Fluorescence-detected linear dichroism imaging in a re-scan confocal microscope equipped with differential polarization attachment

Abstract

Confocal laser scanning microscopy is probably the most widely used and one of the most powerful techniques in basic biology, medicine and material sciences that is employed to elucidate the architecture of complex cellular structures and molecular macro-assemblies. It has recently been shown that the information content, signal-to-noise ratio and resolution of such microscopes (LSMs) can be improved significantly by adding different attachments or modifying their design, while retaining their user-friendly features and relatively moderate costs. Differential polarization (DP) attachments, using high-frequency modulation/demodulation circuits, have made LSMs capable of high-precision 2D and 3D mapping of the anisotropy of microscopic samples—without interfering with their ‘conventional’ fluorescence or transmission imaging (Steinbach et al. in Methods Appl Fluoresc 2:015005, 2014 ). The resolution and the quality of fluorescence imaging have been enhanced in the recently constructed Re-scan confocal microscopy (RCM) (De Luca et al. in Biomed Opt Express 4:2644–2656, 2013 ). In this work, we developed the RCM technique further, by adding a DP-attachment modulating the exciting laser beam via a liquid crystal (LC) retarder synchronized with the data acquisition system; by this means, and with the aid of a software, fluorescence-detected linear dichroism (FDLD), characteristic of the anisotropic molecular organization of the sample, could be recorded in parallel with the confocal fluorescence imaging. For demonstration, we show FDLD images of a plant cell wall ( Ginkgo biloba ) stained with Etzold’s staining solution. Electronic supplementary material The online version of this article (10.1007/s00249-019-01365-4) contains supplementary material, which is available to authorized users.

Structure and principles of self-assembly of giant “sea urchin” type sulfonatophenyl porphine aggregates

“…The image acquisition was controlled by the Nikon EZ-C1 software. The images were further processed using EZ-C1 v3.91 (Nikon, Japan) and ImageJ v1.53a software (NIH, USA) [49].…”

Differential Polarization Imaging of Plant Cells. Mapping the Anisotropy of Cell Walls and Chloroplasts

“…The first DP-LSM which was extended to the fluorescence light path, and, thus, in addition to LB and LD, was capable of confocally determining FDLD, r, and P, was designed and constructed in our laboratory [ 26 ], and was first used to characterize the anisotropic molecular organization of Alexa-phalloidin-stained actin-based Drosophila melanogaster nurse cell samples via mapping of r [ 27 ]. In this device, and in similar LSMs of other types and makes, different DP attachments and different configurations were used, but employing the same basic principles and high-frequency modulation and demodulation modules, and passive polarization optical components [ 10 , 12 ]. In all of these DP-LSMs, DP images are recorded parallel with the ‘conventional’ intensity images, and the DP images, after normalization, are saved in the same format as the conventional LSM image.…”

“…On the other hand, DP-LSM measurements provide 2D or 3D microscopic maps of the anisotropy parameters. The use of these devices has been demonstrated on a number of highly organized structures in biological samples and intelligent materials (see [ 10 , 12 ] and references therein).…”

“…A recently constructed DP-RCM is capable of confocal DP imaging using fluorescence detection [ 12 ]. Since the RCM employs camera-based imaging—instead of using a photomultiplier (PMT) or any other point detection method—the PEM and lock-in modules cannot be used.…”

“…However, some of these measuring techniques cannot readily be combined with the ‘conventional’ laser-scanning excitation and the confocal detection of the fluorescence of the sample. We have shown earlier that differential polarization (DP) measurements can be relatively easily conjoined with the ‘conventional’ imaging regimes of LSM [ 10 , 11 ] and RCM [ 12 ]. This can be performed by using the theory and technologies of spectropolarimetry and DP measuring procedures [ 13 , 14 ].…”