Diagnostics of vascular reactions triggered by weak allergens using the laser speckle imaging with the long exposure time Vyacheslav Kalchenko MD, PhD In Vivo Optical Imaging Unit, Department of Veterinary Resources Weizmann Institute of Science, Rehovot, Israel

Motivation An allergy is a hypersensitivity disorder of the immune system. A variety of tests exist to diagnose allergic conditions. http://www.aaaai.org

Motivation Challenge Tests: A very small amount of an allergen is inhaled or taken by mouth. IgE Skin Tests: A very small amount of certain allergens is put into skin. Blood Tests: This test involves drawing blood, so results are not available as rapidly as with skin tests. http://www.aaaai.org

Skin Testing for Allergies Scratch test Intradermal test Patch testing http://www.webmd.com/allergies/guide/skin-test

Allergy testing 21th Century Clinical studies wikipedia.org Patch test

Patch test Allergy testing 21th Century Clinical studies http://www.mnn.com/health/allergies http://www.skindermatologists.com Patch test

Allergy testing 21th Century Preclinical studies Mouse Ear Swelling Test (MEST)

Goal To develop an imaging system for assessment of vascular changes induced by immune reaction (allergens) as well as contact irritants Preclinical studies

Hypothesis - Itching - Redness - Burning - Swelling Histamine SENSITISATION Acute INFLAMATION Contact irritant Allergen Trigger Methyl salicylate 72 - 96h

In Vivo Imaging Center Multiple modalities

Hypothesis Acute inflammation (two components): Alterations in vascular caliber that lead to an increase in blood flow Structural changes in the microvasculature that permit plasma proteins and leukocytes to leave the circulation I. Laser Speckle Imaging (LS) might be used to asses blood flow alterations II. Fluorescence Imaging (FI) might be used to asses plasma proteins extravasation (VERIFICATION, ONLY FOR PRECLINICAL USE) Mouse ear model

Laser Speckle Imaging LSI Motion Contrast t frame Laser light source CCD camera Blood vessel Kalchenko et al., Laser Physics Letters. V7., Issue 8. 2010.

Calculation of Laser Speckle (motion) Contrast frame EXPOSURE TIME: 50 ms Where σ is a standard deviation of the CCD intensity counts at pixel (x,y) during n frames. n is the number of frames acquired and <x,y> is the mean value of CCD intensity counts at pixel (x,y) over the n frames Kalchenko et al., Laser Physics Letters. V7., Issue 8. 2010.

Setup of LSFI Methyl salicylate PC Workstation CCD camera Fluorescent microscope Methyl salicylate Area of a weak contact irritant application Laser module Mouse external ear Lab mouse

Long Exposure Speckle Imaging Lymphatic vessels (650 ms) Blood vessels (50 ms) Kalchenko et al., JBO. V17., Issue 5. 2012.

Extended exposure time frame EXPOSURE TIME: 350 ms Where σ is a standard deviation of the CCD intensity counts at pixel (x,y) during n frames. n is the number of frames acquired and <x,y> is the mean value of CCD intensity counts at pixel (x,y) over the n frames Kalchenko et al., Laser Physics Letters. V7., Issue 8. 2010.

Results Long Exposure LS imaging (350 ms) Methyl salicylate (MS) Before 30 min after MS aplication

Results Color coded (PCA) FI imaging of FITC extravasation Principle Component Analysis FITC (Fluorescein isothiocyanate–dextran) was administrated 30 min after Methyl salicylate application

Conclusions We have developed an imaging approach for assessment of vascular changes induced by contact irritants based on laser speckle imaging with multi-exposure time Further development is required to test sensitivity of the system to different weak contact allergens

Coauthors and collaborators Dr. Yuri Kuznetsov Department of Veterinary Resources Weizmann Institute of Science, Rehovot, Israel Prof. Alon Harmelin Department of Veterinary Resources Weizmann Institute of Science, Rehovot, Israel Prof. Igor Meglinski Department of Physics, University of Otago, Dunedin, New Zealand