1. Detection of the presence of Chlamydia trachomatis bacteria using diffusing wave spectroscopy with a small number of scattering centers.

2. Sergey Ulyanov1,2,4, Nadezhda Filonova2,3, Irina Subbotina2,3, Yulia Moiseeva5, Sergey Zaitsev2, Yury Saltykov2, Tatiana Polyanina2, Anna Lyapina2, Irina Kalduzova4, Onega Ulianova2, Olga Larionova1,3, Sergey Utz5, Valentina Feodorova1,2,3   1Laboratory for Molecular Biology of Chlamydia, Federal Research Center for Virology and Microbiology…………………………………………………………………. 2Laboratory for Molecular Biology and NanoBiotechnology, Federal Research Center for Virology and Microbiology, Branch in Saratov 3Department for Microbiology, Biotechnology and Chemistry, Saratov State Agrarian University…………………………………………………………………………. 4Department for Medical Physics, Saratov State University 5Department of Dermatology and Venerology , Saratov State Medical University  

3. 1 Federal Research Center for Virology and Microbiology,
601120, Pokrov, Vladimir region, Russia
2 Federal Research Center for Virology and Microbiology,
Branch in Saratov, Ap. 9, Proviantskaya Street, Box 1580,
410028, Saratov, Russia
3 Saratov State Agrarian University, 1, Theatralnaya Square,
410012, Saratov, Russia
4 Saratov State University, 83 Astrakhanskaya Street,
5 Saratov State Medical University, named after N.I. Razumovsky, Ministry of Health of the Russian Federation, Bolshaya Kazachia st., 112, , Saratov, Russia

4. Abstracts
Theory of diffusing wave spectroscopy has been adapted to problem of identification of Chlamydia trachomatis bacteria in aqueous suspension. Formula for correlation function of temporal fluctuations of speckle intensity is derived for the case of small number of scattering events. Dependence of bandwidth of spectrum on average number of scatterers is analyzed. Set-up for detection of the presence of Chlamydia trachomatis bacteria inside the epithelial cells is designed. Possibility of detection of Chlamydia trachomatis bacteria in suspension of epithelial cells is demonstrated. Good agreement between theoretical results and experimental data is shown.

5. General theory is presented in [*]:
S. Ulyanov. Diffusing wave spectroscopy with a small number of scattering events: An implication to micro flow diagnostics
PHYSICAL REVIEW E 72,052902, 2005

6. A photon, which propagates through the microvolume, scatters N times
A photon, which propagates through the microvolume, scatters N times. The single photon, after passing from a laser source to the detector acquires the total phase shift

8. As it has been shown [*], for independent scatterers, the fields from the different paths are not correlated

10. are randomly distributed values
(displacements), corresponding to
Brownian motion of scattering centers, i.e.
Chlamydia trachomatis bacteria
To make further progress it is necessary to calculate
and
in the expression, presented on slide# 8

11. As it has been demonstrated in [*], the number of
scattering events obeys Poissonian distribution in the
case of small number of scatterers and the distribution
of a fraction of scattered intensities in diffraction
paths of length s is described by a simple formula:
where μs is a scattering coefficient

12. The autocorrelation function of the fluctuations of
complex amplitude of scattered light may be expressed as ds
where n, as before, is a number of scattering centers (number of Chlamydia trachomatis bacteria in a probing volume).

13. Taking into account, that
And assuming that Siegert relation is fulfilled

14. Then the correlation function of the intensity
fluctuations of scattered light has the following form
Regression analysis, based on the results of Monte
Carlo simulations allows us to find out the relation
between the averaged phase and averaged number of
scatterers (number of Chlamydia trachomatis bacteria)

15. Dependence of
on average number of
bacteria in probing volume. Dots are results of Monte
Carlo simulation, solid (red) line is the curve of linear
regression. Case of Brownian motion of bacteria

16. Dependence of
on average number of
bacteria in probing volume. Dots are results of Monte
Carlo simulation, solid (red) line is the curve of linear
regression. Case of scanning of motionless bacteria

17. Theoretical analysis, based on DWS theory, shows
Bandwidth of the spectrum of intensity fluctuations is linearly proportional to average velocity of motion of bacteria or velocity of scanning of bacteria fixed in the volume (this result is absolutely trivial)
Bandwidth of the spectrum is linearly proportional to the concentration of bacterial cells in suspension
Curves (see slides #15 and #16) are identical if velocity of scanning of “frozen” sample is matched with averaged velocity of Brownian motion of bacteria in suspension

18. Methods and materials Four types of suspensions of
- Chlamydia trachomatis bacteria
McCoy cells
McCoy cells incubated with Chlamydia trachomatis cells
McCoy cells incubated with Chlamydia trachomatis cells and dyeing by Romanowsky- Giemsa
have been prepared.
Initial concentration of cells in suspension has been measured using Goryaev’ camera. It was equal to 8 x106 cells per ml.

19. Methods and materials
Pendent drop has been applied on the cover glass. Bacterial loop #3 has been used. Volume of the drop was 3.5 μl. Cover glass has been fixed by Vaseline on microscope slide with a hole.
Solution with 10 concentrations have been tested. Suspension have been dissolved by physiological solution with concentrations of 100% (non-dissolved solution), 90%, 80%,70%…,10%

20. Sample preparation Droplets of suspension of Chlamydia trachomatis
cells

21. Optical scheme Hanging drop of suspension Speckles Microobjective
90x (LOMO)
Photodetector
PDA10, (Thorlabs),
connected
to DAC, (NI)
He-Ne laser beam
LGN 205
Microscope slide with a hole,
covered by cover glass,
fixed by Vaseline
Motorized linear stage
(Standa)

22. Signal from moving bacteria is not detectable.
Output signal of photodetector. No bacterial sample. Own noise of detector.
Output signal of photodetector.
Non-dissolved solution of Chlamydia trachomatis bacteria
Signal from moving bacteria is not detectable.

23. Signal from moving bacteria is very slow.
Spectrum of output signal of photodetector.
Sample is non-dissolved solution of Chlamydia trachomatis bacteria.
Sample is motionless.
Spectrum of own noise of detector
Signal from moving bacteria is very slow.
Brownian motion of Chlamydia trachomatis bacteria
is not detectable. Additional scanning of the sample is required.

24. Scanning of micro volume with bacterial suspension.
Output signal and its spectrum.
Signal is clearly detectable.

25. Scanning of hanging droplet of bacterial suspension.
Bandwidth of spectrum essentially depend on concentration
of Chlamydia trachomatis bacteria
dissolved solution.
80 μl of initial bacterial suspension
+ 20 μl physiological solution
non-dissolved bacterial solution

26. Conclusions
New technique of scanning DWS with a small number of scattering centers with application to detection of Chlamydia trachomatis cells is suggested.
Possibility of detection of Chlamydia trachomatis bacteria in suspension of epithelial cells and in physiological solution is demonstrated.
Good agreement between theoretical results and experimental data is shown.

27. Acknowledgments
This research has been supported by Russian Scientific Foundation, grant #