Investigation of the Nonlinear Refractive Index of MCF7 and MCF10A Breast Cell Lines for Optical Diagnosis

Bahareh Khaksar Jalali ¹ ( Department of physics, Kharazmi University, Tehran, Iran )
Somayeh Salmani Shik ² ( 1. Department of physics, Kharazmi University, Tehran, Iran. 2. Applied Sciences Research Center, Kharazmi University, Karaj, Iran. )
Latifeh Karimzadeh Bardee ³ ( Department of animal biology, Faculty of biological sciences, kharazmi university, Tehran, Iran. )
Sharife Shahi ⁴ ( 1. Department of Biomedical Engineering, Islamic Azad University of Isfahan (Khorasgan) branch, Isfahan, Iran. 2. Laser and Biophotonics in Biotechnologies Research Center, Islamic Azad University of Isfahan (Khorasgan) branch, Isfahan, Iran. )

Submited date : 2022-10-12 Accepted date : 2023-01-31

Keywords: MCF7, Z-scan technique, Cancer Diagnosis, Linear Absorption, MCF10A, Optical Coefficients,

Abstract :

To demonstrate that the Z-scan approach is a reliable tool for diagnosing normal and cancer cells, the absorption coefficient (α) and nonlinear refractive index (n2) of normal (MCF10A) and cancer cell lines (MCF7) were examined as linear and nonlinear optical properties. Cancer cells had a higher absorption coefficient than normal cells, according to the findings. Cancer cells exhibit a greater nonlinear refractive index (n2) and distinct signs, despite being in the same order (10-7 cm2/W). This method is a quick method for differentiating between normal and cancerous breast cells, making it suitable for use in clinical stages in the future. This is because of the obvious fundamental difference between the optical behaviour of normal and cancer cells, as well as the affordability, the potential for reproducibility without compromising sample quality, and the high accuracy.

Full-Text:



        

Investigation of the Nonlinear Refractive Index of MCF7 and MCF10A Breast Cell Lines for Optical Diagnosis

 

B. Khaksar Jalali a, b, *, S. Salmani Shik a, c, L. Karimzadeh Bardeei d, Sh. Shahi b, e

 

a Department of physics, Kharazmi University, Tehran, Iran.

b Laser and Biophotonics in Biotechnologies Research Center, Islamic Azad University of Isfahan (Khorasgan) branch, Isfahan, Iran.

c Applied Sciences Research Center, Kharazmi University, Karaj, Iran.

d Department of animal biology, Faculty of biological sciences, kharazmi university, Tehran, Iran.

e Department of Biomedical Engineering, Islamic Azad University of Isfahan (Khorasgan) branch, Isfahan, Iran.

 

 

 

 

 

Abstract: To demonstrate that the Z-scan approach is a reliable tool for diagnosing normal and cancer cells, the absorption coefficient (α) and nonlinear refractive index (n2) of normal (MCF10A) and cancer cell lines (MCF7) were examined as linear and nonlinear optical properties. Cancer cells had a higher absorption coefficient than normal cells, according to the findings. Cancer cells exhibit a greater nonlinear refractive index (n2) and distinct signs, despite being in the same order (10-7 cm2/W). This method is a quick method for differentiating between normal and cancerous breast cells, making it suitable for use in clinical stages in the future. This is because of the obvious fundamental difference between the optical behaviour of normal and cancer cells, as well as the affordability, the potential for reproducibility without compromising sample quality, and the high accuracy.

Keywords: Cancer Diagnosis, Linear Absorption, MCF7, MCF10A, Optical Coefficients, Z-scan Technique

 

 

I. Introduction

Today, no one is unaware of the biology behind a common illness like cancer. In order to improve and increase the effectiveness of therapies, there has to be more information and study in this area [1]. This will lead to a more precise, trustworthy, and early diagnosis. In the world, cancer is a highly widespread illness. The recognized causes of cancer exacerbation include a number of risk factors, including as environmental influences, inherited traits, and gene abnormalities resulting in mutations [2]. Female breast cancer, lung cancer, and prostate cancer are the most commonly diagnosed cancers worldwide, according to a compilation of International Agency for Research on Cancer (IARC) GLOBOCAN cancer statistics based on reliable data from population-based cancer registries (PBCRs) and the World Health Organization (WHO) mortality database for the year 2020 [3]. Furthermore, after lung cancer, breast cancer is the second greatest cause of cancer death in women [4]. As a result, prevention, early detection, and high-quality therapy can help minimise mortality [5].

Many ways to diagnosis and therapy have been proposed, including the use of lasers and the investigation of the optical behaviour of materials [6, 7], because the analysis of the optical interaction between the laser beam and the tissue can lead to a wide range of applications [8].

The accuracy of the diagnosis is crucial for determining the best course of therapy, lowering mortality, and affecting the disastrous statistics brought on by cancer [9, 10]. As a result, diagnosis is of particular relevance in today's cancer research. One frequent technique for detecting breast cancer is the examination of chromatin interactions at the genome level. This approach is suggested because each chromosome prefers a specific location within the nucleus that is not always stable and because gene-dense chromosomes typically live there whereas gene-poor chromosomes are found close to the nucleus' periphery. In addition, HER2 (human epidermal growth factor receptor 2) protein status can be determined using the immunohistochemistry (IHC) technique. By examining the correlation between HER2 status and morphological characteristics, chromogenic in situ hybridization (CISH) and fluorescence in situ hybridization (FISH) of HER2 copy number variations are methods to assess gene amplification. However, all of these procedures are time intensive, necessitate specialised training, incur health-care costs, and necessitate additional peripheral equipment and particular filters that are not readily available in many places [11-13]. As a result, it appears ideal to have an accurate, quick, and low-cost diagnostic approach for all forms of cancer.

The Z-scan technique, developed by Sheikh Baha'i et al. in 1990 to assess nonlinear optical characteristics, is one of the most exact, practical, and dependable optical techniques utilized in biomedical diagnostics today [14]. This optical method has been demonstrated to be a sensitive technique for quantifying a wide range of LDL (low-density lipoprotein) values. The nonlinear optical response is influenced by different fractions of LDL, such as free and esterified cholesterol, triglycerides, fatty acids, and phospholipids. The phospholipid molecules from the outer shell, of course, appear to be the principal contributors to the nonlinear optical observations on LDL. Because oxLDL does not exhibit a Z-scan signal, the oxidation process has significantly affected this fraction of LDL, affecting the size of the nonlinear response. The amplitude of the nonlinear response of LDL particles, on the other hand, increases with temperature. These findings suggest that this method could be used as a spectroscopic tool to analyse the structure of LDL. Another method of diagnosing heart disease was proposed in 2006 by analysing the thermal nonlinear optical response of human normal and copper-oxidized low-density lipoproteins (LDLs) with the Z-scan technique as a function of temperature and LDL particle concentration. This method permits the detection of changes related to inflammatory stress and can thus indirectly measure the stability and susceptibility of LDL, associated to the presence of heart disease risk [15]. The amplitude of the Z-scan signal rose linearly with LDL concentration in accordance with the Beer-Lambert rule [16]. In 2010, Dhinaa and Palanisamy used the Z-scan approach with 532 nm Nd:YAG CW and 633 nm He-Ne lasers to measure total protein and albumin. Their findings exactly matched those of the traditional colorimetric method. Additionally, they demonstrated in the other study that the 532 nm Nd:YAG CW method provided adequate results in comparison to the traditional colorimetric method for displaying the nonlinear optical properties of some other bioanalytes, such as urea and uric acid in standard and blood samples [17, 18].

Creatinine levels in the blood rise as a result of renal cleansing failing. Renal problems are more likely to occur with an increase in creatinine. Renal failure is always evaluated by measuring the blood creatinine level using conventional assays, the Jaffe technique, and enzymatic procedures. Ghader et al. made the first nonlinear refractive index calculation of the plasma creatinine levels in humans. They demonstrated that their results had sufficient precision and were equivalent to the conventional Jaffe approach when they were repeated numerous times [19]. Ghader et al. (2018) looked at the possibility of the Z-scan approach as a highly accurate, low-cost, and quick diagnostic optical method to define abnormalities in two different breast cancer cell lines, SK-BR-3 and MCF7. They asserted that each cell line's distinct nonlinear refractive index can be used as a marker to distinguish between various breast cell lines [20]. Additionally, certain validated data from the Majles ara research group's application of the Z-scan approach for early diagnosis of specific human brain (U87MG) and breast (mda-mb-231) malignant tumour cells were published in 2019 and are compatible with these findings [21, 22].

For the first time, a very precise and repeatable diagnostic method for distinguishing between healthy breast cells and several cancer cell types was presented in this study. Despite the fact that MCF10A and MCF7 are both epithelial, research into the relative arrangement of chromosomes in the interphase nucleus has allowed for the detection of both their normal and abnormal conditions. In this regard, a higher background interaction frequency has been detected in the MCF7 genome compared to the MCF10A genome; this may be due to an increase in the variety of interactions within this genome.  Higher-order chromatin structure is messed up in cancer cells. The intra-chromosomal connections between breast cancer cells and breast epithelial cell types varied significantly as well. Additionally, due of structural variations, some elasticity or subcellular structures developed [23, 24]. These breast epithelial cells were classified as normal cells and the malignant breast epithelial cell line (MCF7) since the MCF10A cell line is a non-tumorigenic cell line [25, 27]. The major goal of this work is to assess the optical characteristics of cells to make a non-invasive in-vitro distinction between breast cancer cells and the matching normal cells.

 

II. MATERIAL AND METHODS

A.       Sample preparation

 

 

 

Fig. 1 Preparation of transparent samples by fixing cells on lamellar.

 

Iranian Biological Resource Center provided the human mammary cell lines MCF7 and MCF10A. They were cultivated in individual cell culture flasks with Dulbecco's Modified Eagle's medium (DMEM-Gibco), 10% fetal bovine serum (FBS-Gibco), 100 units/ml penicillin, and 100 μg/ml streptomycin antibiotic (Sigma-Aldrich). DMEM culture medium at 37°C in an incubator with 5% CO2. Each cell line was given its own plate with six wells, into which a sterile layer was inserted. Following cell counting, 10 × 103 cells were added to each well and cultured for 24 hours to allow for cell growth, proliferation, and adherence to the slide. The samples were then submerged in a 1 ml solution of 4% paraformaldehyde. After leaving the solution, the layers were allowed to cool for 15 minutes at ambient temperature. Finally, the samples were washed three times with PBS for 5 minutes each before being transferred in fixed form to the optical apparatus shown in Figs. 2 and 3. Fig. 1 depicts the full stabilising procedure.

 

Fundamental of optical method

1)        Measurement of linear absorption

The 532 nm Nd:YAG laser is put in a basic optical setup (see Fig. 2) to investigate the linear absorption coefficient in Eq (1).

 

 

(1)

 

In this equation, ,  and  are, respectively, initial intensity, the length of sample, and the linear absorption coefficient of sample. According to this law, the absorption coefficient depends on the intensity of the incident beam, path length, concentration of the absorbing species (chromophores) and the extinction coefficient [28].1

 

 

Fig. 2 Schematic of optical setup to investigate linear absorption coefficient of fixed layers of cell lines.

 

2)        Closed-aperture Z-scan setup

Fig. 3 depicts the schematic diagram of the closed-aperture optical Z-scan. The approach is used to determine the sign and magnitude of a material's nonlinear refractive index (here cell layers; biomaterials). The focused laser beam passes through the sample by passing through the aperture centred on the beam's axis. The strength of the incident beam varies as the sample moves across the Z-axis around the focal point from -Z to +Z. This tendency may produce a change in the material's refractive index, resulting in a change in the beam radius in the aperture plane. As a result, the transmitted power can be used to calculate the nonlinear refractive index of any material.

 

Fig. 3 Schematic of experimental closed-aperture Z-scan setup.

 

A 532 nm Nd:YAG laser with a maximum output power of 110 mW was employed as the coherent light source in this optical system [20, 29, 31]. The laser beam has a beam waist of 44 m and is focused on an 80 mm focal length lens. This beam passes through a nonlinear sample, causing a change in the overall refractive index proportional to the intensity of the beam and the material's nonlinear optical behaviour. In the simplest case, under the third-order nonlinearities condition, this leads to the change  [32], where , , , and  are total refractive index, linear refractive index, the third-order nonlinear refractive index, and incident intensity, respectively. The intensity of the incident laser beam with a Gaussian profile creates a nonlinear refractive index in the sample, causing it to operate as a positive or negative lens to change the wavefront, a phenomenon known as self-focusing and self-defocusing nonlinear optical phenomena [33]. These modifications can be estimated using n2 and Eq. (2):

 

(1)

In this equation, , , , and  are the wavelength of the laser beam, the difference between maximum (peak) and minimum (valley) of the normalized transmittance power, the effective length of sample, and the maximum intensity at the focal point, respectively [34]. In addition,  is linear transmittance of the aperture calculated by Eq. (3);  is its radius and  is the Gaussian beam radius on the aperture plate.

(2)

 

 can be calculated by Eq. (4), where  and  are the beam waist and input power, respectively. The  value is also determined by Eq. (5),  is the linear absorption coefficient and  is the sample length.

(3)

 

 

 

(4)

 

It is noteworthy that in the closed-aperture Z-scan curve, the sign of  is negative when the transmission peak is observed before the valley, and this parameter would be positive when the transmission peak comes after the valley [14], [34].

III. RESULT AND DISCUSSION

In this investigation, eight samples were divided into two major biological layer groups. MCF10A refers to normal breast cells, while MCF10B refers to cancer breast cells (MCF7). To increase the accuracy of the results, each group has four separate samples, and all data has been replicated three times in different portions of each layer. Because the changing arrangement of cells will certainly give various outcomes with all of these adjustments, all values reported are averages. In many circumstances, cell samples cannot be examined immediately after collection and must be fixed using chemical procedures. Fixation not only inhibits cell autolysis, retains cell components, maintains cell morphology and structural integrity, and enhances the microscopic appearance of cells, but it also prevents unexpected changes by maintaining the essential chemical and physical properties of cells, and its main purpose is to keep cells or cell components alive. One of the most commonly utilised chemical agents for cell samples is paraformaldehyde (PFA). PFA creates covalent cross-links between molecules, thereby bonding them into an insoluble meshwork. However, investigations have demonstrated that fixation at 4% corresponds to optimum cell fixation in biological protocols, and fixation in protein cross-linking reagents like paraformaldehyde retains cell structure better than organic solvents [35, 36]. Notably, no paraformaldehyde solution, PBS solution, culture medium, or lamellar slide exhibited nonlinearity. As a result, all nonlinear reactions are caused by cellular activities. Fig. 4 depicts transmission measurements of MCF10A and MCF7 bio-layers exposed to a low-power laser at 532 nm. These samples' linear absorption coefficients (α) were computed using a 1 mm sample length and the slope of each line. Table 1 shows the linear absorption coefficients (α) of all samples and the mean of both cell lines. Cancer cells are obviously stronger attenuators than normal cell lines since their linear absorption coefficient (α) is substantially higher [37].

 

Table 1 Linear absorption coefficient values of all MCF10A and MCF7 samples and average values assigned to each cell line.

Cell line	Sample number	Linear absorption coefficient (𝛼), (𝑐𝑚−1)
MCF10A	1	1.58	Mean
	2	1.65	1.8
	3	1.83
	4	2.14
MCF7	1	3.45	3.66
	2	3.62
	3	3.67
	4	3.81

 

 

 

 

Fig. 4 Linear absorption coefficient (α) for 8 samples. a) MCF10A, b) MCF7 cell lines.

The nonlinear refractive index (n2), which is measured using the close-aperture Z-scan technique, is one of the third-order nonlinear coefficients. All samples in this section were put in the arrangement illustrated in Fig. 3 to test their nonlinear optical properties. Fig. 5 depicts all of the nonlinear refractive indices of the MCF10A and MCF7 layers. The sign of n2 is positive in all MCF10A cell lines, and the cell behaves like a convex lens, which is known as self-focusing. While the sign of the nonlinear refractive index of all MCF7 layers is negative, similar to a concave lens, this process is known as self-defocusing. Table 2 shows the nonlinear refractive index values for all samples. These cell lines showed considerable genome-wide interaction differences, with MCF10A showing a strong physical closeness of gene-rich, tiny chromosomes (chr16-22) compared to MCF7. As a result, there is a considerable rise in inter-chromosomal connections between chr16 and chr22 in the MCF10A genome; this region contains the most gene-dense chromosomes in the human genome. According to research, the interaction frequency of tiny and gene-rich chromosomes chr16 through chr22 in MCF7 breast cancer genome representation is lower than in MCF10A epithelial cells, which is connected with the presence of more open compartments in MCF7 chr16-22. Furthermore, structural changes dictate the unique elasticity of whole cells or subcellular structures, implying that individual tumour cells are roughly twice as soft as matching normal tissue cells. These changes in elasticity between normal and tumour cells are caused by variances in the size of the actin cortex linked to the cellular membrane, the makeup of the cytoskeleton, and varying levels of nuclear proteins. MCF7 cells are bigger and have poorer cytoplasm and nucleus flexibility (based on Young's modulus) than normal MCF10A breast cells [23, 24]. Actin is a relatively abundant intracellular protein that plays an important functional role in determining cell shape and architecture, hence modulating tensions locally. Actin is a relatively abundant intracellular protein that plays an important functional role in determining cell shape and architecture, hence modulating tensions locally. As a result, the diameter of the actin cortex of MCF7 cancer cells is clearly smaller than that of MCF10A normal cells [38]. Table 3 contains a list of these parameters. The nonlinear refractive index of the cell environment will alter as a result of the biological changes that have occurred in the structure of normal and malignant cells. These modifications will also result in nonlinear optical effects such as self-focusing and self-defocusing.

Table 2 Nonlinear refractive index values of all MCF10A and MCF7 samples and average values assigned to each cell line.

Cell line	Sample number	Nonlinear Refractive Index (𝑛2 × 10-7 cm2/W)
MCF10A	1	+ 4.68	Mean
	2	+ 4.77	+ 4.79
	3	+ 4.84
	4	+ 4.89
MCF7	1	- 9.71	- 9.78
	2	- 9.76
	3	- 9.82
	4	- 9.86

 

 

Fig. 5 Normalized data of Close aperture Z-scan (n2) for 8 samples. a) MCF10A cell line, b) MCF7 cell line.

The trend of the optical behaviour of the cells in respect to each other is visualized in Fig. 6, which is based on the average values of the samples. As can be observed, the linear absorption coefficients differ greatly. Furthermore, Fig. 6.b. clearly illustrates that the nonlinear refractive index has the potential to differentiate cancer cells from healthy cells, as the signs of these parameters differ, as shown + 4.79 × 10-7 cm2/W for MCF10A and - 9.78 × 10-7 cm2/W for MCF7 cells.

Table 3 Different dynamic parameters of normal MCF10A normal breast cells and MCF7 cancer breast cells [23].

Parameter / unit	MCF7	MCF10A
Cell volume / μm3	3375 - 16873	678 - 1317
Cytoplasm Young’s modulus / kPa	0.47	0.7
Nucleus Young’s modulus / kPa	4.7	7
Actin cortex diameter / μm	1.27 – 2.17 (13.5%)	1.37 – 2.47 (20%)

 

Fig. 6 Comparative optical behaviour of MCF10A and MCF7 cells. a) Linear absorption coefficients (𝛼), b) Nonlinear refractive index (n2).

 

The nonlinear optical behaviour of normal and cancerous human cells was indicated by various indications in a prior 2016 investigation on the nonlinear behaviour of normal and cancerous ovarian cells using the Z-scan technique, with the magnitude of n2 being on the order of 10-8 (cm2/W) [39]. Another 2018 investigation of benign and malignant brain tissue discovered that the mean nonlinear refractive index for each tissue was on the order of 10-5 (cm2/W) and had opposite signs [29].

The nonlinear refractive index of the U87MG brain cell line is estimated to be on the order of 10-7 (cm2/W) [21]. The nonlinear refractive index of the mda-mb-231 cell line is 1.83×10-7 (cm2/W) [22], whereas the SK-BR-3 and MCF7 cells had nonlinear refractive indexes of 18.14 × 10-7 and - 9.73 × 10-7 (cm2/W), respectively [20]. In comparison to the current investigation, the order and sign of the nonlinear refractive index for the MCF7 cancer cell are close (- 9.78 × 10-7 (cm2/W)). As a result, it appears that in the optical method of cancer diagnostic tests, the varied signs and orders of distinct cell lines are more essential than the amount of the nonlinear refractive index. These results suggest that the optical approach is not only reliable for discriminating between normal and malignant cell lines, but it can also differentiate the different cell lines by a specific amount of nonlinear refractive index, with n2 for breast cell lines being around 10-7 (cm2/W). Aside from that, this approach can diagnose and distinguish any sort of cell, including cancer cells (brain or breast in this case). Because of the local metabolic and structural changes that occur at the cellular and subcellular levels of malignant tissue, such as changes in chemical components, cell size, and shape, which are likely to affect the optical properties of the tissue, such as scattering, absorption, and fluorescence. Hemoglobin, melanin, proteins, DNA, genetic variations, and dynamic behaviour affect the majority of light absorption, and many cellular and subcellular components of living tissues also contribute to light scattering [23, 29]. The samples can be reused because PBS protects samples even under these experimental fixation conditions and Nd:YAG laser power no burns or photo destruction which are observed on the surface of the slides and in the cell morphology, and the method is simple, affordable, and time-consuming for the user. As a result, the cells survive the irradiation [40].

IV. CONCLUSION

In conclusion, nonlinear optical behaviour can be seen in both healthy and cancerous breast cells. Despite the fact that all samples have nonlinear refractive indices of the same order (10-7 cm2/W), MCF10A and MCF7 have opposite signs. MCF10A bio-layers exhibit self-focusing behaviour, whereas MCF7 bio-layers exhibit self-defocusing behaviour. The optical behaviour might help distinguish between MCF7 and MCF10A. It might be a means of assessing the nonlinear optical qualities in future medical diagnosis. Because nonlinear activity is more accurate than linear in identifying healthy cells from cancer cells. Additionally, this straightforward procedure can be used again without affecting the samples' characteristics. The interactions between the laser and the materials (in this case, the cells) may expose the materials' optical properties, making this parameter crucial in nonlinear optics.

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