Holographic Interferometry Real Time Imaging of Refraction Index

2D Distribution and Surface Deformations in Biomedicine

N. A. Davidenko

, X. Zheng

, I. I. Davidenko

, V. A. Pavlov

, N. G. Chuprina

, N. Kuranda

S. L. Studzinsky

, A. Pandya

, H. Mahdi

, A. Ladak

, C. Gergely

, F. Cuisinier

and A. Douplik

2,4

Taras Shevchenko National University, 64/13, Volodymyrska Street, City of Kyiv, 01601, Ukraine

Photonics Group, Department of Physics, Ryerson University, 350 Victoria Street, Toronto, M5B 2K3, Canada

Université de Montpellier, 34090, Montpellier, France

Keenan Research Centre of the LKS Knowledge Institute, St. Michael Hospital, Toronto, M5B 1W8, Canada

Keywords: Holography, Interferometry, Imaging, Holography Interferometry, Double Exposure Holography,

Holography Interferometry Imaging, Photodynamic Therapy, Dentistry, Biomedicine.

Abstract: Holographic Interferometry of 2D Imaging of Refraction Index and Surface Deformations were recorded in

in real time video: (1) monitoring of local refraction index perturbation at accuracy of 10

-4

in transmission

mode during heat and photochemical reactions with human hemoglobin using methylene blue, protoporphyrin

IX and rhodamine as the photosensitizers and (2) monitoring in reflectance mode of human tooth local

mechanical pressure at accuracy of 10

-7

1 INTRODUCTION

In this study, we explored potentials of holography

interference imaging in transmission and reflectance

modes. The transmission mode was exploited to map

the refraction index modifications inside the phantom

while the reflectance mode was employed to monitor

the surface displacement map of the human tooth.

We explored photochemical and photothermal

reaction due to interaction of light and hemoglobin at

presence of a photosensitizer. Hemoglobin was

selected as a component of blood that is the main

absorber of light in visible range. Blood is the main

oxygen supplier of biotissues and oxygen is one of the

key components of photodynamic therapy (PDT).

Blood is also a main carrier of photosensitizers (PSs)

administrated systemically – orally or intravenously.

PDT is a non-surgical treatment modality based on

photochemical reactions and administrated as a part

of clinical routine in Europe, Japan, Australia, North

and South America (Wilson B.C., Patterson, M.S.,

2008). PDT is an extremely precise and controllable

light-based therapy targeting malignant lesions,

mostly focusing on those that develop across

significant areas (e.g., esophageal cancer) or

relatively shallow depth (GI tract, prostate), or

applied as an antimicrobial therapy (Hamblin MR,

Hasan T., 2004). Within the biophotonics market

sector, PDT demonstrates the highest growth, almost

40% annually (Biophotonics Market, Tematys,

2013). Pharmaceutical companies, such as Axcan,

Parmaceuticals Inc, Nigma, Glaxo-Welcome Inc,

DUSA, Photocure, Galdderma and others have

become important players in PDT.

The physical processes involved in the PDT

interaction of laser beam and material are divided into

three parts: (1) absorption of the light energy; (2)

transformation of this energy into chemical energy

and/or into heat; (3) ultimate chemical reaction and/or

phase transformation. In some cases, it is difficult to

distinct hyperthermal and photochemical reactions,

hence in the study at the current stage we consider the

both effects taking place.

PDT is a photochemical reaction involving (1)

light, (2) photosensitive molecules (photosensitizer)

absorbing light and (3) ambient molecular oxygen

) generating reactive oxygen species (ROS),

which in turn destroy biotissue cells or extracellular

matrix (Dougherty, T.J. et al, 1998). These include

the Type I (sensitizer-substrate) and Type II

(sensitizer-oxygen) reactions. Type II photochemical

reactions (Fig. 1) represent the transfer of light energy

to the molecular oxygen conversion into singlet

oxygen (

). In this study, we presume only the

216

Davidenko, N., Zheng, X., Davidenko, I., Pavlov, V., Chuprina, N., Kuranda, N., Studzinsky, S., Pandya, A., Mahdi, H., Ladak, A., Gergely, C., Cuisinier, F. and Douplik, A.

Holographic Interferometry Real Time Imaging of Refraction Index 2D Distribution and Surface Deformations in Biomedicine.

DOI: 10.5220/0010390802160220

In Proceedings of the 14th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2021) - Volume 2: BIOIMAGING, pages 216-220

ISBN: 978-989-758-490-9

second type of oxygen dependent photochemical

reaction which comes with release of

taking into

account the photosensitizer used in the study –

Methylene Blue. Methylene Blue is an FDA-

approved clinical dye, which also can be used as a

photosensitizer (Foster TH et al., 2010). The II type

of PDT reaction leads to both depletion of dissolved

molecular oxygen and eventually to conversion of

oxygenated to reduced form of hemoglobin (Douplik

A., Stratonnikov A., Loschenov V et al., 2011), and

to photobleaching or photodegradation of the

photosensitizers. An example of such conversions

from the previous studies is demonstrated in Fig. 2

a,b. The transformation of hemoglobin from the

oxygenated to deoxygenated form is clearly observed,

confirming oxygen consumption during PDT (Fig.

2a). The photosensitizer photobleaching during PDT

without a substantial change in the spectral line shape

is demonstrated in Fig. 2b. Absorption and

fluorescence spectroscopy and imaging have been

successfully applied in reflectance mode for PDT

monitoring (Dougherty et al., 1998). However,

currently there are no monitoring techniques that can

be applied for PDT in optically transparent tissues

and organs such as eye. This study is an attempt to

develop a PDT monitoring technology capable to

based on local alterations of the refractive index,

exploiting the model reaction with human

hemoglobin, which was previously successfully

studies and described by absorption and fluorescence

methods.

Figure 1: Jablonski diagram of the processes during second

type of PDT reaction (via singlet oxygen generation

mechanism) (Modified from Douplik A., Stratonnikov A.,

Loschenov V et al, 2011).

Holographic interferometry monitoring of

hyperthermal and photochemical reactions with

human hemoglobin have been conducted under

control of the sample absorption spectra.

Figure 2: a)The absorption spectra variation in blood

sample incubated with Methylene Blue during laser

irradiation. The initial two-hump spectrum of oxygenated

hemoglobin (HbO

) transformed into a single peak

spectrum of reduced hemoglobin (RHb) as a result of PDT

reaction. b)Fluorescence spectra from a blood sample

incubated with Phtalocyanine Aluminum (Photosence) as

the photosensitizer at a concentration of 20 μM. Laser

fluence rate is 25 mW/cm

at 670 nm. The time interval

between spectra is 20 s. (Modified from Douplik A.,

Stratonnikov A., Loschenov V et al, 2003, 2011).

In dentistry, there is a demand to assess how the

tooth undergoes by mechanical pressure in case of

counter implants. Identification of particular points of

high mechanical pressure and pressure gradients is

required.

2 MATERIALS AND METHODS

2.1

Materials

Lyophilised human hemoglobin powder (H7379

Sigma-Aldrich, USA) was used for this study

component, replacing red blood cells, as it is readily

available and easily handled. Due to the process of

lyophilisation approximately 80-85% of the

hemoglobin is methemoglobin (MetHb). Sodium

dithionite (S310-100 Fisher Scientific) was added to

MetHb in phosphate buffered solution (PBS)

(Multicell sterile, Wisent inc., Canada) to form

reduced haemoglobin (HbR) which was then

oxygenated to form oxyhaemoglobin (HbO

)

according to the procedure described in our previous

paper (Zhernovaya, Sidoruk, Tuchin, Douplik, 2011)

providing ultimate concentration 0.5 g/L.

Holographic Interferometry Real Time Imaging of Refraction Index 2D Distribution and Surface Deformations in Biomedicine

217

Photosensitizers Protoporphyrin IX (PPIX, Sigma-

Aldrich, USA) and Rhodamine (Sigma-Aldrich,

USA) were added to the solution creating

concentrations of 0.05g/L and 0.02 g/L accordingly.

Half of the samples with PPIX were created in liquid

form, and the other half was prepared on 300-Bloom

gelatin derived from acid-cured porcine skin (G2500,

Sigma-Aldrich Corp., St. Louis, MO) forming

polymerized samples. The samples with Rhodamine

were created only in liquid form. The control samples

included: (1) PBS, (2) PBS/10%-gelatin, (3)

PBS/0.5g/L Hb, (4) PBS/0.05g/L PPIX, (5)

PBS/10%-gelatin/0.5 g/L Hb, (6) PBS/10%-

gelatin/0.05g/L PPIX, (7) - PBS/0.02g/L Rhodamine

3 samples were used in each experimental group

including both the control and PPIX experiments. For

Rhodamine control and experiment were used 2

samples per group.

2.2 Experimental Setup

The absorption spectra were measured on Shimadzu

UV-3600 spectrophotometer (Japan). The

Holography Interference setup is depicted in Fig. 3

and described in (Derzhypolska L., Davidenko N.,

2006).

Figure 3:

Double exposure real time Holography

Interferometry setup.

A black-and-white web camera was used for

experiment with PPIX and for the Rhodamine

experiment we used a RGB camera (Basler ac-

A2000-165uc, Basler AG, Germany). Red laser diode

was employed for holography (660 nm, 4mW,

INTEGRAF LLC, USA) and green laser diode (532

nm, 50 mW, Thorlabs, USA) used for irradiation

providing the irradiance of 62 mW/cm

. A long pass

interference filter (Comar Optics, UK) was exploited

to facilitate both the irradiation of the sample and

holography interference imaging acquisition.

The monitoring of PDT reactions was carried out

in transmission mode, while monitoring of the surface

displacement map of the human tooth was conducted

in reflectance mode.

2.3 Data Processing

Fringe images was processed with carrier

frequencies. Fringe patterns with carrier frequencies

are processed using the Fourier transform method

discussed earlier in introduction. A Matlab function

that includes an automatic and a manual

implementation of the method was obtained from

Matlab File Exchange (mathworks.com/

fileexchange/53421). The user of the GUI is given the

option of manually selecting the side peak in the

Fourier spectrum representing c(x,y), or an automatic

option can be used but the user has to specify the

width of the side peak in the Fourier spectrum.

Increasing the width avoids negating phase

information but allows for more noise to be included.

Figure 4: (a) noisy fringe pattern; (b) noisy extracted phase

using the Fourier transform method; (c) denoised phase

using WFT.

In addition, the user is given an option to use a

windowed Fourier transform after demodulation in

order to denoise the obtained phase before

unwrapping at the cost of significantly increasing

processing time. The application of a windowed

Fourier transform (WFT) for fringe analysis was first

proposed in (Qian Kemao, 2004). The combination of

the Fourier transform method and the WFT was tested

and proven to be successful. Figure 4 shows the

effectiveness of using the WFT method in denoising

phase.

3 RESULTS AND CONCLUSIONS

The holography interference images for control

experiments are depicted in Fig. 5 (a snapshot from

the video clip at 60th second). A partial formation of

the first interference ring can be observed only in case

of PBS/10%-gelatin/0.5 g/L Hb sample (Fig. 5 c),

which apparently was caused by the hyperthermal

effect, while the control samples with PBS/gelatin

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and Methylene Blue/gelatin (Fig. 5a, b) did not

manifest any registerable photochemical reaction.

a) b) c)

Figure 5: Holography interference images for control

experiment (60

second snapshot): a) gelatin only; b)

gelatin plus Methylene Blue; c) gelatin plus Hemoglobin.

The contours of the cuvette walls are clearly seen.

The holography interference images for

Methylene Blue experiment are shown in Fig. 6

demonstrating a significant reaction forming ca. 5

interference rings, which approximately matches a

modification of the refraction index by 5 folds.

Preliminary we could conclude that contributions

of photochemical reactions are considerably higher

than a pure heating effect. Once the green laser

irradiation was switched off, in the experimental

samples the refraction index returned back to its

original values within 60 seconds.

a) b) c) d) e)

f) g) h) i) j)

Figure 6: Holography interference snapshot from video clip

images for Methylene Blue experiment arranged in seconds

of irradiation: a) 2 s; b) 10 s, c) 30 s, d) 59 s, e) 60s (laser is

switched off), f) 65 s (5 seconds later after laser switched

off), g) 70 s, h) 80 s, i) 90 s, k) 120 s. The long pass filter

was set in front of the camera to let a minor leak of the

irradiation green light to expose onto the side walls of the

cuvette for monitoring the irradiation condition consistency

(a-d). The laser was switched off in the frames e-j. The

white circle in the center of the interference pattern of the

first frame (a) is the irradiation spot location (not scaled, the

diameter of the spot was 1.5 mm).

As a result of the work, the heat and photochemically

induced reactions with human hemoglobin have been

successfully monitored by holography interference

method towards development of PDT monitoring

technology in transparent tissues.

The holography interference images for PPIX

experiment are depicted in Fig. 7. A formation of the

interference ring can be observed. The holography

interference images for Rhodamine C experiment are

depicted in Fig. 8. The control samples with PBS,

gelatin and PPIX did not manifest any photochemical

reaction. The control samples with HbO2 only have

shown some reactions but much less than that of with

PPIX plus HbO2 experiments even after 60 seconds

of irradiation.

a) b) c) d)

Figure 7: Holography interference images for PPIX

experiment: a) liquid sample before irradiation; b) liquid

sample, beginning of the reaction (25th second); c) gelatin

sample before laser irradiation; d) gelatin sample,

beginning of the reaction (12th second).

a) b) c)

Figure 8: Holography interference images for Rhodamine

C experiment: a) liquid sample before irradiation; b) liquid

sample, beginning of the reaction (2nd second), c) liquid

sample, development of the reaction (4th second). The long

pass filter was set in front of the camera to let a minor leak

of the irradiation green light to expose onto the bottom left

corner of the frame for monitoring the irradiation condition

consistency. The light rod shape in the center of the frame

is the irradiation spot.

The reaction without photosensitizer was interpreted

as a pure heating effect. Once the green laser

irradiation was off, in the gelatin samples the RHb

have been completely oxygenated back during 1-2

minutes. Such a fast resurrection of RHb to HbO2 has

not been observed in liquid samples.

Holography interference images in reflectance

mode for tooth before and after applying mechanical

pressure are shown in Fig. 9 a,b. The mechanical

pressure was applied from the top. Estimated

displacement of the surface was ca. 80 nm.

Holographic Interferometry Real Time Imaging of Refraction Index 2D Distribution and Surface Deformations in Biomedicine

219

a) b)

Figure 9: Holography interference images for tooth before

applying mechanical pressure (a) and after (b). The

mechanical pressure was applied from the top. Estimated

displacement of the surface was ca. 80 nm.

The heat, photochemically induced reactions with

human hemoglobin and surface deformation due to

mechanical pressure have been successfully

monitored by holography interference method

towards development of monitoring technology in

both transparent and opaque tissues for biomedicine.

ACKNOWLEDGEMENTS

This study was supported by the Natural Sciences and

Engineering Research Council of Canada (NSERC),

Personal Discovery Grant (Douplik A), Ryerson

University Health Research Grant. Aman Ladak was

supported by NSERC Engage grant

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