Use bright field signal to trace the drift in 3D with nanometer precision.

This is a set of Python tools with command-line interface.

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• python 3.6+
• All dependencies are automatically pulled during installation of BFDC

We recommend using Conda virtual environment to run BFDC. Install Miniconda and type the following instructions in Anaconda promt (Windows) or Terminal (Unix / MacOS)

With Conda create a new environment with python 3.7:

conda create -n pydrift python=3.7 -y

Then activate it and run installation:

source activate pydrift (Unix / MacOS) activate pydrift (Windows)

pip install git+https://github.com/imodpasteur/Brightfield_Drift_Tracking_3D.git

In order to trace the drift in 3D a calibration stack needed (dict.tif).

Acquire bright field images with the same exposure (movie.tif) in order to trace the drift with nanometer precision.

You'll need to select a contrast region say 32x32 pixels from the dict.tif and save as crop.roi (ImageJ ROI format)

python -m bfdc trace dict.tif crop.roi movie.tif

python -m bfdc apply ZOLA_localization_table.csv BFCC_table.csv --smooth 10

python -m bfdc batch data_folder

Expected file structure:

• data_folder
  • FOV1
    • dict_LED_100nm
      • dict_LED_100nm_Pos0.ome.tif
      • 32x32.roi
    • sr_ast_642_LEDskip9
      • sr_ast_642_LEDskip9.Pos0.ome.tif
      • sr_ast_642_LEDskip9.Pos0_1.ome.tif
      • ZOLA_localization_table.csv
  • FOV2
    • ...

The possible arguments include:

• Recursively parsing folders with fields of views (--fov_prefix=FOV)
• Looking for the folder with dictionary stack (--dict_folder_prefix=dict)
• Finding ROI file (--ROI_suffix=roi)
• Opening dictionary stack next to ROI (--dict_suffix=ome.tif)
• Opening STORM movie with bright field frames (--sr_folder prefix=sr, --sr_movie_suffix=Pos0.ome.tif)
• Automatically selects brightfield frames with background more than (--filter_bg=100)

python -m bfdc -h

python -m bfdc trace -h

python -m bfdc apply -h

python -m bfdc batch -h

Super resolution imaging of EDU Alexa 647 in the nucleus: beads photobleaching.

Luckily, we have recorded the bright field movie along with the fluorescence by alternating illumination between illuimination and LED!

How can we use it to trace the drift in 3D?

Using the stack, recorded prior to imaging with the same exposure and same illumination intensity, we can reveal the drift from bright field only in 3D!

32x32 px crop form the dictionary and 64x64 crom form the movie tracks the drift with the precision higher than using beads and better sampling than redundant cross-corellation.

Stack crop Movie crop

Note, how Nikon's Perfect Focus System struggles to maintain the focus!

Fluorescent bead track before and after BFDC.

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V0.2.0 Processing tif file sets

V0.1.4 Batch processing

V0.1.3 Avoid plots ovelaps from diffrent datasets

v0.1.2 Removing localizations from the ZOLA table if they come from bright field.

v0.1.1 Automatically detecting BF frames based on intensity. No more need of skipping parameters to know.