Fluorescence detected linear dichroism and anisotropy imaging using RCM

Session

Poster Session

Authors

Rita Bibok (1), Ábel Garab (1, 3), Gyozo Garab (1, 2), Gabor Steinbach (1, 2)

Affiliations

1. Biofotonika Ltd.
2. HUN-REN BRC Szeged
3. University of Szeged

Keywords

polarized light, DP-LSM, fluorescence detected linear dichroism, anisotropy, molecular organization

Abstract text

Fluorescence microscopy imaging techniques have substantially advanced our knowledge about cellular structures. Laser-scanning microscopy in general – in addition to their high sensitivity, fast data acquisition, and great versatility of 2D–4D imaging – also opened the technical possibilities to combine microscopy imaging with further modalities. 

Hierarchically organized complex molecular macro-assemblies are widespread among biological samples: e.g. cellulose fibres, stacked membranes, ordered macromolecular fibrils, self-assembled aggregates. These complex ordered molecular structures are beyond the resolution of conventional optical microscopy. During the examination of these structures, the interaction between sample and polarized light provides unique information by revealing the anisotropic molecular architecture of the sample.

Using differential polarization attachments for LSMs and Re-scan Confocal Microscopy (RCM) facilitate revealing ordered structures at the molecular level [Steinbach 2019].While examining  subcellular ultrastructures, increasing the available resolution can be fundamental. Re-scan confocal microscopy (RCM) provides 4 times better signal to noise ratio (compared to conventional PMTs) using an sCMOS camera, moreover, due to the second scanner, the lateral resolution of the imaging increases by the factor of 1.4, while the axial resolution is identical with the LSMs [De Luca 2017].

Our DP-RCM (RCM equipped with polarization attachment) contains liquid crystal modulators in both excitation and emission lightpaths in order to determine / analyse the orientation of polarization. This extension enables the 2D and 3D microscopic mapping of the fluorescence-detected linear dichroism (FDLD) and the anisotropy of the fluorescence (r). The use of the liquid crystal modulators is synchronised with the imaging controlled by the microscope software (NIS-Elements): communication is integrated with the acquisition process. The DP-RCM is suitable for obtaining unique structural information on the anisotropic molecular organization of biological samples and intelligent materials [Radosavljević 2021, Pleckaitis 2022] in 2D and 3D.

References

De Luca et al. Re-scan confocal microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity. Methods Appl. Fluoresc. 5, 015002, 2017. doi: 10.1088/2050-6120/5/1/015002

Steinbach et al. Re-scan confocal microscope modified for anisotropy imaging – as a part of a differential polarization system. European Biophysics Journal 48, 457–463, 2019. doi: 10.1007/s00249-019-01365-4

Simonović Radosavljević et al. Anisotropic molecular organization of plant cell walls revealed by differential polarization imaging techniques. Int. J. Mol. Sci. 22, 7661, 2021. doi: 10.3390/ijms22147661

Pleckaitis et al. Structure and principles of self-assembly of giant “sea urchin” type sulfonatophenyl porphine aggregates. Nano Research 15, 5527–5537, 2022. doi: 10.1007/s12274-021-4048-x