Plasmon-induced light concentration enhanced imaging visibility as observed by a composite-field microscopy imaging system

A composite-field microscopy imaging (iCFM) system is constructed to observe the plasmon-induced light concentration (PILC) effect and to enhance imaging visibility.

SUPPLEMENTARY INFORMATION SECTIONS 1 TO 6.

Optical path refit of the condenser for iCFM system.
Prior to the refit of the illumination optical path of dark-field microscopy imaging (iDFM) system to build the composite-field microscopy imaging (iCFM) system, we firstly identified the thickness of the embowed refractive element in the axis of U-DCD dark-field condenser. After ensured the thickness was ~1.0 mm (noted by red double slash in Fig. S1a) which was ease of casting and might bring little influence to the concentrating effect of the condenser, we dug a round hole ( = 5 mm) at the arc of the embowed refractive element (Fig. S1c). In order to adjust the introduced light to a monochromatic light with a controllable intensity and colour, a filter and a set of neutral density attenuators were installed into the inner of the U-DCD condenser through the fixation of the custom optical bracket ( Fig. S1d and e).
The optical lens ( = 6.8 mm) were placed in a pipe which had internal threaded and was connected to outer cylinder by three symmetrical distributed connecting arm. Between the adjacent connecting arms there was 120 degree intervals to reduce the light intensity difference on the light ring of the oblique dark-field illumination as much as possible. After the excavation of the hole, the dark-field effect was obtained by a black lens in the same size and was also fixed to a circular metal frame ( = 7.0 mm) in the same manner with other optical lens (Fig. S1e). The optical parameters of the optical lens were showed in Table S1 and S2.
With the refit condenser, monochromatic light was indeed obtained as shown in Fig.   S2. To better display the illumination principle of the refit optical path, we then make the formed monochromatic background visible with the help of a common coverslip and the light path schematic diagram was showed as Fig. 3a. It was the"Tyndall effect" of the impurities on the coverslip mapped out the formed composite spot, containing a white focal point and a round monochromatic spot (Fig. S3c-e). In the central, the inherent white focal point of dark-field illumination was also seen, suggesting the introduced monochromatic illumination brought a superimposed imaging effect of a monochromatic field (Fig. S2).   Attenuator Actual transmission in 400-800 nm 1% 0.0% < T < 1.7% 5% 4.5% < T < 7.7% 10% 9.8% < T < 10.3%     The used gold nanorods (AuNRs) in Fig. 2c were prepared with a regrowth method 2 .
The seeds used in the regrowth process was firstly prepared according to the previous

Synthesis and characterization of the SiO 2 nanoparticles.
The SiO 2 nanoparticles in size of 118.1 nm were prepared according to the previous reported modified Stöber method and with some modification 4   The size and morphology of these two SiO 2 nanoparticles were characterized by SEM and dynamic light scattering (DLS) measurement (Fig. S5a-c). The average size of these two SiO 2 nanoparticles were 118.1 ± 14.6 nm and 180.5 ± 14.0 nm. From the extinction spectra, both of these SiO 2 nanoparticles had no characteristic peaks in the whole visible area (Fig. S5d). The scattering imaging intensity of the SiO 2 nanoparticles in size of 118.1 nm was weak, and that of the SiO 2 nanoparticles in size of 180.5 nm was much stronger and suitable as the control nonplasmonic nanoparticles ( Fig. S5e and f).

Scattering properties of the AgNPs, AuNRs and the SiO 2 nanoparticles.
The scattering spectra of the single plasmonic or nonplasmonic nanoparticles were measured by connecting a spectrograph (MicroSpec-2300i, Roper Scientific) and an intensified CCD camera (PI-MAX, Princeton Instrument) mounted onto the BX51 darkfield microscope. Both 10 nanoparticles of the AgNPs and AuNRs were measured and their corresponding image and spectra was showed in Fig. S6 and S7. The average maximum scattering wavelength of AgNPs was about 453.6 nm and that of the AuNRs was about 631.8 nm. Both of these maximum scattering wavelength were consistent with the blue and red colour. The measured spectra of these plasmonic nanoparticles suggested that the blue and red filters was suitable.
Different from the scattering spectra of AgNPs and AuNRs with sharp peaks, the scattering spectra of SiO 2 nanoparticles in size of 180.5 nm was bread peak and was consistent with their scattering images with mixed colours in Fig. S5f (Fig. S8).   3. Imaging of plasmonic nanoparticles by the newly developed iCMF system.

Imaging operation by iCMF system.
The iCFM system based on the refitted condenser was used in the same manner with the original U-DCD condenser and the colour and intensity of the introduced monochromatic light were controlled by changing of the optical lens showed in Section 1, as a result, the nanoparticles, for example of the blue AgNPs, could be imaged with different imaging modes as Fig. S9 showed. In the imaging of the same nanoparticles under different imaging modes, the objective lens and the used homemade sample trough made by glass slide and coverslip 6 were kept standing motionless to maintain the focal length along the whole imaging process. No matter imaging with the oil-immersed or the dry type objective lens, the objects above the glass slide remained intact.

Enhanced visibility evaluated by RGB analysis.
Structural similarity (SSIM) system 7 can impersonally reflect the image quality, mainly from the perspective of the luminance measurement ( x ,  y ), the contrast measurement ( x ,

Enhanced visibility evaluated by RGB analysis.
The  (b), (d) Scattering images and RGB line distribution of the red nanoparticles in dark-field (b1, d1) and red, green and blue background imaging (b2-b4, d2-d4).

Scattering imaging of AgNPs with a filtered monochromatic light source.
The imaging with the refit condenser was different from the imaging illuminated by a monochromatic light source which was achieved by the filtration of the light source with a blue broadband filter (Fig. S11). In the monochromatic background imaging with the refit optical path, the scattering intensity of the nanoparticles scattering light in same colour was increased, however, the intensity was decreased by this filtering methods due to the reducing of light source intensity instead of enhancing the utilization efficiency (Fig. 12).

Three-dimensional model and high-pass output of the scattering images.
The three-dimensional model of the scattering images was obtained by the "Surface Plot" function of Image-Pro Plus 6.0 Software. The principle of this function was grayscale conversion of the images and changing the image to a three-dimensional model with a variable angle. A typical rotation model was obtain by two procedure, elevation and rotation, and the process was showed in Fig. S13 in details.
The three-  Then, the whole picture is rotated degree clockwise and the last existing form (red rectangle) was obtained.

Scattering imaging of SiO 2 nanoparticles with the iCMF system.
From the scattering imaging and the RGB line distribution of SiO 2 nanoparticles in size of 180.5 nm, no obvious visibility enhancement was found (Fig. S15). The integral scattering intensity ((R+G+B)/3) of SiO 2 under different monochromatic imaging mode was almost the same as that under the dark-field imaging mode, and this was indeed different from that the imaging visibility of plasmonic nanoparticles was optimal under the same coloured monochromatic imaging mode (Fig. S16).   To further aware the light concentration of plasmonic nanoparticles, the electric field distribution of all the above nanoparticles were calculated and showed (z direction). All the nanoparticles were assumed to be embedded in water with a refractive index of 1.333.

Finite
From the calculated results showed in Fig. 3c-f, the AgNPs and AuNRs indeed had much larger extinction and scattering cross-section area than their physical cross-section area at their corresponding LSPR wavelength. Differently, the scattering cross-section areas of SiO 2 nanoparticles were much smaller than their physical cross section areas. Besides, around the SiO 2 nanoparticles, there were no local field enhancement, however, the field enhancement effect of AgNPs and AuNRs were significant. All the aforementioned results suggested that the light concentration effect was special property of plasmonic nanoparticles.

Wavelength-dependent scattering efficiency of AgNPs and AuNRs.
For spherical nanoparticles, the scattering efficiency could be given by Equation (S1) 8 , where k = 2 ( o ) 1/2 /s was the cross-section area,  1 ,  2 and  3 were the polarizabilities at the long axis direction and two perpendicular short axis. The  n could be described by the following general formula as Equation (S3) 9 , where a, b and c was the semiaxis of the ellipsoid. As the transverse cross-section of nanorod was considered as a circle, so c > a = b. L n was as Equation (S4) 9 , (S4) In L 2 and L 3 , a was replaced by b and c, respectively.

Single nanoparticle spectra of AgNPs under different imaging modes.
The single nanoparticle scattering spectra of a blue and a red nanoparticles under the iDFM and iCFM illumination systems were measured and the results showed in Fig. 5b. It could be seen that the scattering spectra of the blue or red nanoparticles were kept in the nearly similar wavelength. Besides, the RGB value of the dominant colour and the total RGB value showed in Fig. S17 and S18 were consistent with the RGB analysis results is Fig. 3.     In imaging of the nanoparticles in the cancer cell, which was a complex environment, the nanoparticles might be located in different geometric level, so the depth of field was important in such case. Therefore, in imaging of BSA protected AgNPs, the used objective lens was a 100× oil immersion objective with a NA of 1.30, However, a 40× non-oilimmersed objective lens with a NA of 0.60 was used instead of the 100× oil-immersed objective lens in the imaging of the silver nanoparticle aggregates which had a different spot dimension with the dispersed AgNPs. In this way, the coexistence of these two existing state could be easily obtained.
The imaging results were showed in Fig. S21 and the protection of the BSA was obvious from the scattering imaging. Among these imaging modes with the dry type condenser, it was also that the imaging with the corresponding monochromatic background imaging displayed the optimal quality and visibility. The RGB line distribution analysis of a single blue nanoparticle and a single red nanoparticles also revealed the same results (Fig.   S22).