Spectrally resolved intensity maps were acquired for single stripes in a modified Leica DMRX optical microscope with a × 20 objective where an additional photoport allowed for the collection of the light reflected from the specimen into an optical fibre of 50-μm diameter. Spectra were collected from a spot of 2-μm diameter with a collection cone half angle of 33° and the corresponding maps were obtained by laterally scanning the sample in 2-μm steps while maintaining the specimen in constant focus. Quantitative spectral measurements for the determination of the stripes reflectivity in water were performed by using a × 63 water immersion objective with numerical aperture NA=1.0 allowing for the collection of spectra from spots of ~2-μm diameter over a polar angle range of 0–50°. Optical scattering of the samples was studied using a modified Olympus BXFM microscope, where by the incorporation of a Bertrand lens, scattering of a specimen under quasi-plane wave illumination, achieved via an additional photoport, was analysed by imaging the back focal plane of the objective. A high numerical aperture oil immersion objective (Leica PL APO 100 × /1.4 −0.7 OIL) was used to increase the observable angular range. The corresponding scattering angles for water as a medium above the shell surface were then calculated using Snell’s law. These are the angles displayed in Fig. 4c .
Bxfm microscope
Manufactured by Olympus
The BXFM is a high-performance biological microscope designed for a wide range of laboratory applications. It features a stable and ergonomic frame, a high-resolution optical system, and a range of illumination options to support various imaging techniques.
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5 protocols using bxfm microscope
1
Spectral Microscopy and Scattering Analysis
2
Visualizing Sodium Metal Reactivity
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For visual analysis of the Na reactivity, Na metal pieces (0.25 g, Alfa Aesar, 99.95 %) were thoroughly cleaned by scraping the surface layer of the metal with a scalpel in an argon‐filled glovebox to remove any impurities on the metal surface. These shiny metal pieces were added to 5 mL of each of the three different electrolytes. Pictures were taken right after the addition of the metal and three days later.
For optical microscopy studies, the microscopy cell consisted of a polyethylene (PE) film, an O‐ring, and a borosilicate glass, which were clamped between two metal plates to seal the cell. The upper metal plate was perforated to enable optical microscopy through the underlying borosilicate window. The sodium metal was pressed onto the polyethylene film and the cell was filled with the electrolyte (1m NaClO4 in EC/DMC) inside an argon‐filled glovebox. Bright field imaging with an Olympus BXFM microscope at 10× magnification was performed. To increase the depth of field, focus stacks consisting of 25 single CCD images with 8 μm distance between the focal planes were taken. During the experiment, images were recorded every 15 min.
For optical microscopy studies, the microscopy cell consisted of a polyethylene (PE) film, an O‐ring, and a borosilicate glass, which were clamped between two metal plates to seal the cell. The upper metal plate was perforated to enable optical microscopy through the underlying borosilicate window. The sodium metal was pressed onto the polyethylene film and the cell was filled with the electrolyte (1
3
Raman Microspectroscopy Characterization
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Raman spectra were recorded with a LabRam HR800 micro-Raman instrument (Horiba Scientific, FR) equipped with a Peltier-cooled CCD detector at −70 °C, an Olympus BXFM microscope, a 600 groove/mm grating and a 50× objective to collect the Raman scattering signals. The excitation source was a He–Ne laser (632.8 nm line) with a maximum laser power of 20 mW. A minimum spectrum accumulation of 10 times per second was used; if a high background was recorded, the accumulations were increased to a maximum of 100 times per second to improve the signal-to-noise ratio.
4
Micro-Raman Analysis of Mineral Phases
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A LabRam HR800 micro-Raman from Horiba Scientific, equipped with an air-cooled CCD detector at − 70 °C, an Olympus BXFM microscope (objective 10 × and 50 ×), and a 600 groove/mm grating, was used to collect the Raman scattering signals of mineral phases present on the samples. The excitation source was a He–Ne laser (632.8 nm line) with a maximum laser power of 17 mW and the spectrometer was calibrated with silicon at 520 cm−1. Spectra acquisition and treatment were performed using HORIBA Scientific’s LabSpec 6 Spectroscopy Suite Software. Identification of the mineral phases and peaks attribution was done referring to BIO-RAD spectral database, using KnowItAll spectroscopy software.
5
Raman Spectroscopy of E. coli Cells
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A Lab-RAM HR Raman microscope (Horiba Scientific, Japan) was used for Raman analysis. The system was equipped with an integrated Olympus BXFM microscope, a He-Ne-Laser (633 nm, 4 mW at the sample) and a Peltier-cooled CCD detector. A 100× objective (Olympus MPlan N, NA = 0.9) was used to focus the laser beam onto the sample and to collect the scattered light. The light then passes a diffraction grating (600 lines per mm) and a confocal pinhole (100 µm). A laser power of 0.4 mW at the sample and an acquisition time of 1 s were applied for the SERS analysis of E. coli cells. The spectral range of the recorded Raman spectra was set to 50 cm -1 -3750 cm -1 . A silicon wafer with its first-order phonon band at 520.7 cm -1 was used for wavelength calibration.
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