Single-camera multi-color emission anisotropy optical splitter

Session

Poster Session

Authors

Greeshma Pradeep S (2), Charitra Sree Senthil Kumar (2), Abhishek Kumar (1), Thomas S van Zanten (2, 3), Satyajit Mayor (2)

Affiliations

1. Marine Biological Laboratory, Woods Hole, MA
2. National Center for Biological Sciences, Tata Institute of Fundamental Research, Bellary Road, Bangalore, 560065
3. Instituto de Nanociencia y Materiales de Aragon, Zaragoza

Keywords

FRET, Anisotropy, Polarization, Optical Splitter, Multi-wavelength Simultaneous Anisotropy Imaging

Abstract text

Fluorescence emission anisotropy is the ratiometric measure of the polarization of fluorescence emission, which is used to provide insights into molecular organization. In the field of optical microscopy, the typical approach for multi-wavelength emission anisotropy imaging involves either sequential imaging using a single camera [1] or simultaneous imaging using multiple cameras [2]. Given the limitations of the sequential imaging method, particularly in capturing sub-second dynamic processes in live-cell imaging, a viable option is the utilization of simultaneous imaging with multiple cameras. However, when accounting for multiple deformations or aberrations, the process of image registration for estimating anisotropy becomes more cumbersome. For facilitating simplified simultaneous imaging using a single camera, we engineered a novel multi-wavelength, emission anisotropy image-splitter system. The measurement of point spread function (PSF) and dynamic range in emission anisotropy measurements served as critical parameters validating the potential of the constructed configuration for high-resolution cell imaging. We conducted simultaneous visualization and quantification of actin filament orientation using various actin labels, aiming to establish a benchmark for emission anisotropy measurements across three distinct excitation wavelengths: 488 nm, 561 nm, and 640 nm. Homo Förster Resonance Energy Transfer (Homo-FRET) assessments investigating the clustering behavior of Glycosylphosphatidylinositol-anchored proteins (GPI-AP) labeled with distinct fluorophores (GFP and mRuby) facilitated the realization of simultaneous multi-color anisotropy imaging through the utilization of a single camera system. We validated the capability of the system to accurately measure subtle changes in emission anisotropy during photobleaching of homo-FRET competent molecular species. This was demonstrated through a comparative study involving photobleaching of the trimeric-VSVG-EGFP where VSVG-EGFP was diluted with a non-homo-FRET competent monomer (VSVG-tdTurboRFP), showcasing equivalent precision in anisotropy measurements. Simultaneous multi-wavelength, emission anisotropy imaging of photoconversion of tetrameric Kaede and monomeric Dendra fluorescent proteins, transitioning from green to red fluorescence, provided a real time view of the interconversion dynamics inherent in the photoconversion process. Monte Carlo simulations were employed to provide an understanding of the intricate dynamics underlying the photoconversion process of the tetrameric fluorescent protein, demonstrating the power of this system. Simultaneous recording of Fluorescence Anisotropy Reporter (FLARE) sensors of cAMP levels with cytosolic (mVenus-cpVenus-FLARE-AKAR, mCherry-mCherry-FLARE-AKAR) and membrane (Lyn-mVenus-cpVenus-FLARE-AKAR) sensors [3], stimulated by G-protein coupled receptor activation, illustrates the optical splitter’s capability to monitor the transfer of a second messenger signal from the membrane to the cytosol in real time. This substantiates the versatility of the setup, thereby unlocking a diverse array of applications within the realm of multiband emission anisotropy imaging.

A) schematic of the 4-way optical splitter setup. B) intensity images in the 4 different channels from Phalloidin-AF488 and SiR-actin, labelling identical actin filaments. C&D) anisotropy images from the Phalloidin-AF488 and SiR-actin channels, respectively. E) relationship between anisotropy values from different f-actin labels and actin filament orientation.

References

1. Varma and Mayor, Nature, 1998

2. Ghosh et al, Methods in Enzymology, 2012

3. Ross et al, eLife, 2018