[Figure 35: Liquid class parameters and values, showing M, N, Min, Max settings.]

• M (Slopes): Defines generated droplet volume (nL) per 1 mbar*ms during pressure pulse.
• N (Offset): Default 0 nL. Intersection of the graph with the Y-axis.
• Min (Minimum dosing energy): Default 50 mbar*ms. Lower pressure limit for the liquid class.
• Max (Maximum dosing energy): Default 300 mbar*ms. Maximum pressure limit for the liquid class.

Select "Save" and "Finish" to save the liquid class.

The unit mbar*ms describes the relationship of pressure and mechanical valve opening time.

5.3. About Plate Format Library

The plate format library stores target plate formats used with the I.DOT. Standard SBS formats (96-, 384-, 1536-well) are saved by default. New and custom target formats can be created using the plate format editor.

[Figure 36: Target Plate Editor window, showing properties of a plate format.]

Editable Items:

• Name: Insert plate format name.
• Optional: Edit plate manufacturer's name.
• Length/Width/Height: Enter outer dimensions of the plate format.
• Number of Rows/Columns: Enter the respective values.
• Optional: Cavity Volume: Enter the maximum filling volume.
• Distance to First Row/Columns: Enter the distance shown in the editor.
• Distance between Rows/Columns: Enter the distance shown in the editor.

ℹ️ Note: Cavity volume does not affect the plate format itself; exceeding volume is not counted as an error and is only relevant for the user.

5.4. About Advanced Device Settings

Advanced settings are enabled by applying the password "Dispendix" (Figure 37). These settings allow adjustments to position and dispense head configurations.

[Figure 37: Device Settings screen, with the "Show Advanced Settings" button highlighted.]

5.4.1. Reset Liquid Classes

If original Dispendix liquid classes are deleted or modified, they can be restored by clicking the "Reset Liquid Classes" button in the general settings window (Figure 38).

[Figure 38: Restoring original liquid classes via the "Reset Liquid Classes" option.]

Note: Resetting liquid classes does not delete individually created liquid classes.