1. Application of LASCA imaging for detection of disorders of blood microcirculation in chicken embryo, infected by Chlamydia trachomatis 

2. Onega Ulianova2, Irina Subbotina2,3, Nadezhda Filonova2,3, Sergey Zaitsev2, Yury Saltykov2, Tatiana Polyanina2, Anna Lyapina2, Sergey Ulyanov1,2,4, Olga Larionova1,3, 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  

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,

4. Methods of t-LASCA and s-LASCA imaging have been adapted to the problem of monitoring of blood microcirculation in chicken embryo model. Set-up for LASCA imaging of chicken embryo is mounted. Disorders of blood microcirculation in chicken embryo, infected by Chlamydia trachomatis, are detected. Speckle-imaging technique and ovoscopy are compared, advantages of LASCA imaging is demonstrated.

5. t-LASCA
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

6. s-LASCA
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.

7. Optical scheme of LASCA imaging system
Collimated laser beam
650 nm, 5 mW
Microobjective 40x
LOMO
Diverging laser beam
tube
Objective MBS-10
CMOS camera
Chicken egg
polarizer
Linear stage

8. Set-up for LASCA imaging of chicken embryo

9. Single frame. Speckles scattered by egg are clearly visible.

10. Laser ovoscopy

11. Comparison of white light ovoscopy and laser ovoscopy. Alive embryo.
Laser illumination.
More details are visible
White light illumination

12. Scanning of embryo. Egg shell is in subject plane.
Time-averaged speckles.
Averaging over 100 frames
t-LASCA image.
Averaging over 100 images
s-LASCA image.
Averaging over 100 images
Internal structure in s-LASCA and t-LASCA images
is visible

13. Scanning of embryo. Egg-objective distance is reduced on 7 mm.
Time-averaged speckles.
Averaging over 100 frames
t-LASCA image.
Averaging over 100 images
s-LASCA image.
Averaging over 100 images
Internal structure in s-LASCA and t-LASCA images
is visible.

14. Scanning of embryo. Egg-objective distance is reduced on 15 mm.
Time-averaged speckles.
Averaging over 100 frames
t-LASCA image.
Averaging over 100 images
s-LASCA image.
Averaging over 100 images
Internal structure in s-LASCA and t-LASCA images
is visible

15. Dead embryo (it was infected by Chlamydia
trachomatis). Egg shell is in subject plane.
Time-averaged speckles.
Averaging over 100 frames
t-LASCA image.
Averaging over 100 images
s-LASCA image.
Averaging over 100 images
Internal structure in s-LASCA and t-LASCA images
disappears

16. Conclusions
Structure of s-LASCA and t-LASCA images is changing when embryo is scanned. Different structure correspond to different selected layers of the egg.
All minutia of the images completely disappear in the case of monitoring of dead embryo.
LASCA imaging is effective tool of detection of disorders of blood microcirculation in chicken embryo infected by Chlamydia trachomatis
s-LASCA is more informative than t-LASCA technique

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