The PeriCam PSI System by PERIMED AB (Rommerskirchen, Germany) provides a new method for the measurement and imaging of real-time blood flow by utilizing Laser Speckle Contrast Analysis (LASCA). LASCA works according to the following principles: a laser illuminates the tissue of interest, which in turn backscatters light onto a camera. Here the light produces a random interference pattern called a speckle pattern. If there is movement within the tissue (e.g. erythrocytes in blood vessels), the speckle pattern will change correspondingly and produce intensity variations, causing a blurred speckle pattern. Hence, higher amounts of motion result in higher levels of blurring which can be quantified by the speckle contrast. Further analysis of these speckle contrast fluctuations is then performed by the software in order to obtain information on the blood perfusion of the tissue being examined. The CCD camera that is used to capture the fluctuations in the speckle pattern does this at a speed of 113 images per second and up to 1386 x 1036 pixels per image, ensuring an accurate display of the real-time microcirculation. For the measurement of small areas, the high-resolution (HR) version appears most suitable, as the image resolution is 20µm/pixel. Furthermore, the lighting conditions during the procedure are compensated by the software and have no effect on the results [1,2,3,4].
The CAM-Assay is performed as described in former works [1,5,6]. On the 8th day after the start of incubation, the amount of fertilized eggs (usually 2-6 at a time), chosen for the measurement of blood perfusion, are taken out of the egg incubator and placed on a glass tray, bedded with sand. The eggs are then transported to the PeriCam PSI System HR model. Here the tape-sealed window of the first CAM-Assay is opened carefully and placed on a Styrofoam piece, shaped to contain one egg securely for the process of measurement. the CAM-Assay is positioned 9.8-10.2 cm away from the CCD camera of the PeriCam PSI System and adjusted so that there are no disturbances (e.g. the egg shell) seen in the image caught by the camera. If necessary, the window that has been previously cut into the eggshell can be enlarged with scissors. Now the measurement is initiated, generating one image of perfusion every 2 seconds until the procedure is stopped. For precise results the embryo has to be static for at least 5 consecutive measurements after each other (at least 10 seconds in total). Inaccuracies due to the embryo’s movement can be controlled and administered through instant updates of graphs and images displayed by the software during the process. Once a sufficient phase without motion by the embryo is encountered, the measurement can be ceased, the region of interest selected and saved accordingly. Now the egg is carefully sealed with tape again and put back into the glass tray. The other eggs are then measured equivalently.
In adherence to protocol, on the 16th day after the start of incubation, the second measurement is executed in the same way as described for the previous one. Since the second measurement takes place after the inoculation of the CAM, the camera has to be adjusted congruently to the initial measurement in order to ensure an accurate display of the angiogenesis that has taken place during the 8-day period.
Images were taken from the following publication:
Pion E, Asam C, Feder AL, Felthaus O, Heidekrueger PI, Prantl L, Haerteis S, Aung T. Laser speckle contrast analysis (LASCA) technology for the semiquantitative measurement of angiogenesis in in-ovo-tumor-model. Microvasc Res. 2021 Jan;133:104072. doi: 10.1016/j.mvr.2020.104072 . Epub 2020 Sep 17. PMID: 32949573
Images were taken from the following publication:
Pion E, Asam C, Feder AL, Felthaus O, Heidekrueger PI, Prantl L, Haerteis S, Aung T. Laser speckle contrast analysis (LASCA) technology for the semiquantitative measurement of angiogenesis in in-ovo-tumor-model. Microvasc Res. 2021 Jan;133:104072. doi: 10.1016/j.mvr.2020.104072 . Epub 2020 Sep 17. PMID: 32949573.
 Pion E, Asam C, Feder AL, Felthaus O, Heidekrueger PI, Prantl L, Haerteis S, Aung T. Laser speckle contrast analysis (LASCA) technology for the semiquantitative measurement of angiogenesis in in-ovo-tumor-model. Microvasc Res. 2021 Jan;133:104072. doi: 10.1016/j.mvr.2020.104072. Epub 2020 Sep 17. PMID: 32949573
 Ansari, Mohammad Zaheer et al. “Monitoring microvascular perfusion variations with laser speckle contrast imaging using a view-based temporal template method.” Microvascular research vol. 111 (2017): 49-59. doi:10.1016/j.mvr.2016.12.004
 J. David Briers and Sian Webster “Laser speckle contrast analysis (LASCA): a nonscanning, full-field technique for monitoring capillary blood flow,” Journal of Biomedical Optics 1(2), (1 April 1996). https://doi.org/10.1117/12.231359
 Dunn, A. K., Bolay, H., Moskowitz, M. A., & Boas, D. A. (2001). Dynamic Imaging of Cerebral Blood Flow Using Laser Speckle. Journal of Cerebral Blood Flow & Metabolism, 21(3), 195–201. https://doi.org/10.1097/00004647-200103000-00002
 Kauffmann P, Troeltzsch M, Cordesmeyer R, Heidekrueger PI, Schliephake H, Canis M, et al. Presentation of a variation of the chorioallantoic membrane set up as a potential model for individual therapy for squamous cell carcinoma of the oropharynx, Clin Hemorheol Microcirc. 2017;67(3-4):453-7. doi:10.3233/CH-179226
 Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971 Nov 18;285(21):1182-6. doi: 10.1056/NEJM197111182852108. PMID: 4