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

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  

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, 410012, Saratov, Russia

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.

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

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

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

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

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

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).

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

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)

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

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

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

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.

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%

Sample preparation Droplets of suspension of Chlamydia trachomatis cells

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)

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.

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.

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

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

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.

Acknowledgments This research has been supported by Russian Scientific Foundation, grant # 17-16-01099