Smart microscopy targeted imaging

Prerequisites
Before starting this lesson, you should be familiar with:

No programming or engineering knowledge is required

Know how to operate a microscope

Segmentation

Thresholding

Connected component labelling

Learning Objectives
After completing this lesson, learners should be able to:

Understand basic feedback microscopy workflow for automatically finding target objects and re-imaging them at high resolution

Understand essential steps in constructing and testing such a workflow

Learn which use cases would be appropriate for such workflow and possible limitation

Motivation

High-resolution imaging of large samples is a very time-consuming process. In many biological studies high-resolution microscopy data are not required for the entire sample, but only for specific areas. For example, often only sub-population of cells showing particular phenotype need to be imaged at high resolution. Manual selection of target areas is a laborious process and requires constant involvement of experimenters. Automating such experiments with smart (adaptive feedback) microscopy enables running such experiments in high-throughput manner without any user supervision. Image processing is done via pre-defined image analysis routine, which ensures unbiased and reproducible selection of imaged objects.

Concept map

graph TD L("Low resolution image") --> I("Find objects") I --> H("Image objects at high resolution") H --> M("Move to new position") M --> L

Figure

Activities

Basic targeted acquisition workflow

Manual configuration and test at the microscope
- Setup imaging settings for low zoom and high zoom acquisition jobs
- (If available) Save all settings as imaging jobs (observation methods) in microscope software
- Acquire and save example images for testing and debugging image analysis
Image analysis
- Define or re-use image analysis function implementing threshold-based segmentation of nuclei
- Use acquired images to test segmentation parameters
Configure and run feedback microscopy pipeline
- Define default positions for the feedback microscopy (the same as positions for screening)
- Configure selected feedback microscopy tool (see implementations) by linking pre-configured acquisition settings and segmentation protocol as parameters.
- Run the pipeline

Show activity for:

Automic Tools with Zen Blue
Hardware and software requirements This implementation required Zeiss microscope controlled by ZenBlue software. Macro programming module is not required, although preferential to simplify changing parameters of Zen macro. Installation Install Fiji with AutoMicTools package on microscope running PC using these guidelines Install zeiss_zenbLue_automation package using these guidelines Step-by-step guidelines Set up imaging settings Define settings for low zoom and high zoom imaging Each time activate “Tiles” option Save settings as “Experiment setups” in ZenBlue

Set up image analysis Download example Fiji macro and open it in Fiji (drag-and-drop) Take example low zoom image and open it in Fiji (drag-and-drop) Test macro by running it. As a result, segmented ROIs should be added to image overlay. Start AutoMicTools distributor in Fiji [ Plugins › Auto Mic Tools › Applications › Imaging › AF LowZoom HighZoom - script] Parameters of the first dialogue (General JobDistributor parameters): Image File Extension: czi Experiment Folder: {path to experimental folder where data will be stored} Microscope Commander: ZenBlueCommander Other parameters: default values Second dialogue (processing parameters for autofocus job): keep all by default Third dialogue (processing parameters for low zoom job): Script path: path to the Fiji macro tested in step 2 Configure parameters of ZenBlue script and run it (Optional) Simulate acquisition workflow using pre-acquired data

Zen Blue Guided Acquisition
TODO

Assessment

Questions

TODO?
TODO?

Answers

TODO

TODO

Follow-up material

Recommended follow-up modules:

Automatic threshold for binarization

Learn more:

Wikipedia: Binary image