Time and structural crosscorrelation image analysis of microscopic volumes, simultaneously recorded with second harmonic generation, third harmonic generation, and multiphoton excitation fluorescence microscopy

Catherine Greenhalgh; Richard Cisek; Nicole Prent; Arkady Major; Juerg Aus de Au; Jeff Squier; Virginijus Barzda

doi:10.1117/12.628786

14 October 2005 Time and structural crosscorrelation image analysis of microscopic volumes, simultaneously recorded with second harmonic generation, third harmonic generation, and multiphoton excitation fluorescence microscopy

Catherine Greenhalgh, Richard Cisek, Nicole Prent, Arkady Major, Juerg Aus de Au, Jeff Squier, Virginijus Barzda

Author Affiliations +

Proceedings Volume 5969, Photonic Applications in Biosensing and Imaging; 59692F (2005) https://doi.org/10.1117/12.628786
Event: Photonics North, 2005, Toronto, Canada

Abstract

Our newly developed multimodal microscope enables simultaneous collection of second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence (MPF) signals. The signals can be generated within different or the same intercellular structures. In comparing two signals, traditional methods of image crosscorrelation analysis using Pearson's coefficient provide a general parameter as to whether the images are similar, however it does not give detailed information about correlation of different structures inside the images. We present here a new technique that employs a pixel by pixel analysis over an entire area or volume that is used to correlate the structures appearing in the images. The result of the analysis reveals structures within the sample that are generated by both nonlinear signals as well as highlighting the structures that are generated by only one of the nonlinear signals. The algorithm provides a means to colocalize different structures revealed by the different nonlinear contrast mechanisms. Structural correlation maps are useful in identifying the origin of structures in one nonlinear contrast mechanism when the origin of structures in another is known. Image analysis has also been exploited for sequences of images taken in time. The intensity fluctuations in time for each pixel reveal regions of intense physiological activity in biological samples. Correlation of time dependent fluctuations from different pixels in the image time series allows construction of the structural map that undergoes similar time behavior or appears out of phase. These structural correlation analysis techniques are demonstrated based on polystyrene beads and cardiomyocytes.

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Catherine Greenhalgh, Richard Cisek, Nicole Prent, Arkady Major, Juerg Aus de Au, Jeff Squier, and Virginijus Barzda "Time and structural crosscorrelation image analysis of microscopic volumes, simultaneously recorded with second harmonic generation, third harmonic generation, and multiphoton excitation fluorescence microscopy", Proc. SPIE 5969, Photonic Applications in Biosensing and Imaging, 59692F (14 October 2005); https://doi.org/10.1117/12.628786

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