Razoredge

Raman spectroscopy measurements were performed using a Leica microscope and lenses (JY LabRam HR 800, Horiba Jobin Yvon, France) with curve-fitting Raman software (Labspec 5) using a monochromatic green laser (532 nm, 8 mW incident power, ThorLabs, Newton Avenue, New Jersey, USA). This was alongside a long pass filter and a beam splitter (Razor Edge, Semrock). A CCD camera (IXON, Andor, Oxford Instruments) with a spectrograph (SP-2300i, Teledyne Princeton Instruments) was used to collect data over an exposure time of 1 s. An average of 10 measurements were taken at 50 × magnification and a spectral resolution of 1 cm⁻¹ (natural line widths of Raman lines) and a laser power of 4.94mW recorded from different locations. Readings were taken from the central region of the files (n = 5) ending onto the left side of the specimen in 1 µm steps using the x–y–z stage with background-corrected counts.

The measurements
were performed using a bespoke Raman system consisting of a monochromatic
laser (HeNe, ThorLabs) with a beam splitter and a long-pass filter
(RazorEdge, Semrock), an inverted optical microscope (IX71, Olympus),
a spectrograph (SP-2300i, Princeton Instruments), and a CCD camera
(iDus 401, Andor).^{55 (link),68 (link)−70 (link)} A 50×
objective was used to focus the laser (532 nm wavelength, 5 mW incident
power regulated by an attenuator) and collect the Raman and fluorescence
signals with an exposure time of 2 s in an accumulation mode (10 accumulations).
The CCD camera was calibrated over the spectral window using the Raman
spectrum of toluene. To take spatial variability into consideration,
an average signal from 10 different spots on the sample was reported.

Solution (methylene chloride) resonance Raman spectra were collected in either a 180° backscattering (780 nm) or 90° scattering geometry using 407 nm excitation from a Coherent Innova 70 Kr⁺ (1 W) ion laser. The scattered radiation was passed through a longpass filter (Semrock RazorEdge) to remove Rayleigh scattered laser light and then dispersed onto a liquid nitrogen-cooled Infrared Associates CCD detector using a Princeton Acton spectrograph. The laser power at the sample was typically kept between 40 and 100 mW in order to prevent possible photo- and thermal degradation of the sample. The sample was sealed in a glass capillary tube and spun with a custom-made sample holder. All data were scan-averaged, and any individual data set with vibrational bands compromised by cosmic events were discarded.

PEF measurements were undertaken
using a bespoke Raman system that consisted of an inverted optical
microscope (IX71, Olympus), a monochromatic laser (green laser, ThorLabs)
with a beam splitter and long-pass filter (RazorEdge, Semrock), a
spectrograph (SP-2300i, Princeton Instruments), and a CCD camera (IXON,
Andor).^{12 (link),13 (link)} To focus the laser (532 nm wavelength, 5
mW incident power), a 50× objective was used. PEF spectra were
collected with an exposure time of 1 s. A 30 μL sample of the
analyte molecule TMPyP, RhB, or QDs with and without PMMA at a concentration
of 10^–9 M was deposited (drop-casting) above the
aligned FFNTs in the presence and absence of AgNPs. The average of
typically 10 measurements is reported. PEF measurements were performed
during an in situ applied electric field generated
through the application of 0–60 V, in steps of 5 V; the voltage
was applied using a PEW0028 DC power supply, following a process reported
previously.^{12 (link),35 (link)} Electrical cables or bonding
wire was used to connect the microfabricated chip using silver paint,
and then a DC voltage was applied during in situ Raman
measurements as shown in Figure 1a. Relaxation was also recorded after removing the
applied electric field or by applying low electric field values. The
current flow in the microfabricated chip was measured using a TENMA
digital multimeter (72-7725).

Ferrous L16 variants and their complexes with NO were prepared in an anaerobic glove box. Protein was reduced to the ferrous state with excess sodium dithionite. To prepare RR samples of gaseous heme complexes, excess dithionite was removed using a minispin desalting column (Zeba filter, Pierce), followed by introduction of gas into the headspace of septum-sealed capillaries using a gas-tight Hamilton syringe. The identity of RR samples was verified by UV-vis spectroscopy before and after exposure to the laser beam using a modified Cary 50 spectrophotometer. RR spectra were recorded on a custom McPherson 2061/207 spectrograph (set to 0.67 m) equipped with a Princeton Instruments liquid N₂-cooled (LN-1100PB) CCD detector. Excitation wavelengths were provided by the 406.7 nm and 413.1 nm lines of a Kr ion laser and the 441.6 nm line of a He–Cd laser. Rayleigh scattering was attenuated using supernotch filters (Kaiser) or long-pass filters (RazorEdge, Semrock). RR spectra of frozen samples, maintained at 100 K with a liquid nitrogen cold finger, were obtained using a ∼150° backscattering geometry and laser powers of 5–25 mW (at the sample). A 90° scattering geometry was used for RR spectra of room temperature samples. RR spectra were typically measured for periods of 2–5 min with indene and aspirin used to calibrate Raman shifts to an accuracy of ±1 cm^–1.