SYSTEM FOR DETERMINING THE CONCENTRATION AND VISUALIZATION OF THE SPATIAL DISTRIBUTION OF PHOTOSENSITIZERS BASED ON TETRAPYRROLE COMPOUNDS IN THE TISSUES OF THE FUNDUS

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SYSTEM FOR DETERMINING THE CONCENTRATION AND
VISUALIZATION OF THE SPATIAL DISTRIBUTION OF
PHOTOSENSITIZERS BASED ON TETRAPYRROLE COMPOUNDS
IN THE TISSUES OF THE FUNDUS

• Sergey S. Model
• Laser Biospectroscopy Lab.,A.M. Prokhorov
  General Physics Institute,Russian Academy of
  Sciences,Moscow, Russia

E-mail biospec_at_nsc.gpi.ru Web
www.nsc.gpi.ru/lbs.html, www.biospec.ru
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A.M. Prokhorov General Physics Institute RAS

• Laser Biospectroscopy Lab.
• research of spectral fluorescent properties of
  biological tissues
• development and manufacture of equipment for
  fluorescence diagnostics and photodynamic
  therapy
• development of methods for targeted drug
  delivery using nanoparticles and cellular
  technologies

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Introduction

• In modern ophthalmology there are a number of
  diseases whose treatment is difficult. One such
  disease is senile macular degeneration (SMD) with
  neovascularization, have a devastating effect on
  the central vision. The probability of loss of
  vision in the SNM varies greatly and depends on
  the stage of disease, age, race, and sex.
• Currently, there are multiple optical,
  conservative (drug therapy), surgery (removal of
  subretinal membrane translocation of the retina)
  and laser treatment of the severe group of
  patients, indicating the absence of a common
  approach to the treatment of disease.
• A few years ago, the only available method of
  treatment, which is quite debatable could be
  called a success was retinal laser
  photocoagulation.
• Currently the most successful method of
  influence on SMD with subfoveal
  neovascularization is photodynamic therapy (PDT)
  because of the lack of laser coagulation. PDT is
  a much more secure than laser coagulation because
  the energy levels used for PDT is significantly
  lower.
• Thus, the development of equipment for the FD
  and PDT is the current trends in modern medical.
  During the creation of such systems must overcome
  a number of difficulties.

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Interaction of laser radiation with the eye
tissues

• Tissues of the human eye is a multicomponent
  structure. It is necessary to consider the nature
  of their interaction with radiation of different
  spectral composition.
• Even a transparent fabric, the cornea of ??the
  human eye, diffuse light, and therefore the total
  and axial (collimated) transparency are not
  identical. Water absorption peaks are clearly
  visible at 300, 980, 1180, 1450, 1900, and 2940
  nm due to the weak scattering. They provide low
  transmission through the cornea in the UV and IR
  spectral regions.
•   In the visible region the normal crystalline
  lens is less transparent than the cornea, because
  in addition to the scattering absorption by
  different chromophores, including
  3-hydroxy-L-kynurenine-O-ß-glucoside and age
  protein (responsible for the yellowing of the
  lens with age) is important.
•   The sclera is weakly transparent fabric due to
  the strong scattering of light on the structural
  elements (polydisperse package system of
  irregular collagen cylinders embedded in a base
  material with a lower index of refraction).
• For effective treatment and diagnosis of
  diseases of the fundus absorption and
  fluorescence spectra of photochemical agents
  (photosensitizers (PS)) should be consistent with
  the corresponding spectra of chromophores and
  fluorophores in the eye tissues.

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The absorption spectra of major chromophores and
fluorophores, and the absorption spectrum of PS
"Photosense"
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The fluorescence spectra of the major
fluorophores and PS "Photosense"
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Quantitative information obtaining methods

• We use the following method to improve
  efficiency. To obtain accurate quantitative
  information about the concentration of
  photosensitizer in certain points of the
  biological tissue is a series of point
  measurements by micro spectral fluorimetric
  confocal method is used. Concentration map is
  constructed by illuminating the surgical field by
  laser beam with constant power density in the
  cross section and shooting a fluorescent cell
  tissue response in the appropriate spectral range
  on a highly sensitive camera.
• Confocal microspectrofluorometers produces
  quantitative information about the concentration
  of the photosensitizer in the small localized
  amount of tissue. As it was shown above, the
  precise position information of diagnostic spots
  in fundus is very important because of its
  complex composition.
• We developed a system that enables the
  operational control of the spatial distribution
  of the concentration of PS "Photosence" and other
  PS based on tetrapyrrole compounds in the fundus.
• The drug "Photosens" was developed in State
  Research Center "NIOPIK for FD and PDT in
  different fields of medicine including
  ophthalmology. This drug is better in comparison
  with other FS because diagnosis and treatment can
  be performed in one procedure, which reduces
  treatment time. Traditionally, FD and PDT were
  conducted with different PS. Because of this it
  was necessary to wait for one PS will be excreted
  from the organism before entering another PS.

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Phantom medium

• As a phantom medium for construction of
  calibration curves we used a solution of
  "Photosence" in Intralipid 1 (Fresenius Kabi
  Austria (Austria)).
• Concentrations used E(-5) g / L, 5 E(-5) g /
  l, E(-4) g / l, 5 E(-4) g / l, E(-3) g / L, 5
  E(-3) g / l.

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System setup

• System for determining the concentration and
  visualization of the spatial distribution of
  photosensitizersbased on tetrapyrrole compounds
  in the tissues of the fundus is based on slit
  lamp XCEL 250 (Reichert, USA) with a laser
  adapter for FD and PDT and fundus imaging system.
  System setup of the complex is shown below.
• To obtain quantitative information on the
  concentration of PS we made an additional
  measurement channel in the visualization system
  (dashed line).

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Equipment

• SPECTROMETER
• Fluorescent radiation is incident on the
  spectrometer LESA-2 (ZAO "BIOSPEC"). The
  integrated power in the range of fluorescence in
  relative units is calculated by using the
  software of the spectrometer. To make these
  measurements correct, it is necessary to
  construct the calibration curves in the same
  relative units. Thus, the calibration curves were
  constructed using a set of operational and
  diagnostic equipment, only the eye tissues were
  replaced by phantom media.
• Video Analytics
• To analyze the video images we used monochrome
  camera Videoskan 415-USB (NPO "Videoskan") witch
  allows to set the exposure time and gain.
  Interference filter that suppresses the exciting
  laser radiation and broadband emission slit lamp
  and transmits fluorescence in the red and
  near-infrared region of the optical spectrum was
  installed before the camera .
• To determine the optimal reception performance,
  we analyzed the images obtained with different
  exposure times and gain values.

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Algorithm of calculation of the penetration depth

• The depth of light penetration into the tissue
  can be calculated using the following expression
• ,
• where µa - the total absorption coefficient, µs
  - transport coefficient of the medium. For the
  retina it is 230 cm-1.
• The absorption coefficient of Photosence
  varies depending on its concentration from 0.24
  cm-1 to 120 cm-1. Aspect Ratio
• .
• Introduce a dependence on the concentration of
  PS, now the formula for calculating the depth of
  penetration of radiation into the fabric of the
  fundus is
• ,
• Where ?aft 24 103 cm-1 dm3/g, µs 230
  cm-1.

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The calibration curves
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Results

• For the construction of calibration curves, we
  performed measurements of the fluorescence power
  for different concentrations of "Photosence" in
  the phantom medium.
• The calibration curve obtained micro spectral
  fluorimetric method allows to determine the
  concentration of photosensitizer "Photosence" in
  the range of 10-5 g/l to 5 10-3 g/l. To improve
  the accuracy of the calibration curve one needs
  to perform more detailed measurements. The
  accuracy of this method of determining the
  concentration of 10-5 g/l.
• In the analysis of spectrally-resolved images
  we shooted all the surgical field. Since each
  pixel of the matrix is gained information about
  the power of fluorescence from a small volume of
  tissue. However, the sensitivity and accuracy of
  this method is lower than that of the previous
  method because each pixel gets some radiation
  from other areas of tissue, as well as its depth.
  The accuracy of this method is 10-4 g/l.
• Optimal use of the spectral-analysis method
  allowed us to get generalized distribution of
  photosensitizer in the fundus, and using the
  micro spectral fluorimetric method provided us
  with accurate measurement of borders of tumors.

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Conclusion

• A system for the analysis of the spatial
  distribution of the concentration of
  photosensitizer "Photosense" in the fundus, which
  allows you to control the accumulation of PS in
  the affected and healthy tissues was developed.
  The combination of micro spectral fluorimetric
  method implemented in one of the receiving
  channels of the system, with analysis of the
  spectral-resolution images, implemented in the
  other channel, enables the precise delimitation
  of the affected area and the study of the
  interaction of PS with biological tissue at the
  molecular level. For both methods, the
  calibration curves are constructed on the basis
  of experiments with phantom media. Knowing the
  concentration of PS in each point of the
  operative field, we can determine the depth of
  penetration of radiation in biological tissue by
  the proposed algorithm. The system is easy to
  operate and can be used in daily medical
  practice, to speed up diagnosis and improve the
  safety of treatment.

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• Work was performed under the grant RFBR "Study
  of interaction of laser and broadband light with
  tissues fundus in hypoxia
• Equipment was produced on manufacturing
  facilities of ZAO "BIOSPEC"