Lasers in Medical Science

, Volume 25, Issue 4, pp 545–550

Laser-induced effects in different biological samples

Authors

    • Biophotonics LaboratoryNational Institute of Lasers and Optronics
  • S. Firdous
    • Biophotonics LaboratoryNational Institute of Lasers and Optronics
  • M. Nawaz
    • Biophotonics LaboratoryNational Institute of Lasers and Optronics
Original Article

DOI: 10.1007/s10103-010-0760-6

Cite this article as:
Atif, M., Firdous, S. & Nawaz, M. Lasers Med Sci (2010) 25: 545. doi:10.1007/s10103-010-0760-6

Abstract

Experiments were carried out on cancerous HeLa cells and blood serum using a double integrating sphere and a He-Ne laser to investigate the optical properties and cellular effects due to photodynamic therapy (PDT). In the first experiment, HeLa cells were exposed to Photofrin at concentrations of 0, 10, 20, 30, 50 and 112.4 µg/ml at an irradiance of 0.2 W/cm2 using diode laser light. Using a confocal microscope, cell debris and morphological changes in HeLa cells were recorded at different Photofrin concentrations. The results showed cell debris in HeLa cells at the highest concentration of Photofrin. In a second experiment, photobleaching was observed in HeLa cells in the presence of various concentrations of 5-aminolaevulinic acid ranging from 0–50 µg/ml. There was progressive degradation of the 635 nm peak during continuous laser irradiation at an irradiance of 0.2 W/cm2. We conclude that cells demonstrating high initial fluorescence undergo bleaching at a faster rate than those with lower fluorescence. Finally in a third experiment, cancerous and noncancerous blood serum was irradiated at an irradiance of 0.1 W/cm2 using a He-Ne laser in conjunction with a double integrating sphere system. Forward and back scattering of normal and malignant serum showed an exponential decrease in fluorescence amplitude. The results indicate that there is notable amplitude difference between malignant and normal blood serum with malignant blood serum showing decreased scattering. These results have important implications for photodiagnosis and photodynamic therapy.

Keywords

Photodynamic therapyHeLa cellsPhotobleaching effectConfocal microscopyCell debris

Introduction

The optical properties of cancerous and noncancerous cells and blood serum are an important factor in the success of laser treatments such as photodynamic therapy (PDT) and optical diagnostic measurements. A knowledge of the optical properties is very important for dose calculations as well as for the effectiveness of laser treatment. As well as often changing the optical properties of the tissue, laser irradiation also changes the temperature of the tissue affecting its behaviour. Hence laser light propagation in cells and its transformation into thermal energy due to absorption by photons is governed by the optical properties. The optical properties are monitored by measuring the forward and back scattering in the tissues/serum.

One of the simplest and most widely used devices for measuring optical radiation is the double integrating sphere due to its ability to redistribute nonuniform incident radiation into uniform radiation through multiple reflections and scattering. Over the last few decades, there has been a continuous effort in the field of biomedical optics to determine the optical properties of different biological materials, not only for diagnostic purposes but also for therapeutic applications. The optical properties of interest are those that primarily guide light through the tissue or blood serum, namely forward and back scattering coefficients, the transmission coefficient, and the absorption coefficient [16]. These measurements have many important biomedical applications in cancerous and noncancerous blood serum and tissues for PDT.

In the present study, the photobleaching effects of the photosensitizer aminolaevulinic acid (ALA) at different concentrations and the single photon-induced photodynamic activity of Photofrin were determined in HeLa cells, and the optical properties of malignant and premalignant blood serum were measured using the double integrating sphere system. The morphological changes in HeLa cells following incubation with Photofrin at different concentrations were recorded using microscopy to observe cell debris. The principal objective of the research was to characterize the response to the PDT photosensitizer Photofrin using laser irradiation in HeLa cells in vitro at a wavelength of 630 nm.

Materials and methods

Experiments were performed on samples of normal and malignant breast cancer blood serum. The efficiency of PDT photosensitizer including its photobleaching effects were also determined in cells of the malignant HeLa cell line derived from human cervical adenocarcinoma.

Culturing of HeLa cells

The cells were revived from liquid nitrogen trap and thawed for 20 min in water at 37°C. The cell suspension was taken from the ampoule and poured into a culture flask. The flask was incubated for 4 h at 37°C after addition of 10 ml of 10% MEM. Then fresh 7.5% MEM was added and the flask again incubated for 24 h. The cell line was harvested three times weekly.

Photofrin and 5-aminolaevulinic acid administration

A 10 ml stock solution of Photofrin at a concentration of 112.4 µg/ml was prepared in phosphate-buffered saline (PBS) in a culture tube. Using this stock solution, 5 ml solutions with concentrations of 50, 30, 20, 10, 0 µg/ml were prepared in PBS. Proliferating cells were then taken from the incubator and 400 µl of each of the above solutions was poured into different culture flasks. These flasks were then incubated for 3 hours at 37°C. To observe the effects of photobleaching, HeLa cells were incubated in the presence of ALA at concentrations in the range 0–50 µg/ml.

Cell viability

Cell viability was calculated using the trypan blue dye exclusion test. Dead cells absorb the dye, turn blue, and can be counted under a microscope using a haemocytometer. To calculate the total number of cells per ml of suspension, the following formula was used
$$ C1 = \frac{t}{4} \times TB \times {10^4} $$

Where ‘t’ is the total number of cells in each of the four quadrants of the haemocytometer, TB (=2) is the correction factor for trypan blue and 104 is the conversion factor for the counting chamber. The calculated cell viability was 87.5%.

Confocal microscopy

A commercially available laser scanning confocal microscope (LSM 510; Carl Zeiss, Jena, Germany) was used to image the HeLa cells. The 633 nm He-Ne laser radiation was directed onto the sample using a ×10 objective lens (Zeiss; numerical aperture 0.3). Confocal images are produced by raster scanning. HeLa cells are adherent and attach to the surface of the flask. They have a specific shape that can be clearly observed by confocal microscopy. A laser scanning confocal microscope image of the cells is presented in Fig. 1. Photomicrographs of cell debris (no whole cells) at different photosensitizer concentrations were also obtained.
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Fig. 1

Photofrin-treated HeLa cells showing whole cells (×10)

Experimental set-up

The experimental arrangement used for the measurement of the optical properties of tissue consisted of a double integrating sphere, the He-Ne laser, a spectrometer and a computer. The diameter of each sphere is 30 cm and the inner surface is coated with BaSo4 which results in a nearly perfect diffuse reflectance surface.

The excitation source used in the illumination set-up was a 17 mW He-Ne laser emitting radiation at a wavelength of 632 nm which was directed to the entrance slit of the first integrating sphere. By placing the sample at the position shown in Fig. 2, the light intensity can be measured at different detection points on the spheres. This light was taken to an Avantes spectrometer (having a grating with 600 lines/mm and a range from 200 nm to 800 nm) for detection via an optical fibre. The output of the spectrometer was fed to a computer. The recorded data was analysed using Avasoft-Basic software. The other two experiments were performed using a 630-nm diode laser as the excitation source to observe photobleaching effects and single photon-induced photodynamic activity in HeLa cells.
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Fig. 2

Illumination setup for the measurement of the optical properties of tissue

Results

Drug uptake by HeLa cells at different concentrations of Photofrin

HeLa cells were seeded into a 96-well plate at a density of 1×105 cells/well, although not all the wells were filled in any of the experiments. The cells were incubated with the drug at concentrations in the range 0–50 µg/ml. After incubation Photofrin uptake was quantified by measuring the absorbance with a 620-nm filter using a microplate reader (AMP PLATOS R-496). Photofrin uptake as a function of incubation time for the various sensitizer concentrations is shown in Fig. 3.
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Fig. 3

Photofrin uptake against incubation time

Photofrin uptake was very low at the beginning and then reached a maximum and appeared to be saturated at 24 h of incubation. From these results it is possible to adjust the incubation time of HeLa cells such that the uptake of Photofrin is optimized for PDT. These results are in good agreement with those of previous studies [7, 8]. However for different cell lines, effective Photofrin uptake is different and hence the optimum incubation time of Photofrin will be different for different cell lines. Also for different PDT photosensitizers, the incubation times for HeLa cells are different [9] indicating that drug uptake might be structure-dependent.

Scattering effects

The normal and malignant breast cancer blood serum samples to be studied were placed one by one between two integrating spheres. The samples were illuminated by a collimated He-Ne laser light. With the double integrating sphere set-up, forward and back scattering intensities from normal and malignant breast cancer blood serum could be measured by collecting photons at the respective points of the spheres. The novelty of the set-up is that it enables measurement of light globally propagating from a sample integrated over all solid angles using one detector on the inner sphere wall. This is made possible by the highly reflecting inner surfaces of the spheres, and with the spherical geometry, the reflected power is homogeneously distributed over the entire inner sphere surface. Accordingly, the power measured by the detector is proportional to the total reflected/transmitted power, which can be calculated by comparing it to a reference measurement. Malignant breast cancer blood serum showed a slight decrease in the intensity of back scattering (Fig. 4) and an exponential decrease in the intensity of forward scattering (Fig. 5) as compared to normal breast blood serum. Proteins in blood serum are the important particles that show scattering. Most patients suffering from cancer have hypoproteinaemia due to tumour lysis syndrome, and the decreased scattering is the result of the low protein concentration.
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Fig. 4

Back scattering in normal and malignant serum

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Fig. 5

Forward scattering in normal and malignant serum

Photobleaching effects

The excitation source was a 630-nm diode laser. The laser output was focused onto the culture flask and the fluorescent light emitted by the sample was fed to a spectrometer (Avantes) via an optical fibre. The output signal from the spectrometer was fed to a computer to analyse the effects of photobleaching. The 635-nm peak progressively degraded with increasing concentrations of ALA during continuous laser irradiation (Fig. 6). Hence, cells demonstrating high initial fluorescence undergo bleaching at a faster rate than those with low fluorescence. This is consistent with the results reported by Atif et al. [1014] and Schneider et al. [15] for mTHPC.
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Fig. 6

Photobleaching effect of ALA at different concentrations. Irradiance at the sample cell was 0.1 W/cm2

Single-photon-induced photodynamic activity in HeLa cells

The photodynamic activity of the Photofrin at various concentrations in HeLa cells was investigated using one photon excitation at 630 nm. The laser output was incident to the 6.6-mm diameter wells at an average irradiance of 0.2 W/cm2. To verify whether laser exposure could induce any damage to HeLa cells alone (i.e. in the absence of Photofrin) untreated cells were illuminated using the same experimental protocol. The Photofrin concentrations were 0, 30, 50 and 112.4 µg/ml and the total exposure times were 0, 4, 8 and 12 min, respectively.

Whole cells were seen after exposure of the cells to the diode laser in the absence of Photofrin (Fig. 1). HeLa cells irradiated for 12 min in the presence of Photofrin showed no whole cells, only cell debris (Fig. 7). This means that the drug had an effect on cell killing when the cells were exposed to laser light. We use the term cell killing, as we were unable to differentiate between necrosis and apoptosis (programmed cell death) in these experiments. In untreated cells, laser exposure in the absence of photosensitizer did not induce any damage to HeLa cells. We also observed no loss of viability with the longest (12 min) laser exposure time. Hence, in the absence of Photofrin, irradiation with the diode laser did not lead to cell death via a direct damage mechanism and there was greater activity at the highest drug concentration used in the experiment. Therefore cell killing was dose- and time-dependent. There was almost complete loss of viability with the highest concentration of photosensitizer and the longest laser exposure. Moreover, the difference between the irradiated cells and dark control cells at different drug concentration and exposure times proves that Photofrin is an effective PDT photosensitizer.
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Fig. 7

HeLa cells irradiated with diode laser light at an irradiance of 0.2 W/cm2 in the presence of different concentrations of Photofrin (a, b, c 30, 50, 112.4 µg/ml, respectively). One photon excitation of Photofrin induces a strong photodynamic effect at the highest concentration (c) as shown by the destruction of the cell monolayer

Hence it has been verified by the current study that one-photon excitation of the Photofrin photosensitizer induces a photodynamic effect. Exposure of cells to 630-nm diode laser light in the absence of photosensitizer confirm did not induce either killing or inhibition of cell growth. These findings are in agreement with the results reported by Atif et al. [1014] and Schneider et al. [15].

Discussion

The main objective of the current study was to determine the optical properties of normal and cancerous blood serum with the double integrating sphere by simultaneously measuring the back scattering and forward scattering intensities. Due to laser irradiation, the molecular alterations in cancerous blood serum caused changes in tissue optical properties which were determined by measuring both forward-scattered and back-scattered light from a double integrating sphere. There were consistent differences between the spectra of abnormal and normal breast cancer serum. Therefore, fluorescence spectroscopy has the potential to improve the noninvasive diagnosis of breast cancer. Further studies will better define the role of this technique in the detection of premalignant and malignant breast cancer. Moreover, the changes in scattering caused by laser heating of the cancerous blood serum resulting in its coagulation and a number of normal and cancerous blood serum samples with unknown optical properties were measured with encouraging results. We do not have facility to measure coagulation in this experimental set-up and there is considerable scope for expanding this study toward the photocoagulation process to determine the significance of changes in the scattering properties of normal and cancerous blood serum coupled with photothermally induced chemical and structural changes. The normal and cancerous blood serum study showed that spectroscopy of serum may be used for diagnostic purpose. But further studies are needed in this field for precise and accurate photodiagnosis. The findings of the current study support those of the previous study by Black and Barton [16].

On irradiation of HeLa cells, Photofrin was rapidly photobleached by a phenomenon attributed to a singlet oxygen-mediated process and a rapid fall in signal amplitude was seen in HeLa cells (Fig. 6) with increasing photosensitizer concentration. This provides further support for the argument of singlet oxygen-induced reactions being involved in the progressive loss of photosensitizer molecules.

Hence, we can conclude that singlet oxygen plays a significant role in the rapid photosensitizer photobleaching which in turn causes the Photofrin degradation rate to vary as a function of the rate of photodynamic 3O2 consumption (i.e. photodynamic 3O2 consumption increases with the available drug concentration). The HeLa cells with higher drug concentration exhibit faster bleaching than those with lower concentration, which is in excellent agreement with the results reported by Atif et al. [12], Bagdonas et al. [17] and Moan et al. [18].

It is interesting and relevant that Photofrin at the highest concentration retained an evident photodynamic effect in HeLa cells irradiated with the 630-nm wavelength diode laser with a clear loss of cell viability shown by destruction of the cell monolayer fragments via a type II (triplet oxygen) process as seen in the confocal microscope images (Fig. 7). The fact that untreated cells were found to remain viable when exposed to the laser appears to rule out the possibility that the laser pulses produce localized heating that contributes directly to cell death. Hence, we cannot fully rule out local hyperthermic effects in the cell monolayers containing the photosensitizer. Further work in different cell lines and with different photosensitizers is needed to optimize effective PDT behaviour.

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© Springer-Verlag London Ltd 2010