2.1.

Cell Culture

Human neuroblastoma dopaminergic cells (SHSY5Y; ATCC, Virginia) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, New York) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, Missouri) and 1% penicillin-streptomycin (Invitrogen, New York) in a T75 flask at 37°C and 5% ${\mathrm{CO}}_2$ in an incubator. The cells were incubated in serum-free conditioning medium and were treated with 300 μM of MPP+ for 24 h in the model of PD. MPP+-induced PD cells were treated with 100 μM of FAC (ferric ammonium citrate, Sigma-Aldrich, Missouri) for either 1 or 6 h to induce iron deposition. Each cell preparation was then washed three times with 1 ml of PBS. The cells were seeded on a cover glass coated with laminin (Roche Diagnostics GmbH, Mannheim, Germany) and grown in a 12-well plate in a 5% ${\mathrm{CO}}_2$ incubator.

2.2.

Sample Preparation

The neural cells on cover glass were fixed for 20 min with a 4% paraformaldehyde solution (Biosesang, Seongnam, South Korea) and rinsed three times with PBS at room temperature. The cells were mounted on a slide using mounting medium (Dako Cytomation, California). The samples were stored at 4°C before imaging.

2.3.

Imaging

A hyperspectral nanoscale optical imaging system was assembled with a VNIR spectral range hyperspectral imager (CytoViva, Inc., Alabama), a high-resolution dark-field condenser (Numerical Aperture; $\mathrm{NA}=1.2-1.4$; CytoViva, Inc., Alabama) and an illuminator system (metal halide light; Welch Allyn Inc., New York) on an Olympus microscope (BX-53, Japan). The construct diagram of imaging system is shown in Fig. 1(a). This system provided $\sim 90\mathrm{nm}$ resolution with an improved contrast and signal-to-noise ratio.¹³ The high-resolution dark-field condenser was directly positioned on the slide glass upon the workstage with the immersion solution and focused until to get a donut ring type in pattern of light. The dark-field optical image of sample was acquired and then hyperspectral absorption signal was recorded in the same position of sample. The images in the slide were obtained and recorded with a CCD camera (Olympus Microsystems, BX-53, Japan).

Fig. 1

Hyperspectral fluorescence signals and mapping images of iron [ferric ammonium citrate (FAC)] dispersed in water. (a) A schematic of the hyperspectral imaging system with visible/near infrared (VNIR) spectral range. (b) VNIR hyperspectral absorption profile of iron. (c) High-resolution optical image of iron with a dark-field nanoscale optical condenser. (d) The mapped hyperspectral signal is shown in red. Almost all points are identically mapped with the iron deposits shown in (c).

2.4.

Data Analysis and Statistics

Region of interest (ROI) of cells tracing and analysis of the hyperspectral absorption signal were performed using CytoViva imaging software and Olympus microscope software (BX-53, Japan). Statistical evaluation was performed using an independent t-test (SPSS19, International Business Machines Corp., New York). Significant difference, *; $p$-value $<0.1$.

3.1.

High-Resolution Hyperspectral Optical Imaging in Cells

We used the high-resolution dark-field imaging and hyperspectral fluorescence signal imaging system for the detection of cellular iron in a cellular model of PD. The imaging system has several compartments for high-resolution imaging up to the several nanometer range, which we have designated nanoscale resolution. These include a VNIR spectral range hyperspectral imager, a dark-field nanoscale optical condenser (numerical aperture; $\mathrm{NA}=1.2–1.4$), and a metal halide light illuminator [Fig. 1(a)]. This imaging system, which was developed by CytoViva, Inc., can visualize dark-field optical images of the sample and record the VNIR hyperspectral fluorescence signal absorption from the intrinsic signal. It can also visualize the fluorescence-tagged biosample. Using this system, we collected the fluorescence absorption signals from iron (FAC) to build a reference signal profile [Fig. 1(b)]. The signals showed peak absorption near 600 nm and varied in intensity; the intensity difference was due to the variable size of the iron aggregates. We also obtained the dark-field optical image of FAC iron [Fig. 1(c)]. This image was scanned with the reference signal, and then mapped in a red color [Fig. 1(d)]. The mapping results were highly correlated in Figs. 1(c) and 1(d).

We mapped the reference signal from the FAC iron deposits in water onto the dark-field image of cells treated with FAC [Fig. 2(a)]. The cells were prepared on a laminin-coated glass slide. The numerous extracellular iron aggregates (bulk iron) that were detected are shown in red color [Fig. 2(a)]. The hyperspectral fluorescence absorption signal profiles from the cells and iron deposits were compared in Fig. 2(b). Each square box was an ROI designated in Fig. 2(a). The extracellular bulk iron exhibited a very-high-intensity signal compared to the cell (cytoplasm, nucleus) or glass. The magnified images of the center of ROIs, including the high-intensity signal from the bulk iron, are shown in Fig. 2(c).

Fig. 2

Spectral profiles of different regions of the cells. (a) Dark-field images of the SHSY5Y cells [1-methyl-4-phenylpyridinium (MPP+)] incubated with FAC for 1 h, with the hyperspectral signal mapping areas marked by colored boxes. (b) The hyperspectral signal intensity of each area. All bulk iron areas have a peak absorbance near 600 nm, whereas the peak signal in the cells is near 500 nm and the signal on the glass plate is almost zero. (c) Magnified images ($17\times$) of the hyperspectral fluorescence signal mapped from the glass, cytoplasm, nucleus, and bulk iron.

3.2.

Cellular Iron Imaging in PD Cell Model

To investigate the distribution of cellular iron in the PD condition, we used the MPP+-induced SHSY5Y cells for an in vitro cell model of PD.⁹ Cells were imaged as above, and the hyperspectral signal of iron was mapped in the cells conditioned using an $\mathrm{MPP}+/\mathrm{FAC}$ treatment. We studied the variation in iron aggregation between a transient exposure (1 h) and a heavy exposure (6 h) to FAC. Figure 3 shows the hyperspectral mapping images of the cellular iron detected in the cells.

Fig. 3

Iron detection in the MPP+-induced Parkinson’s disease (PD) cell model and the dependence of iron aggregation on the time of exposure. The dark field optical images (first row) and iron detection images (second row) in the SHSY5Y cells exposed with MPP+ only (without FAC) and with FAC for 1 or 6 h as a sign in right side. The hyperspectral iron signal on the mapping images indicated by red color (third row). All scale bars: 40 μm.

The magnified mapping images show the detailed cellular location of the iron (Fig. 4). Each cell was exposed to FAC for 1 h. The mapped location of iron was near the edge of most of the cells. Points a, b, c, d, and e are the yellow boxes in Figs. 4(a) and 4(b). The large bulk iron aggregates were dispersed in the extracellular area and several were detected on the cell surface. Many small particle-like iron deposits were located in the cells. Specifically, small iron clusters were found in the dendrites of cells, indicated by blue arrows in the images of d and e in Fig. 4.

Fig. 4

Iron deposition in the MPP+-induced PD model after 1 h of exposure to FAC. The hyperspectral iron signals (a) were mapped onto the cell images (b) with red color. The magnified ($4\times$) and representative images show in the points a, b, c, d, and e image set marked with yellow boxes in (a) and (b). Each a, b, c, d, and e image set has iron signal image (left) and mapped image on a cell (right). The blue arrows in d and e show very small particles. Scale bars (a and b): 40 μm. Scale bars (a, b, c, d, and e): 10 μm.

In addition, we observed an even higher iron signal after 6-h exposure (Fig. 5). Dense iron deposits were detected in the MPP+-induced PD cells, and some of the cells shrank. The high iron accumulation was spread uniformly throughout individual cells and was found in most of the imaged cells [Figs. 5(a), 5(b), and 5(d)]. Locally deposited iron existed along the cell membrane of the shrunken cells [Figs. 5(a), 5(c), and 5(e)].

Fig. 5

Iron deposition in the MPP+-induced PD model after 6 h exposure to FAC. The hyperspectral iron signals (a) were mapped onto the cell images (b) with red color. The representative images of points a, b, c, d, and e show the magnified images ($4\times$) from the yellow boxes in (a) and (b). Scale bars (a and b): 40 μm. Scale bars (a, b, c, d, and e): 10 μm.

3.3.

Quantification of Cellular Iron in the Cellular Model of PD

The hyperspectral signal intensity measurement of iron is easy for quantification method compare with routine iron measuring method of the colorimetric ferrozine-based assay.^10,11 Also, this hyperspectral signal imaging provides the location with quantity of iron which was not offered from colorimetric ferrozine-based assay. Using this intensity measurement, we can relatively quantify the intensity differences between sample groups without cell lysis (Fig. 6). To quantify the iron signal in single cells, the hyperspectral absorption intensity was analyzed for ROIs in each cell [Fig. 6(b) and 6(c)]. The ROI was excluded outside the area of cells. In Fig. 6, the average intensity for a single cell (total intensity for 20 cells) after 1 or 6 h of FAC exposure was 198.6 a.u. (3973 a.u.) and 278.6 a.u. (5572 a.u.), respectively. The 6-h group showed statistically high cellular iron deposition compared with little accumulation in the 1-h group.

Fig. 6

Comparison of the total iron signal intensity (left) and mean iron signal intensity (right) between exposure time of 1 and 6 h (a). The regions of interest (ROIs) analyzed are shown in the hyperspectral iron signal image of (b) (1 h) and (c) (6 h).

Cellular iron was dispersed in a small, particulate form, whereas the extracellular iron was detected in an aggregated form. Additionally, iron particles were found to be concentrated around the edge of the cell membranes of shrunken cells. Cellular iron accumulation was easily triggered in MPP+-induced cells, mirroring the previous studies and demonstrating the elevated iron levels in the SN of PD.^2–7,9,19

In further studies, we will investigate which transporting pathway is mainly related to cellular uptake of iron in the cellular PD model. The extracellular iron can be transported into the cell via iron transporting proteins such as divalent metal transporter 1, ferroportin 1, transferrin receptor, and these transporters were upregulated in the PD models.^9,20,21 Intracellular iron uptakes and overloads might be correlated with overexpression of these proteins. Therefore, the colocalization study of various iron transporters and cellular iron mapped using hyperspectral fluorescence signal imaging will give a better understanding of the pathophysiological processes in PD progression.