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WIREs Nanomed Nanobiotechnol
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Multiphoton microscopy applications in nanodermatology

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Abstract Multiphoton microscopy is being used as a tool for tracking nanoparticles and assessing endogenous autofluorescent molecules. This technology is founded on femtosecond lasers that are capable of exciting unlabeled metal nanoparticles, quantum dots, and upconverting nanoparticles. The addition of time‐correlated single‐photon counting detectors enables fluorescence lifetime imaging. Fluorescence lifetime measurements result in high‐resolution, quantitative data that can be used to distinguish nanoparticle signals from those of endogenous fluorophores. This application of multiphoton microscopy is capable of simultaneous nanoparticle and NAD(P)H imaging, resulting in the capacity for dye‐free assessments of treatment‐induced metabolic changes. The stage is set for advanced, clinical imaging focused nanoparticle trials that can directly address critical issues in nanomedicine and nanotoxicology. WIREs Nanomed Nanobiotechnol 2012, 4:680–690. doi: 10.1002/wnan.1189 This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials

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Schematic of multiphoton microscope (MPM) and energy transfer diagrams. MPMs generally have tunable 80 MHz titanium:sapphire laser sources for infrared excitation light. Scanning galvanometers are used to raster scan the excitation beam over the imaging area. The excitation beam passes through a dichroic mirror and an objective before reaching the specimen. Emission light then passes back through the objective and is reflected to one or more detectors by the dichroic mirror. Photomultiplier tube and time‐correlated single‐photon counting detectors (PMT and TCSPC, respectively) can be stationary or be selected depending on the application. These components are shown in schematic form within panel (a). Panel (b) shows energy transfer diagrams for single photon fluorescence, two‐photon fluorescence, second harmonic generation, and energy transfer upconversion.

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Identifying ZnO‐NP specific signals within living skin. MPM images of ZnO‐NP alone (a) and the stratum granulosum in living human skin (b) using identical settings: 740 nm excitation, 350–450 nm emission, 21 mW at the rear of the 40×/1.30 NA oil immersion objective, 646 contrast, and 540 brightness on a DermaInspect system with a MaiTai laser. If the nanoparticles were within this skin layer, there would be no way to separate the signals based on intensity. Panels (c)–(e) show the FLIM photonic characteristics of untreated stratum granulosum (untreated‐SG), ZnO‐NP alone (ZnO‐NP), vehicle at the stratum corneum depth (CCT‐SC), vehicle at the stratum granulosum depth (CCT‐SG), skin treated with ZnO‐NP for 4 and 24 h at the stratum corneum depth (4 h ZnO‐NP‐SC and 24 h ZnO‐NP‐SC, respectively). Panel (f) shows a standard concentration curve generated with ZnO‐NP alone that was diluted with a CCT vehicle. (Reprinted with permission from Ref 10. Copyright 2011 Springer)

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In vivo multiphoton images of nonlesional and lesional volunteer skin of different depths after 2 h treatment with ZnO‐NP. Each image is 214 × 214 × 1 µm3. These color images depict the autofluorescence from NAD(P)H as blue (α1% 0–85) and ZnO‐NP as yellow/red (α1% 90–100) in volunteer skin. All bars indicate 100 µm. The color scale bar represents α1% 85–100, blue to red. (Reprinted with permission from Ref 10. Copyright 2011 Springer)

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The 3 × 3 mm2 RCM mosaic of Au‐NP treated skin at the spinous layer. Panel (a) shows a 9 mm2 mosaic of skin treated with Au‐NPs in an aqueous vehicle. Panel (b) shows skin of the same size, but treated with Au‐NPs in a toluene vehicle. Both examples were treated for 24 h and took seconds to acquire. This type of mosaic acquisition is possible with motorized stages on MPMs; however, this moving stage approach cannot be readily applied for clinical imaging. In contrast, the Lucid VivaScope RCM is currently the gold standard for clinical confocal imaging because of the ease of use and rapid acquisition, among other features. Whether or not multiphoton devices will be engineered for this type of robust clinical use is yet to be seen.

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Dermoscopy, RCM, and MPT‐FLIM images of treated human skin. Dermoscopy and RCM images showing the surface of skin specimens treated with aqueous solution, toluene, AuNP‐Aq, and AuNP‐TOL for 24 h are shown in panels (a)–(d) for dermoscopy and panels (e)–(h) for RCM images, respectively. The black dashed line indicates abnormal reflectance structure within the toluene treated skin in panels (f) and (h); the white dashed line in panel (g) shows highly reflective particles on the surface of the skin. FLIM images from the stratum granulosum layer of the epidermis from skin treated for 4 and 24 h in panels (i)–(l) and (m)–(p), respectively. Scale bars in panel (d) is 4 mm and in panels (h), (l), and (p) indicates 50 µm; the pseudocolored MPT‐FLIM images are α1% 50–100 from blue to red. The blue‐green coloration indicates cellular autofluorescence, i.e., NAD(P)H and Au‐NP luminescence is orange to red in panels (k), (l), (o), and (p). (Reprinted with permission from Ref 15. Copyright 2011 Springer)

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Scanning electron micrographs of in vivo porcine skin. Panel (a) shows low magnification of CM 643 sunscreen on a hair at the surface of UVB‐exposed skin. Note agglomerated Zn (rectangle) near the base of the hair (H). The bar indicates 50 µm. Panel (b) shows a higher magnification of heterogeneous Zn (arrows) within the area denoted by the rectangle. The inset in (b) shows X‐ray diffraction analysis of areas positive for Zn (arrows). The bar indicates 600 nm. (Reprinted with permission from Ref 33. Copyright 2011 Oxford University Press)

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TEM (a,b,d,e) and MPM (c,f) nanoparticle imaging. All panels show images of the same ZnO‐NP containing sunscreen and the TEM panels show low and high magnification. Panels (a) and (d) show conventional preparation and TEM imaging. Panels (b) and (e) show an improved sample processing method using high‐pressure freezing followed by TEM imaging. Panels (c) and (f) show MPM imaging of unprocessed sunscreen and a sample processed with high‐pressure freezing. This figure (Sunscreen 5) was adapted from the Antaria sunscreen,32 and the preparation is the same as used in other publications.6,10,17 (Reprinted with permission from Ref 32. Copyright 2012 Future Medicine, Ltd.)

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