Pixis 400

Samples were optically pumped at room-temperature with a λ = 532 nm pulsed laser (TEEM Microchip, pulse width 500 ps, spot diameter ~160 μm). The emission was spectrally analysed using a grating spectrometer (Princeton Instruments Isoplane-320) equipped with a 1800 gr mm⁻¹ holographic grating (0.05 nm resolution) and CCD camera (Princeton Instruments Pixis 400). For hyperspectral imaging, a lens in front of the spectrometer was mounted on a translating stage attached to a step motor. The lens was scanned in 25 μm steps and spectral data was recorded for each lens position, resulting in a 3D data set (one spatial dimension × wavelength × lens position). Hyperspectral images were reconstructed from the data set by selecting a particular wavelength of interest and spectra from the entire scanning area was extracted by summing the 3D data set across the two spatial dimensions. Mode control experiments by patterned pumping were carried out using a digital micromirror device (DMD, Ajile AJD-4500), which was inserted in the incoming beam pathway. The illumination pattern was then specified by the pattern projected on the DMD.

The Au nanotriskelions were deposited on cleaned Si substrates with a 300-nm-thick SiO₂ layer. An optical system for single-particle CDS measurements was established based on an Olympus microscope (BX53). The light generated from a quartz-tungsten-halogen lamp (100 W) was adjusted to pass through a linear polarizer (Union Optic, 550–900 nm) and a quarter-waveplate (Union Optic, 550–750 nm). The circularly polarized light then passed through a 100× dark-field objective (Olympus, NA 0.9) to create annular excitation with an incidence angle of 64°. The scattered photons were collected through the same objective and directed to a spectrometer (Acton SpectraPro 2360i) connected to a liquid-nitrogen-cooled charge-coupled-device camera (Princeton Instruments, Pixis 400, cooled to −70 °C). The measured scattering spectrum of a nanoparticle was corrected by first subtracting the background spectrum taken from the adjacent region without any nanoparticle and then dividing the obtained spectrum with the pre-calibrated response curve of the entire optical system. A pattern-matching method was employed to capture the same nanoparticle on the SEM and scattering images. The scattering response of an individual nanoparticle can therefore be correlated with its morphology.

An optical microscope (Olympus,
BX53M) equipped with a monochromator
(Acton, SpectraPro 2360i) and a charge-coupled device camera (Princeton
Instruments, Pixis 400, cooled to −70 °C) was used to
measure the dark-field scattering spectra. A 100× dark-field
air objective (Olympus, numerical aperture: 0.9) was used for scattering
measurements. Light from a halogen lamp passed through the objective
and illuminated the sample obliquely. The backward scattered light
passed through the same objective. Dark-field differential scatterometry
can provide richer information than optical measurements of the solution
samples containing the randomly orientated CGNCs. Circularly polarized
excitation in our experiments was realized by a linear polarizer and
a quarter-wave plate, both of which were purchased from Union Optic.
The working wavelength of the quarter-wave plate (WPA4420–550–750)
is 550–750 nm. The polarization handedness convention used
in this work is such that the RCP and LCP vectors rotate clockwise
and counterclockwise along the propagation axis, respectively. The
wavelengths of the scattering peaks were extracted by fitting the
scattering spectra with Gaussian functions. Extinction spectra were
measured on a PerkinElmer Lambda 950 ultraviolet/visible/near-infrared
spectrophotometer. SEM imaging was carried out on an FEI QF400 field-emission
scanning electron microscope operated at a rate of 20 kV.

A cover glass substrate was cleaned by immersing in a piranha solution (H₂SO₄: H₂O₂ = 3:1) over 30 min to remove organic impurities from the surface. The SERS substrate was prepared by dropping concentrated 100 nm gold nanoparticle colloid onto the cover glass and drying at room temperature. For SERS characterization of the exosomes, 100 μl of the exosome sample (about 10¹² particles/ml) were dropped onto the substrate and thoroughly dried. We measured the SERS spectra with an inverted microscope (Axio Observer D1, Zeiss) and a spectrometer (PIXIS400 and SP2300, Princeton Instruments). A 785-nm wavelength laser was irradiated to the SERS substrate and the reflected spectral signal was detected through a 50x objective lens (NA = 0.70). The laser power was 5 mW and the acquisition time was 10 s. All spectral data were preprocessed for denoising, baseline correction, and normalization. The PCA was performed using the built-in function of MATLAB 2018. Three hundred Raman signal data per sample were used for the classification.

All SERS measurements were performed using an inverted‐type microscope (Axio Observer D1) purchased from Zeiss. The microscope was equipped with a spectrometer (Acton SP2300) from Princeton Instruments. The SERSIA substrate was irradiated with a 1.5‐mW 785‐nm laser, and then scattered light from the substrate passed through the 785 nm filter. A cooled spectrograph detector (PIXIS400, Princeton Instruments) with a resolution of 1340 × 400 pixels was used to scan the Raman spectra. For protein quantification, the acquisition time was 10 s. For protein imaging, the acquisition region was 2 × 2 µm, and the acquisition time was 5 s. The spectral signals were adjusted and denoised using chromatogram baseline estimation and denoising using the sparsity (BEADS) method.^[22] All numerical calculations, including the preprocessing of the spectral data and similarity analysis, were performed using MATLAB R2017a.