Natural light is essential for the adjustment of circadian clocks and the coordination of cellular physiology. Nevertheless, emerging evidence suggests that increased exposure to artificial light can significantly influence important characteristics of cells. It has been shown that blue light at lower doses (≤300 mJ/cm2
) triggers the synthesis of hydrogen peroxide in T-cells and affects cell motility [1
]. Other studies suggest that blue light changes the redox state of the cell causing oxidative stress and alter mitochondrial activity [2
] or that green light can change gene expression [4
]. As recent technological advances brought televisions, LEDs, and displays, blue light emitters have become a part of our everyday life and blue light is also used for polymerization of dental resin in dentistry [5
], its effect on the behavior of cells should be deeply studied. Studies on model organisms suggest that visible light can exert a range of detrimental effects. For example, a single acute blue light exposure causes cell death of photoreceptors in the retina of mice and flies [6
]. Blue light is also used in photodynamic therapy (PDT) of cancer. PDT represents an important treatment approach as it uses a photosensitizer and visible light to induce reactive oxygen species (ROS) to kill cancer cells [8
]. For example, Sullivan et al. have reported the antitumor effect of an agent dequalinium and light of a 366 nm wavelength. A combination of specific light and an agent inhibits protein kinase C and induces inhibition of metastatic behavior of melanoma cells [10
]. However, the effects of blue light itself on the essential characteristic of cancer cells, such as motility or cell division is not known.
The light with 488 nm is commonly used as an excitation light in fluorescence microscopy (FM) which allows precise imaging of cellular structures or even certain molecules in cells [11
]. Despite the widespread of FM in cell research in the last 40 years, the impact of such techniques on basic cell features was not well characterized yet. In this study, we focused on the effect of light with 485 nm wavelength on the dynamical and morphological properties of the three human cancer cell lines established from different tissue (PC-3, A2780, G361) and one non-malignant human prostate cell line (PNT1A), respectively. In our experiments, we use a multimodal holographic microscope (MHM) that combines holographic microscopy with fluorescence microscopy. The MHM used in this study is based on an off-axis setup with an incoherent source of light which enables high-quality quantitative phase imaging, avoiding disruptive interferences [13
]. Holographic microscopy (HM) has been demonstrated as a powerful tool for long-term and label-free imaging of cell samples in vitro, as it works on the principle of quantitative phase imaging (QPI). HM provides information about many cellular processes and parameters such as cell cycle [14
], cell kinetic [15
], viability [16
], cell morphology [17
], and cellular uptake [18
]. This approach enables also to measure cell mass and its distribution in cells [19
]. Based on QPI, a new approach of distinction of apoptosis and lytic types of cell death was developed [22
]. HM also enables effort-less and more accurate tracking of cells and their motility patterns compared to other microscopic techniques [24
]. Our study aims to present QPI and holographic microscopy as a suitable method for the evaluation of cellular morphological and dynamical parameters. The results suggest that HM within advanced image analysis is capable of correctly distinguishing between changes in cell motility, cell dry mass, cell density, and cell death induced by blue light and that the impact of light is dependent on the exposure time and light intensity.
2. Materials and Methods
2.1. Chemical and Biochemical Reagents
RPMI-1640 medium, Ham’s F-12 medium, penicillin/streptomycin, fetal bovine serum (FBS) (mycoplasma-free), ethylenediaminetetraacetic acid (EDTA), were purchased from Sigma Aldrich Co. (St. Louise, MO, USA). Trypsin was purchased from PAA Laboratories Gesellschaft M.B.H. (Pasching, Austria) and phosphate buffer saline (PBS) from Invitrogen Corp. (Carlsbad, CA, USA).
2.2. Cell Lines
The PC-3, A2780, PNT1A, and G361 cell lines were purchased from HPA Culture Collections (Salisbury, UK). PC-3 prostate cancer cell line was derived from bone metastasis of a 4-grade prostatic adenocarcinoma of a 62-year-old Caucasian male [26
]. The A2780 cell line was derived from the ovarian carcinoma of a nontreated patient according to ECACC. PNT1A cell line was established from prostatic epithelial tissue of healthy 35-years old male and immortalized by plasmid transfection containing the SV40 genome with defective replication origin [27
]. The G361 cell line was established from a malignant melanoma of a 31-year-old male Caucasian. The G361 cells produce melanin for up to 50 population doublings. As the aim of this study is to compare the effect of blue light on the cell lines differing by morphology, transformation state, sensitivity to cell death, and origin, we decided to use the cell lines listed above. PC-3 cells are larger in comparison with small A2780 cells. Benign PNT1A cell line differs from malignant PC-3, and all four cell lines are derived from diverse tissues of origin. Furthermore, melanoma G361 cells expressing melanin may differ in the reaction of cells to blue light exposure.
2.3. Cell Cultivation
All four cell lines were cultivated in 25 cm2 flasks with 5 ml of media at 37 °C in a humidified incubator (60%) with 5% CO2 (Sanyo, Osaka City, Japan). Cell lines A2780, PNT1A and G361 were cultured in RPMI-1640 medium with phenol red indicator, L–glutamine, FBS and antibiotics penicillin/streptomycin (Sigma Aldrich Co., St. Louise, MO, USA). For the PC-3 cell line cultivation, Ham´s F-12 medium with FBS and antibiotics (Sigma Aldrich Co., St. Louis, MO, USA) was used. The same supplementation with antibiotics (penicillin 100 U/mL and streptomycin 0.1 mg/mL) and 10% FBS was used in both media. The cell medium was changed two times per week. Cell subculturing was done with 10% of trypsin solution (PAA, Pasching, Austria) with previous washing with EDTA (0.02% in PBS buffer).
2.4. QPI and Holographic Microscopy and Fluorescence Setting
QPI was performed by using a Q-PHASE multimodal holographic microscope (Telight, Brno, CZ). The Q-PHASE is equipped with fluorescence module using a halogen lamp as a non-coherent source of blue light. In this work, the module was used as a source of blue light for treatment of observed cell lines. The 485 nm light waves are emitted by the fluorescence light source of the attached module. The Q-PHASE microscope optical setup can be seen in Figure 1
of the article [22
]. Before the imaging experiment, cells were cultivated overnight in a concentration of 7000 cells/mL in flow chamber µ-Slide I Lauer Family (Ibidi, Martinsried, Germany). During the measurements, the chamber with cells was incubated in 37 °C humidified, 5% CO2
atmosphere in H201–for Mad City Labs Z100/Z500 piezo Z-stages (Okolab, Ottaviano NA, Italy). Images and holograms were captured with lens Nikon Plan 10/0.3 and CCD camera (XIMEA MR4021 MC-VELETA, Münster, Germany) respectively. The fluorescence mode used was a plasma light source (Sutter Instrument Lambda XL Novato, CA, USA). Cells were irradiated with a 485 nm light with a 25 nm bandwidth. Light doses 0 mJ/cm2
, 24 mJ/cm2
and 208 mJ/cm2
were achieved by the combination of time exposition and light intensity shown in Figure 1
The images were acquired automatically from seven positions every 3 min for 24 h. Holographic images were collected by custom software and raw data were numerically reconstructed. The numerical reconstruction was performed by custom software where the established methods of the fast Fourier-transform [29
] and phase unwrapping [30
] are implemented. The output from the software is an unwrapped phase image. This image has high intrinsic contrast and can be processed by an available image processing software. The unwrapped phase image is integrated phase shift through the cell and it is proportional to integrated cell dry mass density [19
]. The values of unwrapped phase image φ
(rad) are linearly transformed into cell dry mass density image m
) with the transformation
where α (0.18 μm3
/pg) is specific refraction increment and λ (0.65 μm in Q-PHASE) is a wavelength, where both values are assumed to be constant. This enables measurements of cell dry mass and analysis of cell mass distribution inside cells.
2.5. Image Analysis and Statistics
Image analysis was performed in customized Matlab R2019b (MathWorks, Natick, MA, USA) algorithm developed by our laboratory. The analysis process consists of segmentation, the interconnection of matching cells in adjacent time frames and extraction of the analyzed dynamical and morphological cell features. For cell segmentation in each frame, Loewke’s Iterative Thresholding (LIT) method [33
] was combined with Empirical Gradient Thresholding (EGT) technique [34
]. EGT was shown to be a very robust and parameter-free foreground cell segmentation method (semantic segmentation) across various microscopical modalities [35
]. On the other hand, LIT is a powerful technique for single-cell separation (instance segmentation), which is specifically designed for QPI images. LIT iteratively separates objects in “intensity valley” if the mass of separated objects is larger than the selected cell mass threshold. Our segmentation procedure is summarized in Figure 2
QPI image foreground can be segmented with simple thresholding; however, we found that a combination of EGT and thresholding can lead to even better results. First, EGT (with default setting) is used for foreground segmentation and its resulting foreground segmentation mask (FSM) is further improved by the inclusion of pixels brighter then foreground_threshold_1 and exclusion of pixels darker than foreground_threshold_2. Furthermore, holes smaller than hole_area_threshold and objects smaller than mass_threshold are removed.
In LIT, each grayscale foreground object masked by FSM (containing one or more cells) is iteratively thresholded, where the threshold increases in every iteration. When thresholding creates two (or more) objects and newly created objects have a larger mass than mass_threshold
, they are used as new individual objects, which can be further split with the increasing threshold (and the original object will not be used). Finally, the object in FSM is separated by watershed lines, which are created by the application of the watershed algorithm [36
] on Euclidean distance transform [37
] of masks of objects created with thresholding. In original LIT [33
], threshold grows until it reaches maximum image intensity, which leads to over-segmentation in some cases, thus we have introduced a maximal_LIT_threshold
parameter and iterations are stopped when the threshold reaches this value.
Parameters foreground_threshold_1, foreground_threshold_2 and maximal_LIT_threshold were set for all cell lines to value 0.07, 0.4 and 2, respectively. Hole_area_trehold and mass_treshold were manually adjusted for each cell line (but stay fixed for all light doses).
Sufficiently, precise tracking of individual cells during the whole sequence is a very hard problem [23
]; however, for analysis of our dynamical features statistics across the whole-cell set, we require only to correctly cross-connect cells between each two consecutive time frames and larger tracks are not required. This was achieved by searching for the largest overlap with respect to Intersection over Union (IoU) of segmentation masks, where everything with IoU smaller than 0.7 was discarded as segmentation errors or cell divisions and thus these cells were not used for calculation of dynamical features. As dynamical features describing cell motility, we have extracted cell speed (Euclidian distance between cell centroid in two consecutive frames) and Cell Dynamic Score (CDS) defined in [23
] (Euclidean distance of cell pixel values in two consecutive frames). Cell dry mass and cell density are calculated as a sum and a mean of cell pixel values, respectively.
Statistics was performed in R version 3.6.1 (R Core Team, 2020) [38
] using ggpubr 0.2.4 (CRAN, 2020) package [39
] with Wilcoxon test. Unless noted otherwise, p
level < 0.05 was considered significant.
Light emitters such as LEDs, TV and other electronics are presently used daily. Also, photodynamic therapy and fluorescence microscopy (FM) uses light to cause-effect on biological samples. Despite the widespread of FM in cell research, the effect of light on basic cell features was not well characterized yet. In this study, we evaluated the effect of blue light (485 nm) on cell parameters such as proliferation activity, cell motility, and cell mass in three human cancer cell lines (A2780, PC-3, G361) and one non-malignant human cell line (PNT1A) using MHM. The light doses in our study were 24 or 208 mJ/cm2
and were achieved by a combination of two different times of expositions 500 and 1000 ms and specific light intensity (Figure 1
). In the last 15 years, holographic microscopy hand in hand with advanced image analysis made significant progress to become a suitable technique for conducting label-free time-lapse experiments with biological specimens.
Our results show that blue light 208 mJ/cm2
× 1000 ms negatively affect malignant cell motility and does not change the motility of benign PNT1A cells. Phan et al. have described the positive effect of 470 nm light on the motility of T-cells. Enhanced motility may be caused by the H2
signaling pathway [1
]. Our finding follows Oh et al., where authors concluded that blue light inhibits cell migration, thus invasivity of cancer cells. This may be mediated by the effect on MAPK and NF-kB activity and down-regulation of the ERK1/2 signaling pathway which leads to a decrease in protein expression of MMP-2 and MMP-9 [40
]. On the other hand, Lan et al. postulated that blue light gradient could have an impact on cancer cell migration. According to their study, ROS production is proportional to light exposition. Cancer lung cells A549 exposed to 473 nm blue light gradient reported higher ROS production but also higher motility driven by increasing light gradient [41
]. However, this study employed a gradient of light which may have a different effect on cells as the light dose on cells varied during the exposition. It was shown that blue light generates oxidative stress [42
], induce cell damage and affects the gene expression [43
Our results also show that blue light significantly affects cell proliferation, in most cases negatively. Although some light doses such as 24 mJ/cm2
× 500 ms in A2780 and PC-3 cells showed some proliferative effects these effects showed no statistical significance. Some proliferative effects of light have previously been described. Moore et al. registered a positive effect of light on the proliferation of endothelial cells and fibroblasts but those results are not comparable with our results since a low-intensity laser with wavelength 665 nm was used [44
]. The higher light doses affect cell proliferation more than lower ones. The least harmful dose for our cells was 24 mJ/cm2
× 500 ms, therefore this dose can be used for fluorescence microscopy of these cells. Oh et al. [45
] described the reverse effect of light on cell proliferation, where blue light exposition leads to the activation of caspase-3 and PARP inducing a caspase-dependent apoptotic pathway [45
To correctly evaluate the effect of the blue light used in our experiment, it is necessary to correlate with cell mass distribution. Interestingly, the cell mass in A2780 decreased quickly after exposure to blue light dose 208 mJ/cm2
× 1000 ms, whereas the cell mass in PC-3 increased. The increase of cell mass implies higher anabolic activity in the metastatic PC-3 cells, which can serve as a mechanism of survival in harmful conditions. As the number of PC-3 cells remains the same from the beginning of the experiment, cells were not undergoing cell death, as the results could suggest, but only suppress their proliferation activity. This finding supports a study from Gilchrest et al. which reports that UV light induces mitosis via the creation of protecting factors which may lead to avoiding apoptosis [46
] and also Tolde at. el. indicate that changes in cell mass distribution may play a role in cell motility [47
]. On the contrary, A2780 cells showed a significant decrease in cell mass even though the number of cells decreased only slightly. The disproportionality of these two parameters is evidence of cell death because the automatic algorithm of the MHM tracks also dead cells during the experiment as previously described in Vicar et al. [23
]. This is following our observations where is evident that A2780 cells were killed during the first hours of the experiment (Supplementary Video S2
Since QPI has recently been used for detection and classification of cell death [16
], based on these results, we hypothesized that light dose 208 mJ/cm2
× 1000 ms induce different types of cell death in A2780 and G361 cell lines. Based on the time-lapse videos (Supplementary Videos S2 and S4
) and real-time quantitative parameters shown in Figure 6
, it is evident that light dose 208 mJ × 1000 ms induces specific changes in cell dry mass, CDS, and density in A2780 and G361 cells. For the A2780 cells, all three parameters steeply decreased in the first six hours of the experiment and remained low. The data correspond with the videos, as it shows that the effect of the light dose on A2780 cells is fast and cells are dead. The dead cells are still adherent to the surface with the disrupted plasma membrane and there is an absence of blebbing or releasing of apoptotic bodies. This may suggest the cells underwent necrosis as these parameters are characteristic for this type of cell death [49
]. On the other hand, G361 cells show a stable slight increase in cell dry mass and density, which decreased in the last two hours. CDS decreased after eleven hours of the experiment. The results for G361, supported by the video (Supplementary Video S4
), imply that cells underwent apoptosis. These cells showed gradual cell rounding and a loss of surface contact followed by intensive membrane blebbing. It corresponds with known facts describing morphological changes during apoptosis [51
]. Recently, several studies have described the characterization of cell death based on cell morphology using quantitative phase imaging [16
]. In particular, mapping of the quantitative phase changes during the time seems to be very promising in the determination of various morphological phenomena related to cell death, because the responsible cell death molecular machinery typically changes the distribution of the cell mass (cell content cleavage by caspases and loss of attachment during apoptosis connected with increased density; cell swelling during some kinds of lytic cell death—decrease in cell density) [23
This study could also contribute to a better understanding of how light affects cells during fluorescence microscopy experiments, as the wavelength employed in our study is widely used. From our results, it is evident that cells were not damaged with the lower doses of light with a shorter time exposure. Thus, there is a need for low fluorescence doses and time exposures to achieve less destructive conditions for live samples during fluorescence microscopy. On the other hand, fluorescent microscopy always compromises between fluorescence damage to live samples and image quality.