In Vivo Imaging in Alopecia Areata Patients Using Multiphoton Microscopy
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This document discusses using multiphoton microscopy (MPM) to non-invasively image hair follicles in patients with alopecia areata. MPM utilizes endogenous fluorophores and second harmonic generation to visualize hair follicles, keratinocytes, collagen, and elastin without staining. Images from patients with alopecia areata showed miniaturized and inflamed hair follicles compared to normal follicles. Measurements of follicles in alopecia areata found decreased follicle and shaft sizes. MPM has potential for bedside diagnosis of alopecia, monitoring treatment effects, and avoiding biopsies by salvaging hair follicles. Further work is needed to standardize the imaging protocol and establish diagnostic criteria
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In Vivo Imaging in Alopecia Areata Patients Using Multiphoton Microscopy
- 1. In Vivo Imaging in Alopecia Areata Patients Using Multiphoton Microscopy Jessica Lin,BSc1, Inga Saknite,PhD2, Manuel Valdebran,MD2, Mihaela Balu,PhD2, Griffin Lentsch,MSc2, Joshua N. Williams,BSc2, Karsten Koenig,PhD2, Bruce J. Tromberg,PhD2, and Natasha Atanaskova Mesinkovska,MD, PhD1,2 1Department of Dermatology, University of California, Irvine, California 2Laser Microbeam and Medical Program, Beckman Laser Institute, Irvine, California
- 2. Financial Disclosure 2 I have no financial interests to disclose in relation to this presentation
- 3. Clinical Diagnosis • Pattern of hair loss • Exclamation point hairs • Dermoscopy – Yellow dots 3 Soutor C, Hordinsky MK. Clinical Dermatology; 2017
- 4. Diagnosis of Alopecia Areata by Histology – Gold Standard 4 Normal Acute Chronic Sperling, Leonard C., Shawn E. Cowper, and Eleanor A. Knopp. An atlas of hair pathology with clinical correlations. CRC Press, 2012.
- 5. Non-invasive Optical Imaging in Trichology • Ultrasound • Optical Coherence Tomography • Laser Scanning Microscopy 5 MPM 200 μm 1100 μm
- 6. Multiphoton Microscopy (MPM) • Laser Scanning Microscopy • Illumination: – Titanium sapphire laser – Spectral wavelength between 710 – 920 nm – Focused femtosecond laser pulses • Endogenous autofluorophores 6
- 7. Endogenous Autofluorophores 7Pena A, Strupler M, Boulesteix T, Schanne-Klein M. Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy. Opt Express. 2005 Aug 8;13(16):6268-74.
- 8. Contrast Mechanism: intrinsic fluorescence combined with second harmonic generation 8M G Blanchin 2015 IOP Conf. Ser.: Mater. Sci. Eng. 80 012027 2 photon emission (2PE) 2nd harmonic generation (SHG)
- 9. Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, Webb WW. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7075-80. Epub 2003 May 19. Autofluorophores - 2 photon emission (2PE)
- 10. Collagen is a nonlinear medium – 2nd harmonic generation 10
- 11. 11 z = 0 μm z = 20 μm z = 40 μm z = 80 μm z = 100 μm z = 140 μm Images from Patient 1 (36 Male Alopecia Universalis) Epidermis Dermal-Epidermal Junction Dermis
- 12. Hair Follicle 12 Z=65µm collagen Hair shaft keratinocytes elastin keratinocytes Hair follicle Z=5µm
- 13. Cellular Details of Keratinocytes nucleus cytoplasm Z=15µm
- 14. Exclamation point hair Image from Patient 1 (36 Male Alopecia Universalis)
- 15. Horizontal and vertical sections of same area SC SS SB 30 μm z = 0 μm z = 20 μm z = 40 μm z = 80 μm z = 100 μm z = 140 μm Image from Patient 1 (36 Male Alopecia Universalis)
- 16. Normal vs. Alopecia Universalis Miniaturized Hair 16 Z = 65 AUNormal Z = 65
- 17. Same Pattern in 3 separate areas 17 Z = 65 Z = 65Z = 65 Images from 41 Female with AU
- 18. Collagen patterns in scarring vs. non-scarring alopecia at same depth 18 Z = 75 Z = 75 FFA AU
- 19. Perifollicular inflammation 19 Z = 95 Image from Patient 1 (36 Male Alopecia Universalis)
- 20. Dilated follicular infundibula filled with orthokeratin 20 Müller CS, El Shabrawi-Caelen L. 'Follicular Swiss cheese' pattern--another histopathologic clue to alopecia areata. J Cutan Pathol. 2011 Feb;38(2):185-9. Khopkar U, Jain N. Chapter 4 – Trichoscopy: The Dermatologist's Third Eye. Imaging in Dermatology, 2016, 29–41. Dermoscopy MPM Histology SC SS SB
- 22. Follicle and Shaft Sizes 22
- 23. Clinical Implications • Bedside Diagnosis • Salvage of Hair Follicles from Biopsy • Monitor Treatment 23
- 24. Next Steps • Standardize imaging protocol • Establish diagnostic criteria • Creating nomenclature and vocabulary • Unique and concurrent features • Tuning for other tissue components 24
- 25. Conclusion • MPM is useful for research and clinical use – Dermatology Trichology – Morphological tool – Diagnosis of alopecia – Research
- 26. Thank you Jessica Lin Email: linj19@uci.edu