Raster Image Correlation Spectroscopy and Brightness Measurements of AtLEA proteins from Arabidopsis thaliana

Temporal sequences of various fluorescent leaves were captured using a confocal scanning microscope (Olympus FV1000 inverted microscope), equipped with a 1.3 NA oil immersion 60X objective and the photon counting detection mode. Utilizing a 488 nm laser at 0.1% power and GFP filters/cubes, each temporal sequence involved the acquisition of 100 frames of 64x64 pixels, with a dwell time of 10 μs (1.76 ms per line, 130.24 ms per frame) and a pixel size of 66 nm (50X digital zoom). The interval between frames was set at 131.6 ms.

Five plants were analyzed, each expressing one of four distinct genetic constructs fused to complementary fragments of Yellow Fluorescent Protein: pYFN-4-/5pYFC-4-5 (representing the complete AtLEA4-5 protein), pYFN-4-51-77/pYFC-4-51-77 (associated with the N-terminal region of AtLEA4-5), pYFN-4-578-158/pYFC-4-578-158 (relating to the C-terminal region of AtLEA4-5), and pYFN-pYFC (serving as the control). The raw data (*.oib files) were collected during three imaging sessions within a one-week period:

- 220618 raw oib dataset.zip

- 220622 raw oib dataset.zip

- 220623 raw oib dataset.zip

Images were converted to *.tif format using FIJI/ImageJ for further analysis and were archived in "tif dataset RICS NB LEAs.zip," excluding files with excessive movement of biological specimens. These images were then subjected to "Raster Image Correlation Spectroscopy" and "Number and Brightness" techniques for analysis.

Notation:

- h1, h2, h3, h4, h5: Replicates (plants) expressing one of four specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein.

- 45: Fused to the full-length AtLEA4-5 protein (pYFN-4-/5pYFC-4-5).

- 4h: Fused to the N-terminal region of AtLEA4-5 (pYFN-4-51-77/pYFC-4-51-77).

- rc: Fused to the C-terminal region of AtLEA4-5 (pYFN-4-578-158/pYFC-4-578-158).

- ct: The control condition (pYFN-pYFC).