Characterization of human cutaneous tissue autofluorescence: implications in topical drug delivery studies with fluorescence microscopy

Maiko Hermsmeier; Sinyoung Jeong; Akira Yamamoto; Xin Chen; Usha Nagavarapu; Conor L. Evans; Kin F. Chan

doi:10.1364/BOE.9.005400

Characterization of human cutaneous tissue autofluorescence: implications in topical drug delivery studies with fluorescence microscopy

Maiko Hermsmeier, Sinyoung Jeong, Akira Yamamoto, Xin Chen, Usha Nagavarapu, Conor L. Evans, and Kin F. Chan

Biomedical Optics Express
Vol. 9,
Issue 11,
pp. 5400-5418
(2018)
•https://doi.org/10.1364/BOE.9.005400

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Maiko Hermsmeier, Sinyoung Jeong, Akira Yamamoto, Xin Chen, Usha Nagavarapu, Conor L. Evans, and Kin F. Chan, "Characterization of human cutaneous tissue autofluorescence: implications in topical drug delivery studies with fluorescence microscopy," Biomed. Opt. Express 9, 5400-5418 (2018)

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Related Topics
Table of Contents Category
- Microscopy
Optics & Photonics Topics
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The topics in this list come from the OSA Optics and Photonics Topics applied to this article.

About this Article
History
- Original Manuscript: August 6, 2018
- Revised Manuscript: September 27, 2018
- Manuscript Accepted: October 5, 2018
- Published: October 12, 2018

Accessible

Open Access

Abstract

In pharmacokinetic studies of topical drugs, fluorescence microscopy methods can enable the direct visualization and quantification of fluorescent drugs within the skin. One potential limitation of this approach, however, is the strong endogenous fluorescence of skin tissues that makes straightforward identification of specific drug molecules challenging. To study this effect and quantify endogenous skin fluorescence in the context of topical pharmacokinetics, an integrating sphere-based screening tool was designed to collect fluorescence yield data from human skin specimens. Such information could be utilized to select specific donors in the investigation of drug uptake and distribution. Results indicated human facial skin specimens from a group of more than 35 individuals exhibited an at least 6-fold difference in endogenous fluorescence. In visualizing drug distributions, the negative impact of autofluorescence could be exacerbated in cases where there are overlapping spatial distributions or spectral emission profiles between endogenous fluorophores and the exogenous fluorophore of interest. We demonstrated the feasibility of this approach in measuring the range of tissue endogenous fluorescence and selecting specimens for the study of drug pharmacokinetics with fluorescence microscopy.

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2018 (2)

S. Jeong, M. Hermsmeier, S. Osseiran, A. Yamamoto, U. Nagavarapu, K. F. Chan, and C. L. Evans, “Visualization of drug distribution of a topical minocycline gel in human facial skin,” Biomed. Opt. Express 9(7), 3434–3448 (2018).
[Crossref] [PubMed]

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[Crossref] [PubMed]

2017 (4)

T. Losanno and C. Gridelli, “Recent advances in targeted advanced lung cancer therapy in the elderly,” Expert Rev. Anticancer Ther. 17(9), 787–797 (2017).
[Crossref] [PubMed]

C. C. Wang, Y. C. Wang, G. J. Wang, M. Y. Shen, Y. L. Chang, S. Y. Liou, H. C. Chen, A. S. Lee, K. C. Chang, W. Y. Chen, and C. T. Chang, “Skin autofluorescence is associated with inappropriate left ventricular mass and diastolic dysfunction in subjects at risk for cardiovascular disease,” Cardiovasc. Diabetol. 16(1), 15 (2017).
[Crossref] [PubMed]

F. E. van Dooren, F. Pouwer, C. G. Schalkwijk, S. J. Sep, C. D. Stehouwer, R. M. Henry, P. C. Dagnelie, N. C. Schaper, C. J. van der Kallen, A. Koster, J. Denollet, F. R. Verhey, and M. T. Schram, “Advanced Glycation End Product (AGE) Accumulation in the Skin is Associated with Depression: The Maastricht Study,” Depress. Anxiety 34(1), 59–67 (2017).
[Crossref] [PubMed]

R. P. van Waateringe, S. N. Slagter, A. P. van Beek, M. M. van der Klauw, J. V. van Vliet-Ostaptchouk, R. Graaff, A. D. Paterson, H. L. Lutgers, and B. H. R. Wolffenbuttel, “Skin autofluorescence, a non-invasive biomarker for advanced glycation end products, is associated with the metabolic syndrome and its individual components,” Diabetol. Metab. Syndr. 9, 42 (2017).

2016 (4)

R. P. van Waateringe, S. N. Slagter, M. M. van der Klauw, J. V. van Vliet-Ostaptchouk, R. Graaff, A. D. Paterson, H. L. Lutgers, and B. H. R. Wolffenbuttel, “Lifestyle and clinical determinants of skin autofluorescence in a population-based cohort study,” Eur. J. Clin. Invest. 46(5), 481–490 (2016).
[Crossref] [PubMed]

C. Cardoso-Palacios and I. Lanekoff, “Direct Analysis of Pharmaceutical Drugs Using Nano-DESI MS,” J. Anal. Methods Chem. 2016, 3591908 (2016).
[Crossref] [PubMed]

L. McDonald, B. Liu, A. Taraboletti, K. Whiddon, L. P. Shriver, M. Konopka, Q. Liu, and Y. Pang, “Fluorescent flavonoids for endoplasmic reticulum cell imaging,” J. Mater. Chem. B Mater. Biol. Med. 4(48), 7902–7908 (2016).
[Crossref] [PubMed]

J. Guyotat, J. Pallud, X. Armoiry, V. Pavlov, and P. Metellus, “5-Aminolevulinic Acid-Protoporphyrin IX Fluorescence-Guided Surgery of High-Grade Gliomas: A Systematic Review,” Adv. Tech. Stand. Neurosurg. 43, 61–90 (2016).
[Crossref] [PubMed]

2015 (3)

E. G. Solon, “Autoradiography techniques and quantification of drug distribution,” Cell Tissue Res. 360(1), 87–107 (2015).
[Crossref] [PubMed]

M. S. Ahmad, Z. A. Damanhouri, T. Kimhofer, H. H. Mosli, and E. Holmes, “A new gender-specific model for skin autofluorescence risk stratification,” Sci. Rep. 5, 10198 (2015).

A. Nongnuch and A. Davenport, “The effect of vegetarian diet on skin autofluorescence measurements in haemodialysis patients,” Br. J. Nutr. 113(7), 1040–1043 (2015).
[Crossref] [PubMed]

2014 (5)

S. F. El-Mashtoly, D. Petersen, H. K. Yosef, A. Mosig, A. Reinacher-Schick, C. Kötting, and K. Gerwert, “Label-free imaging of drug distribution and metabolism in colon cancer cells by Raman microscopy,” Analyst (Lond.) 139(5), 1155–1161 (2014).
[Crossref] [PubMed]

V. N. Du Le, Z. Nie, J. E. Hayward, T. J. Farrell, and Q. Fang, “Measurements of extrinsic fluorescence in Intralipid and polystyrene microspheres,” Biomed. Opt. Express 5(8), 2726–2735 (2014).
[Crossref] [PubMed]

U. A. Gamm, C. L. Hoy, F. van Leeuwen-van Zaane, H. J. Sterenborg, S. C. Kanick, D. J. Robinson, and A. Amelink, “Extraction of intrinsic fluorescence from single fiber fluorescence measurements on a turbid medium: experimental validation,” Biomed. Opt. Express 5(6), 1913–1925 (2014).
[Crossref] [PubMed]

M. Kubicek, G. Holzlechner, A. K. Opitz, S. Larisegger, H. Hutter, and J. Fleig, “A novel ToF-SIMS operation mode for sub 100 nm lateral resolution: application and performance,” Appl. Surf. Sci. 289(100), 407–416 (2014).
[Crossref] [PubMed]

2012 (1)

A. J. Walsh, D. B. Masters, E. D. Jansen, A. J. Welch, and A. Mahadevan-Jansen, “The effect of temperature on the autofluorescence of scattering and non-scattering tissue,” Lasers Surg. Med. 44(9), 712–718 (2012).
[Crossref] [PubMed]

2011 (3)

R. T. Zaman, N. Rajaram, A. Walsh, J. Oliver, H. G. Rylander, J. W. Tunnell, A. J. Welch, and A. Mahadevan-Jansen, “Variation of fluorescence in tissue with temperature,” Lasers Surg. Med. 43(1), 36–42 (2011).
[Crossref] [PubMed]

D. C. Bos, W. L. de Ranitz-Greven, and H. W. de Valk, “Advanced glycation end products, measured as skin autofluorescence and diabetes complications: a systematic review,” Diabetes Technol. Ther. 13(7), 773–779 (2011).
[Crossref] [PubMed]

C. A. Thorling, Y. Dancik, C. W. Hupple, G. Medley, X. Liu, A. V. Zvyagin, T. A. Robertson, F. J. Burczynski, and M. S. Roberts, “Multiphoton microscopy and fluorescence lifetime imaging provide a novel method in studying drug distribution and metabolism in the rat liver in vivo,” J. Biomed. Opt. 16(8), 086013 (2011).
[Crossref] [PubMed]

2010 (3)

R. Bazin, F. Flament, A. Colonna, R. Le Harzic, R. Bückle, B. Piot, F. Laizé, M. Kaatz, K. König, and J. W. Fluhr, “Clinical study on the effects of a cosmetic product on dermal extracellular matrix components using a high-resolution multiphoton tomograph,” Skin Res. Technol. 16(3), 305–310 (2010).
[Crossref] [PubMed]

E. G. Solon, A. Schweitzer, M. Stoeckli, and B. Prideaux, “Autoradiography, MALDI-MS, and SIMS-MS Imaging in Pharmaceutical Discovery and Development,” AAPS J. 12(1), 11–26 (2010).
[Crossref] [PubMed]

W. Y. Sanchez, T. W. Prow, W. H. Sanchez, J. E. Grice, and M. S. Roberts, “Analysis of the metabolic deterioration of ex vivo skin from ischemic necrosis through the imaging of intracellular NAD(P)H by multiphoton tomography and fluorescence lifetime imaging microscopy,” J. Biomed. Opt. 15(4), 046008 (2010).
[Crossref] [PubMed]

2008 (1)

C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R. Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie, “Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy,” Science 322(5909), 1857–1861 (2008).
[Crossref] [PubMed]

2006 (3)

L. A. McDonnell, R. M. A. Heeren, R. P. J. de Lange, and I. W. Fletcher, “Higher sensitivity secondary ion mass spectrometry of biological molecules for high resolution, chemically specific imaging,” J. Am. Soc. Mass Spectrom. 17(9), 1195–1202 (2006).
[Crossref] [PubMed]

V. Raufast and A. Mavon, “Transfollicular delivery of linoleic acid in human scalp skin: permeation study and microautoradiographic analysis,” Int. J. Cosmet. Sci. 28(2), 117–123 (2006).
[Crossref] [PubMed]

G. N. Stamatas, R. B. Estanislao, M. Suero, Z. S. Rivera, J. Li, A. Khaiat, and N. Kollias, “Facial skin fluorescence as a marker of the skin’s response to chronic environmental insults and its dependence on age,” Br. J. Dermatol. 154(1), 125–132 (2006).
[Crossref] [PubMed]

2005 (3)

L. H. Laiho, S. Pelet, T. M. Hancewicz, P. D. Kaplan, and P. T. C. So, “Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra,” J. Biomed. Opt. 10(2), 024016 (2005).
[Crossref] [PubMed]

A. N. Bashkatov, E. A. Genina, V. I. Kochubey, and V. V. Tuchin, “Optical properties of human skin, subcutaneous and mucous tissues in the wavelength range from 400 to 2000 nm,” J. Phys. D Appl. Phys. 38(15), 2543–2555 (2005).
[Crossref]

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Côté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. U.S.A. 102(46), 16807–16812 (2005).
[Crossref] [PubMed]

2004 (1)

J. Sandby-Møller, E. Thieden, P. A. Philipsen, J. Heydenreich, and H. C. Wulf, “Skin autofluorescence as a biological UVR dosimeter,” Photodermatol. Photoimmunol. Photomed. 20(1), 33–40 (2004).
[Crossref] [PubMed]

2003 (1)

S. Schneider, M. O. Schmitt, G. Brehm, M. Reiher, P. Matousek, and M. Towrie, “Fluorescence kinetics of aqueous solutions of tetracycline and its complexes with Mg2+ and Ca2+.,” Photochem. Photobiol. Sci. 2(11), 1107–1117 (2003).
[Crossref] [PubMed]

2002 (2)

J. Ling, S. D. Weitman, M. A. Miller, R. V. Moore, and A. C. Bovik, “Direct Raman imaging techniques for study of the subcellular distribution of a drug,” Appl. Opt. 41(28), 6006–6017 (2002).
[Crossref] [PubMed]

N. Kollias, G. Zonios, and G. N. Stamatas, “Fluorescence spectroscopy of skin,” Vib. Spectrosc. 28(1), 17–23 (2002).
[Crossref]

2001 (1)

E. G. Solon and L. Kraus, “Quantitative whole-body autoradiography in the pharmaceutical industry. Survey results on study design, methods, and regulatory compliance,” J. Pharmacol. Toxicol. Methods 46(2), 73–81 (2001).
[Crossref] [PubMed]

2000 (1)

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A. Nongnuch and A. Davenport, “The effect of vegetarian diet on skin autofluorescence measurements in haemodialysis patients,” Br. J. Nutr. 113(7), 1040–1043 (2015).
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Cardiovasc. Diabetol. (1)

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E. G. Solon, “Autoradiography techniques and quantification of drug distribution,” Cell Tissue Res. 360(1), 87–107 (2015).
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Fig. 1 Fluorescence emission screening experimental setup. Concurrent image capture of tissue section within the same FOV (same as 386-nm excitation cross-section) was performed with conventional fluorescence microscopy. Lower left inset indicates Intralipid-10% photocurrent measurements for a 7-day period to monitor excitation source stability. PD: Photodetector; BP: Bandpass filter; LP: Longpass filter; BS: Dichroic Beamsplitter; xyz translation stage not shown diagrammatically; 40 × objective lens, 0.65NA.

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Fig. 2 Geometry of autofluorescence collection with an integrating sphere from tissue sample. Dimension of tissue sample in this figure is exaggerated for illustration purpose only, while the remainder of the drawing is to-scale. The ray-tracing in this figure is for illustration purpose only. The integrating sphere transfer function (Φ_i /Φ_d) value was analytically estimated to be 947 using Eq. (4), and confirmed with a measured value of 969 using a 532-nm diffused source, as indicated in Fig. 4 below.

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Fig. 3 Total fluorescence emission detection geometry. Fluorescence was collected within the angular acceptance as shown at wavelengths above 450 nm (≥ OD4.0 below cut-on). Excitation wavelength is centered at 386 nm with a passband of 27 nm at FWHM (≥ OD6.0 above cut-off) delivered with a 40 × objective lens, 0.65NA. LP: 450-nm longpass filter.

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Fig. 4 Calibration of integrating sphere with a 532-nm diffused source an integrating sphere transfer function (Φ_i /Φ_d) value of 969.

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Fig. 5 Fluorescence radiance emission ranges 1.09 mW/cm².sr to 6.57mW/cm².sr was measured among specimens from different donors (sample size n = 41 donor n = 36). These quantitative results allow for direct comparison to the qualitative fluorescence intensity among the co-registered images captured during the experiment in Figs. 8, 9, and 10 below.

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Fig. 6 Additional analyses (a) indicate that when grouped into ranges of fluorescence radiance emission (n = 41 samples), the mean autofluorescence in the population was approximately 3.38 mW/cm².sr, with the population spread deviating from a normal distribution. (b) represent further distribution in a box-and-whisker plot where the population median was 3.10 mW/cm².sr. Four measurements exist outside the 95% confidence interval.

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Fig. 7 Confirmation that sections with at least 150-μm intervals from previous measured section of the same donor show consistent measurements. *P≤ 0.05 by Student’s T-test.

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Fig. 8 Images of hair follicles from various donors exhibiting increasing autofluorescence (a) D01; 1.20 mW/cm².sr, (b) D04; 1.69 mW/cm².sr, (c) D12; 2.73 mW/cm².sr, (d) D20; 3.15 mW/cm².sr, (e) D25; 3.58 mW/cm².sr, (f) D28; 4.07 mW/cm².sr, (g) D31; 4.73 mW/cm².sr, (h) D33; 5.78 mW/cm².sr, and (i) D34; 6.35 mW/cm².sr. Qualitative increase in fluorescence in these images are in correlation to quantitative measurements of fluorescence in Fig. 5. Autofluorescence from the blue spectrum appeared to dominate, with slight red-channel fluorescence in the epidermis (e). Fluorescence was measured and images captured with fluorescence originating from within the 500-µm circular FOV using a 40 × objective lens. Scale bar represents 50 μm.

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Fig. 9 Images of sebaceous glands from various donors exhibiting increasing autofluorescence (a) D01; 1.20 mW/cm².sr, (b) D04; 1.69 mW/cm².sr, (c) D10; 2.60 mW/cm².sr, (d) D18; 3.10 mW/cm².sr, (e) D25; 3.58 mW/cm².sr, (f) D28; 4.07 mW/cm².sr, (g) D30; 4.60 mW/cm².sr, (h) D33; 5.78 mW/cm².sr, and (i) D36; 6.57 mW/cm².sr. Qualitative increase in fluorescence in these images are in correlation to quantitative measurements of fluorescence in Fig. 5. Autofluorescence from the blue spectrum appeared to dominate, with no observable red fluorescence under the experimental conditions. Fluorescence was measured and images captured with fluorescence originating from within the 500-µm circular FOV using a 40 × objective lens. Scale bar represents 50 μm.

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Fig. 10 Images consisting of hair follicle with adjacent sebaceous glands from various donors exhibiting increasing autofluorescence (a) D01; 1.20 mW/cm².sr, (b) D17; 3.07 mW/cm².sr, (c) D31; 4.73 mW/cm².sr, and (d) D34; 6.35 mW/cm².sr. Qualitative increase in fluorescence in these images are in correlation to quantitative measurements of fluorescence in Fig. 5. Autofluorescence from the blue spectrum appeared to dominate, with red-channel fluorescence detected in the hair follicles (a,b,c). Fluorescence was measured and images captured with fluorescence originating from within the 500-µm circular FOV using a 40 × objective lens. Scale bar represents 50 μm.

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Fig. 11 Conventional fluorescence microscopy images of tissues from (a) low autofluorescence donor, D06, and (b) high autofluorescence donor, D32, in an ex vivo penetration study of 2.5 × , 8 × , and 25 × daily doses of a 2% topical minocycline gel along with vehicle and untreated controls. * arrows indicate mainly low (blue) autofluorescence of D06, while # arrows indicate mainly high autofluorescence of D32. Left columns in (a) and (b) are raw images, and right columns are segmentation of mainly (red) minocycline fluorescence from 610 to 680 nm. Note that no red fluorescence was detectable in the untreated and vehicle arms of D06, while slight red autofluorescence was noticeable in D32. Yellow dotted line areas in the treated D06 donor tissues indicated perceptible minocycline fluorescence matching the corresponding segmented images to the right. However, in the treated D32 donor tissues, the delivery into the deeper dermal layer, indicated by † arrows, was only perceptible after segmentation of the 610-680 nm signals. 10x objective, scale bar represents 200 μm. Both image sets were captured with the same acquisition parameters.

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Fig. 12 Autofluorescence spectral intensity of 15 donor tissue specimens with TPEF/spectrometer and conventional fluorescence microscopy/integrating sphere measurements. Results from the two data sets were compared using Pearson’s correlation. The analysis indicted fair relative agreement of single- and two-photon excited fluorescence yield (Pearson’s correlation coefficient, r = 0.613, p = 0.015).

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Tables (1)

Table 1 Physical parameters of fluorescence measurement setup. Theoretical estimate based on values in the table below produced an integrating sphere transfer function (Φ_i /Φ_d) value of 947.

View Table

Equations (7)

Equations on this page are rendered with MathJax. Learn more.

Φ_{d} = L_{s p h} A_{d} Ω

ℜ (λ) = \frac{i_{d}}{_{d}}

L_{s p h} = \frac{Φ_{i}}{π . A_{s}} \frac{ρ}{[1 - ρ (1 - f)]} = \frac{Φ_{i}}{π . A_{s}} M

\frac{Φ_{i}}{Φ_{d}} = \frac{A_{s}}{A_{d}} . \frac{1}{M . \sin^{2} θ_{d}}

Φ_{i} = \frac{Φ_{s}}{2} . \frac{r'^{2} \int_{0}^{2 π} d ϕ \int_{0}^{θ_{i}} \sin θ d θ}{r'^{2} \int_{0}^{2 π} d ϕ \int_{0}^{π} \sin θ d θ} = \frac{Φ_{s}}{2} . γ

\frac{Φ_{s}}{Φ_{d}} = \frac{2 A_{s}}{γ A_{d}} . \frac{1}{M . \sin^{2} θ_{d}}

Φ_{s} = 12635 Φ_{d}

INPUT PARAMETERS	VALUES
Output/Detector Port Area (cm²), A_d	0.0707
Photodetector Acceptance Angle (deg), ��_d	10.24
Detector Acceptance Angle, ��	0.099
Detector Responsivity (A/W) @ 532 nm, ℜ	0.33
Detector Responsivity (A/W) @ 550 nm, ℜ	0.35
Integrating Sphere Reflectance, ��	0.99
Input Port Area (cm²), A_i	1.23
Integrating Sphere Surface Area (cm²), A_s	81.07
Sphere Multiplier, M	38.3
Integrating Sphere Acceptance Angle (deg), ��_i	45.7