ECL`

ExperimentImageColonies

ExperimentImageColonies[Samples]⟹Protocol

creates a Protocol for acquiring bright-field, absorbance or fluorescence images of the provided Samples on a plate using a colony handler.

ExperimentImageColonies is an advanced imaging tool designed to acquire high-resolution, multi-type images of samples on solid media using a colony handler. This technique is essential for visualizing colonies with reporter genes (such as lacZ and GFP) or dyes (such as Cy3 and TxRed), facilitating a wide range of microbiological and biochemical analyses. The imaging process involves capturing a default BrightField image along with optional BlueWhiteScreen image and fluorescence images (VioletFluorescence, GreenFluorescence, OrangeFluorescence, RedFluorescence, and DarkRedFluorescence). These images provide detailed insights into colony morphology and fluorescence characteristics. Specific applications of imaging colonies include evaluating colony growth, calculating Colony-Forming Units (CFU), quantitating Phage Titers, and determining Minimum Inhibitory Concentration (MIC), among others. The ability to capture multiple types of images makes this technique adaptable to different experimental needs.

Experimental Principles

Figure 1.1: Procedural Overview of an ImageColonies experiment using a QPix 420 HT ColonyHandler. Step 1: Samples are placed on the light table inside the QPix colony handler, and a BrightField image is captured. For ImagingStrategies including BlueWhiteScreen, an absorbance filter which blocks blue light is placed between the sample and the light source, eliminating blue colonies from the image, even when they are newly formed and appear powder blue. For ImagingStrategies including VioletFluorescence, GreenFluorescence, OrangeFluorescence, RedFluorescence, or DarkRedFluorescence, an LED-based light source with a specific fluorescence excitation wavelength illuminates the sample, and emission is filtered before reaching the CCD camera. The QPix ColonyHandler moves seamlessly between samples and transitions between imaging strategies.

Instrumentation

ColonyHandler with built-in Imager

QPix 420 HT

Figure 2.1.1: Overview of the ColonyHandler WorkCell integrations that are attached to the colony handler instrument deck.

Figure 2.1.2: Overview of the ColonyHandler WorkCell. This figure provides a comprehensive view of the ColonyHandler, showcasing its layout and main components. The QPix instrument is designed to perform multiple tasks including imaging solid media plates, picking colonies from solid media, and plating suspended colonies onto solid media. Its bed features a deck with designated positions for multi-well and single-well SBS plates, a light table, and wash baths for cleaning the pins before and during the process. Above the deck, a robotic gantry moves and is equipped with several components: a pipette nozzle, an LED-based light source, preset filter pairs for fluorescence excitation and emission wavelengths, a monochrome CCD camera, and a position to accept a ColonyHandlerHeadCassette. The robotic gantry moves seamlessly between tasks, and have the capacity to capture multiple types of images.

Experiment Options

General

Instrument

The instrument used to image colonies on solid media.

Figure 3.1: Diagram of the ColonyHandler deck layout. The ColonyHandler is equipped with both transmitted white light and fluorescence imaging capabilities. The deck features two designated positions for single-well plates for each batch of imaging.

Default Value: Model[Instrument, ColonyHandler, QPix 420 HT]

Pattern Description: An object of type or subtype Model[Instrument, ColonyHandler] or Object[Instrument, ColonyHandler]

Programmatic Pattern: ObjectP[{Model[Instrument, ColonyHandler], Object[Instrument, ColonyHandler]}]

Imaging

ImagingStrategies

The end goals for capturing images. ImagingStrategies can be either a single end goal of simple visualization or a list of multiple end goals. The options include BrightField, BlueWhiteScreen, and various fluorescence imaging techniques. BrightField imaging provides essential baseline images and is required if any other imaging strategies are employed. BlueWhiteScreen is used to distinguish recombinant colonies with disrupted lacZ (white colonies) from blue colonies, using an absorbance filter. Fluorescence imaging allows for the visualization of colonies with fluorescent dyes or proteins.

Figure 3.2: Diagram of the imaging module of the ColonyHandler with various imaging strategies. For BlueWhiteScreen, an absorbance filter which blocks blue light between the sample and the light source, eliminating blue colonies from the image, even when they are newly formed and appear powder blue. For VioletFluorescence, GreenFluorescence, OrangeFluorescence, RedFluorescence, or DarkRedFluorescence, an LED-based light source with a specific fluorescence excitation wavelength illuminates the sample, and emission is filtered before reaching the CCD camera. The transition to capture multiple types of images is accomplished by the robotic gantry of the QPix colony handler, which moves seamlessly between imaging strategies and samples.

Default Value: BrightField

Pattern Description: Multiple Imaging Strategies or Single Imaging Strategy.

Index Matches to: experiment samples

ExposureTimes

A single length of time that the camera sensor collects light during image acquisition for each imaging strategy. An increased exposure time leads to brighter images based on a linear scale. When set as Automatic, optimal exposure time is automatically determined during the experiment. This is done by running AnalyzeImageExposure on images taken with suggested initial exposure times. The process adjusts the exposure time for subsequent image acquisitions until the optimal value is found.

Figure 3.3: This table outlines the suggested exposure times for various imaging strategies and lists the corresponding fields where the raw image data is stored for each imaging strategy.

Default Value: Automatic

Pattern Description: Multiple Exposure Times or Single Exposure Time.

Programmatic Pattern: ({(RangeP[1*Millisecond, 2000*Millisecond] | Automatic)..} | (RangeP[1*Millisecond, 2000*Millisecond] | Automatic)) | Automatic

Index Matches to: experiment samples

Post Experiment

SamplesInStorageCondition

The non-default conditions under which the SamplesIn of this experiment should be stored after the protocol is completed. If left unset, SamplesIn will be stored according to their current StorageCondition.

Default Value: Null

Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.

Programmatic Pattern: (Alternatives[SampleStorageTypeP | Disposal]) | Null

Index Matches to: experiment samples

Protocol Options

Organizational Information

Template

A template protocol whose methodology should be reproduced in running this experiment. Option values will be inherited from the template protocol, but can be individually overridden by directly specifying values for those options to this Experiment function.

Default Value: Null

Pattern Description: An object of type or subtype Object[Protocol] or an object of type or subtype of Object[Protocol] with UnresolvedOptions, ResolvedOptions specified or Null.

Programmatic Pattern: (ObjectP[Object[Protocol]] | FieldReferenceP[Object[Protocol], {UnresolvedOptions, ResolvedOptions}]) | Null

Name

A object name which should be used to refer to the output object in lieu of an automatically generated ID number.

Default Value: Null

Pattern Description: A string or Null.

Programmatic Pattern: _String | Null

Example Calls

Basic Examples:

Samples containing microbial cells on solid media plate may be accepted as input:

Plate with only solid agar and without cells can be examined to check contamination:

ImagingStrategies can be specified:

Batching Examples:

If the two samples have the same imaging parameters, they can be grouped in the same batched unit operation:

If the two samples have different imaging parameters, they cannot be grouped in the same batched unit operation:

If more than two samples have the same imaging parameters, up to two samples are grouped together:

Preferred Input Containers

Single-Well OmniTray Plate

Data Processing

If ImagingStrategies include BlueWhiteScreen, the experimental data Object[Data,Appearance,Colonies] has BlueWhiteScreenImageFile in addition to ImageFile:

If ImagingStrategies include GreenFluorescence, the experimental data Object[Data,Appearance,Colonies] has GreenFluorescenceImageFile in addition to ImageFile:

Visualize the BrightField image using PlotImage:

Visualize the all GreenFluorescence images captured with automatic ExposureTimes:

Analyze the colonies using AnalyzeColonies: