1. Development of principles of two-cascaded laser speckle-microscopy with implication to high-precision express diagnostics of chlamydial infection.

2. Nadezhda Filonova2,3, Irina Subbotina2,3, Yulia Moiseeva5, Sergey Zaitsev2, Yury Saltykov2, Tatiana Polyanina2, Anna Lyapina2, Onega Ulianova2, Sergey Ulyanov1,2,4, 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. Principles of two-cascaded laser speckle-microscopy with application to express diagnostics of chlamydial infection are developed. Prototype of two-cascaded speckle-microscope is designed and tested. Specific case of illumination of bacterial cells by dynamic speckles is considered. Express method of detection of epithelial cells, containing defects, which are caused by Chlamydia trachomatis bacteria, is suggested. Possibility of express and high-precision diagnostics of chlamydial infection directly in a clinical specimen of patients with chlamydial infection is demonstrated. Results of improved recognition of Chlamydia trachomatis bacteria are discussed.

5. Schemes of one- and two- cascaded microscope
CMOS camera
Secondary imaging plane
MO 10x
CMOS camera
First imaging plane
Bacteriological sample,
fixed on the microscope slide
MO 90x
MO 90x
Subject plane
Cuvette with milk,
100 um layer
Condenser
Collimated laser beam
650 nm, 5 mW

6. Conventional one-cascaded white light and laser microscope

7. One-cascaded microscope. White light illumination.
Chlamydia

8. Two-cascaded microscope. White light illumination.
As it can be seen,
quality of white light image
is very low

10. t-LASCA microscopy
The principle of speckle-field processing in the t-LASCA method is the following. At each point of the dynamical speckle pattern the contrast of dynamic speckles is calculated using the formula:
where I is the instantaneous intensity of dynamic biospeckless, σI is the standard deviation of temporal intensity fluctuations, the angle brackets mean time averaging

11. s-LASCA microscopy
The second modification (s-LASCA) is based on the analysis of a single realization of static speckles. In this case, the entire two-dimensional implementation of the speckle field is divided into small areas, usually with a size of 5x5 or 7x7 pixels. For each of the selected areas, the local contrast value of the static speckles is calculated, after which a LASCA image is constructed.

12. Speckle dynamics at observation of Chlamydia trachomatis bacteria in LASCA-microscope
One-cascaded LASCA microscopy
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Two-cascaded LASCA microscopy
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13. One-cascaded LASCA microscopy
Averaged speckles.
Averaging over 100 frames
t-LASCA image
Averaging over 100 images
s-LASCA image
Averaging over 100 images
s-LASCA microscopy provides images of very low quality.
It can not be applied for observation of
Chlamydia trachomatis bacteria

14. Comparison of white light and coherent microscopy
White light illumination
Illumination by dynamic speckles

15. Visualization of internal structure of Chlamydia trachomatis
bacteria by two cascaded LASCA microscopy

16. Conclusions
It has been shown, that using of illumination of bacterial samples by dynamic speckles allow to resolve small minutia in the image of bacteria
Application of two cascaded LASCA microscopy allows to visualize internal structure of Chlamydia trachomatis bacteria

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