Research Techniques Made Simple: Two-Photon Intravital Imaging of the Skin

  • Peyman Obeidy
    Affiliations
    The Centenary Institute, Newtown, New South Wales, Australia
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  • Philip L. Tong
    Affiliations
    The Centenary Institute, Newtown, New South Wales, Australia

    Discipline of Dermatology, University of Sydney, Camperdown, New South Wales, Australia

    Department of Dermatology, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia
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  • Wolfgang Weninger
    Correspondence
    Correspondence: Wolfgang Weninger, Centenary Institute for Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown NSW 2042, Australia.
    Affiliations
    The Centenary Institute, Newtown, New South Wales, Australia

    Discipline of Dermatology, University of Sydney, Camperdown, New South Wales, Australia

    Department of Dermatology, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia
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      Over the last few years, intravital two-photon microscopy has matured into a powerful technology helping basic and clinical researchers obtain quantifiable details of complex biological mechanisms in live and intact tissues. Two-photon microscopy provides high spatial and temporal resolution in vivo with little phototoxicity that is unattainable by other optical tools like confocal microscopy. Using ultrashort laser pulses, two-photon microscopy allows the visualization of molecules, cells, and extracellular structures up to depths of 1 mm within tissues. Consequently, real-time imaging of the individual skin layers under both physiological and pathological conditions has revolutionized our understanding of cutaneous homeostasis, immunity, and tumor biology. This review provides an overview to two-photon microscopy of the skin by covering the basic concepts and current applications in diverse preclinical and clinical settings.

      Abbreviations:

      SHG (second harmonic generation), THG (third harmonic generation), TPM (two-photon microscopy)
      CME Activity Dates: 21 March 2018
      Expiration Date: 20 March 2019
      Estimated Time to Complete: 1 hour
      Planning Committee/Speaker Disclosure: All authors, planning committee members, CME committee members and staff involved with this activity as content validation reviewers have no financial relationship(s) with commercial interests to disclose relative to the content of this CME activity.
      Commercial Support Acknowledgment: This CME activity is supported by an educational grant from Lilly USA, LLC.
      Description: This article, designed for dermatologists, residents, fellows, and related healthcare providers, seeks to reduce the growing divide between dermatology clinical practice and the basic science/current research methodologies on which many diagnostic and therapeutic advances are built.
      Objectives: At the conclusion of this activity, learners should be better able to:
      • Recognize the newest techniques in biomedical research.
      • Describe how these techniques can be utilized and their limitations.
      • Describe the potential impact of these techniques.
      CME Accreditation and Credit Designation: This activity has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education through the joint providership of Beaumont Health and the Society for Investigative Dermatology. Beaumont Health is accredited by the ACCME to provide continuing medical education for physicians. Beaumont Health designates this enduring material for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.
      Method of Physician Participation in Learning Process: The content can be read from the Journal of Investigative Dermatology website: http://www.jidonline.org/current. Tests for CME credits may only be submitted online at https://beaumont.cloud-cme.com/RTMS-Apr18 – click ‘CME on Demand’ and locate the article to complete the test. Fax or other copies will not be accepted. To receive credits, learners must review the CME accreditation information; view the entire article, complete the post-test with a minimum performance level of 60%; and complete the online evaluation form in order to claim CME credit. The CME credit code for this activity is: 21310. For questions about CME credit email [email protected] .

      Benefits

      • Depth of light penetration
      • Reduced photobleaching and phototoxicity outside the focal plane
      • Optical sectioning and label-free visualization of autofluorescent molecules and structures

      Limitations

      • Potential phototoxicity in the focal plane after long-term imaging
      • Distorted z-resolution, in particular at higher depths levels
      • Potential thermal damage due to high-laser power pulses
      • High costs of instrument compared with confocal and conventional microscopy
      • High level of expertise required

      Introduction

      The theory underlying two-photon excitation was described initially by Maria Göppert-Mayer in 1931, and the first two-photon microscope was pioneered and patented by Winfried Denk and colleagues almost six decades later in 1990 (
      • Weigert R.
      • Sramkova M.
      • Parente L.
      • Amornphimoltham P.
      • Masedunskas A.
      Intravital microscopy: a novel tool to study cell biology in living animals.
      ). Two-photon microscopy (TPM) enables examination of the deeper layers of live specimens, including the skin, and has many advantages over conventional microscopic imaging methods. Using long-wavelength, ultrashort-pulse laser sources, the excitation volume in TPM is confined to the focal plane, thus excluding out-of-focus background excitation, which is observed, for example, in confocal microscopy. This minimizes photobleaching and phototoxicity. Another useful feature of two-photon excitation relevant to biologic imaging is the capacity of TPM to take advantage of higher-order interactions between light and tissue components, for example, second (SHG) and third (THG) harmonic generation signals, which can provide architectural information of the investigated tissue (
      • Yew E.
      • Rowlands C.
      • So P.T.
      Application of multiphoton microscopy in dermatological studies: a mini-review.
      ).
      Based on its optical features, intravital TPM offers an experimental and diagnostic method that can be used to uncover the homeostatic principles of normal skin and events resulting in skin diseases (
      • Perry S.W.
      • Burke R.M.
      • Brown E.B.
      Two-photon and second harmonic microscopy in clinical and translational cancer research.
      ). The skin is a complex multilayer organ, which imparts optical challenges for imaging. For example, each layer exhibits different optical properties such as the refractive index (i.e., 1.51 in stratum corneum, 1.34 in epidermis, and 1.41 in the dermis). Other potential limitations include the high cost, reduced z-resolution (in particular at depths > 500 μm), and potential thermal tissue damage due to absorption of high-power laser light (
      • Lo W.
      • Sun Y.
      • Lin S.J.
      • Jee S.H.
      • Dong C.Y.
      Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar.
      ,
      • Olivieri D.N.
      • Zenclussen A.C.
      • Tadokoro C.E.
      In vivo tracking of mononuclear cells in the virgin uterus and in implantation sites.
      ). TPM can be expanded by combining it with other optical methods like Forster resonance energy transfer (i.e., FRET) and fluorescence recovery after photobleaching (i.e., FRAP) (
      • Broussard J.A.
      • Green K.J.
      Research techniques made simple: methodology and applications of Forster resonance energy transfer (FRET) microscopy.
      ,
      • Erami Z.
      • Herrmann D.
      • Warren S.C.
      • Nobis M.
      • McGhee E.J.
      • Lucas M.C.
      • et al.
      Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue.
      ).

      The Basic Principles of TPM

      TPM relies on nonlinear photoexcitation of molecules, whereby two low-energy photons are almost simultaneously (within 10–18 to 10–16 seconds) absorbed in the same focal point, resulting in fluorescence emission. Tunable short (femtosecond)-pulsed lasers facilitate such rare collisions. This principle also eliminates the need for a pinhole, which is used in confocal microscopy, because the excitation outside the focal plane is too weak to cause appreciable fluorescence. In addition, short-pulse lasers can keep the average power at the sample low and thereby reduce tissue damage, enabling long-term imaging (
      • Weigert R.
      • Sramkova M.
      • Parente L.
      • Amornphimoltham P.
      • Masedunskas A.
      Intravital microscopy: a novel tool to study cell biology in living animals.
      ). Compared with confocal microscopy, which in the skin is limited to a depth of approximately 50–60 μm, light penetration in TPM goes beyond the epidermis and superficial dermis to about 300–600 μm, depending on site, excitation wavelength, and fluorophores, allowing visualization of endogenous and exogenous fluorophores and structures like collagen or elastin (Table 1) (
      • Nwaneshiudu A.
      • Kuschal C.
      • Sakamoto F.H.
      • Anderson R.R.
      • Schwarzenberger K.
      • Young R.C.
      Introduction to confocal microscopy.
      ,
      • Yew E.
      • Rowlands C.
      • So P.T.
      Application of multiphoton microscopy in dermatological studies: a mini-review.
      ).
      Table 1Skin layers and a selected list of endogenous components and their spectral positions in TPM
      Skin Main LayerSkin SublayerCells in Each LayerEndogenously Detectable ComponentSpectral Positions (Excitation/Emission in nm)
      EpidermisStratum corneum5–6 layers of cornified dead cellsKeratin(760–860/477–503)
      Stratum lucidumDendritic epidermal T cells

      Keratinocytes

      Dendritic cells

      Langerhans cells

      Melanocytes

      Merkel cells
      NADPH (in living keratinocytes)Free

      460 (730–780/460–480)
      Stratum granulosumBound to protein

      440 (730–780/460–480)
      Stratum spinosumMelanin (eumelanin and pheomelanin)440-420-475 (800/550)
      Basal cell layer
      DermisDermal dendritic cells

      Dermal T cells

      Neutrophils

      Macrophages

      Other immune cells

      Stromal cells
      Collagen fibers(800–860/400–430)
      Elastin fibers(730–760/460–480)
      Abbreviation: TPM, two-photon microscopy.
      SHG signals add a unique advantage to TPM by allowing label-free visualization of non-centrosymmetric structural components, such as extracellular matrix proteins (
      • Rehberg M.
      • Krombach F.
      • Pohl U.
      • Dietzel S.
      Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy.
      ). The signal in TPM is generated when excited photons decay to their ground state and emit a photon with a frequency less than double of its original. SHG signal is generated when scattered incident photons recombine into a single photon without energy loss (Figure 1) (
      • Olivieri D.N.
      • Zenclussen A.C.
      • Tadokoro C.E.
      In vivo tracking of mononuclear cells in the virgin uterus and in implantation sites.
      ). SHG is a useful feature of TPM when, for example, studying tissue architecture, for instance, the delineation of boundaries between normal and malignant tissue. Apart from SHG, other less common higher-order processes, THG and fourth harmonic generation, also exist (
      • Yew E.
      • Rowlands C.
      • So P.T.
      Application of multiphoton microscopy in dermatological studies: a mini-review.
      ). THG is induced by changes in refraction index occurring at interfaces such as cell nuclei and cytoplasm or cytoplasm and interstitial fluid (
      • Rehberg M.
      • Krombach F.
      • Pohl U.
      • Dietzel S.
      Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy.
      ). Moreover, fourth harmonic generation signal is the sum of the frequency generation from THG and pump light (
      • Karvonen L.
      • Saynatjoki A.
      • Mehravar S.
      • Rodriguez R.D.
      • Hartmann S.
      • Zahn D.R.
      • et al.
      Investigation of second- and third-harmonic generation in few-layer gallium selenide by multiphoton microscopy.
      ). These signals can be used to obtain further information on structural tissue components.
      Figure 1
      Figure 1Perrin-Jablonski fluorescence diagram describing one-photon excited fluorescence versus two-photon and SHG signals. (a) In one-photon excitation, a higher energy source is required and absorbed to excite the photon from the ground state to the excited state. Visible light is emitted when this photon returns to its ground state, with the emitted photon having slightly lower radiation energy at a frequency of ω than the original light frequency ωi. (b) In two-photon excited fluorescence, this process is replicated with the simultaneous absorption of two lower-energy photons. Both processes involve real energy transition of electrons where emitted light energy is partially lost. (c) In SHG no energy is absorbed, and all the scattered incident photons are recombined into a single photon, without energy lost and at the same frequency as 2ωi. SHG, second harmonic generation.

      Overview of Methodology (Sample Preparation and Filter Setup)

      In this review, we focus on the ear skin model to illustrate the experimental setup and use of TPM in intravital imaging. Mice are appropriately anesthetized, for example, by the intraperitoneal injection of ketamine/xylazine. Hair is then removed from the region of interest using depilatory cream, after which the animal is stably positioned on a custom-built, temperature-controlled mounting platform (Figure 2a). The ear is covered with a coverslip and a solution of glycerin-phosphate buffered saline. Intravenous injection of plasma markers such as Evans Blue dye, high-molecular FITC-dextran, or quantum dots can be used to delineate blood vessels (
      • Li J.L.
      • Goh C.C.
      • Keeble J.L.
      • Qin J.S.
      • Roediger B.
      • Jain R.
      • et al.
      Intravital multiphoton imaging of immune responses in the mouse ear skin.
      ). A mode-locked titanium-sapphire laser at 920 nm wavelength can be used to excite eGFP (excitation/emission = 488/507 nm), Evans Blue (excitation/emission = 620/680 nm), and SHG (excitation/emission = 415/455 nm). Signals are detected with different photomultiplier tubes (filter setup is outlined in Figure 2b). For the imaging presented here, a ×20 water immersion objective was used. TPM imaging sessions commonly produce large-size datasets (giga- to terabyte range), the analysis of which requires powerful hardware and software instrumentation. Postacquisition processing of data can be achieved using commercial software packages such as Imaris (Bitplane, Zurich, Switzerland), Metamorph (Molecular Devices, Sunnyvale, CA), and Volocity (PerkinElmer, Waltham, MA), as well as shareware including ImageJ (National Institutes of Health, Bethesda, MD) (Figure 2c). Publicly accessible newer software like FocusStack and StimServer provide minimal memory footprint and are thus more cost effective. These packages have the capability of performing stack alignment, automated re-randomization of time-lapse data, and automated cell segmentation, with the additional option of direct incorporation into MATLAB (MathWorks, Natick, MA)-based analysis tools (
      • Muir D.R.
      • Kampa B.M.
      FocusStack and StimServer: a new open source MATLAB toolchain for visual stimulation and analysis of two-photon calcium neuronal imaging data.
      ). A variety of parameters such as cell motion patterns, cellular localization, and interactions can be computed to describe the orchestration of immune responses in cutaneous biology (
      • Germain R.N.
      • Robey E.A.
      • Cahalan M.D.
      A decade of imaging cellular motility and interaction dynamics in the immune system.
      ,
      • Li J.L.
      • Goh C.C.
      • Keeble J.L.
      • Qin J.S.
      • Roediger B.
      • Jain R.
      • et al.
      Intravital multiphoton imaging of immune responses in the mouse ear skin.
      ).
      Figure 2
      Figure 2Visualization of mast cells in vivo using the mouse ear skin model. (a) Close-up schematic view of a mouse ear positioned on a temperature-controlled mounting platform designed using Thinkercad (Autodesk, San Rafael, CA). (b) For detection of fluorophores, ear skin was simultaneously exposed to a mode-locked titanium-sapphire laser at 920 nm wavelength for excitation of eGFP (excitation/emission = 488/507 nm), Evans Blue (excitation/emission = 620/680 nm), and SHG (excitation/emission = 415/455 nm). The fluorescence signals were detected using independent photomultiplier tubes after transmitting through or getting transmitted and reflected by dichroic mirrors. Band-pass filters were used to further restrict the wavelength detected to decrease background noise and spectral overlaps. (c) Skin of a c-kit–GFP transgenic mouse was imaged. Mast cells are green. The extracellular matrix in the dermis was detected by SHG signal (blue) and the blood vessels (red) (Evans Blue). The imaging was performed using a ×20 water-immersion objective. SHG, second harmonic generation.

      Experimental Dermatology Research and TPM

      Arguably, the immune system is where multiphoton microscopy has had the greatest impact in basic dermatological studies in both the steady state and during inflammation, where it is possible to study single cell behavior in real time and molecules within an intact living environment (
      • Weninger W.
      • Biro M.
      • Jain R.
      Leukocyte migration in the interstitial space of non-lymphoid organs.
      ). Currently, the most common sites for intravital multiphoton imaging of the skin in mice include the ear (
      • Roediger B.
      • Ng L.G.
      • Smith A.L.
      • Fazekas de St Groth B.
      • Weninger W.
      Visualizing dendritic cell migration within the skin.
      ), hind footpad (
      • Graham D.B.
      • Zinselmeyer B.H.
      • Mascarenhas F.
      • Delgado R.
      • Miller M.J.
      • Swat W.
      ITAM signalling by Vav family Rho guanine nucleotide exchange factors regulates interstitial transit rates of neutrophils in vivo.
      ), and dorsal skin (
      • Amornphimoltham P.
      • Masedunskas A.
      • Weigert R.
      Intravital microscopy as a tool to study drug delivery in preclinical studies.
      ). The footpad is hairless, but hair removal is essential in the ear and back skin because the autofluorescence of hair shafts obscure image acquisition (
      • Li J.L.
      • Goh C.C.
      • Keeble J.L.
      • Qin J.S.
      • Roediger B.
      • Jain R.
      • et al.
      Intravital multiphoton imaging of immune responses in the mouse ear skin.
      ,
      • Roediger B.
      • Ng L.G.
      • Smith A.L.
      • Fazekas de St Groth B.
      • Weninger W.
      Visualizing dendritic cell migration within the skin.
      ). In addition, ear and footpad skin are less affected by respiration, and it easier to produce a stable image. However, it is important that the mouse reaches a stable temperature before image recording, otherwise drift in the x-, y-, or z-axis can occur, resulting in an unstable time-lapse video (
      • Li J.L.
      • Goh C.C.
      • Keeble J.L.
      • Qin J.S.
      • Roediger B.
      • Jain R.
      • et al.
      Intravital multiphoton imaging of immune responses in the mouse ear skin.
      ). Long-term time-lapse imaging can be achieved by exploiting various in vivo cell and tissue labeling techniques and fluorescent reporter mice.
      The application of multiphoton microscopy can, therefore, permit quantitative measurements of spatial distribution, motility, interactions, and response dynamics of leukocytes under homeostatic and inflammatory conditions, as well as host-tumor responses, which otherwise would not be possible. For example, using CXCR6-GFP transgenic reporter mice, differences in EGFP+ γδ T-cell morphology and their density in dermis compared with epidermis was investigated in ear skin (
      • Sumaria N.
      • Roediger B.
      • Ng L.G.
      • Qin J.
      • Pinto R.
      • Cavanagh L.L.
      • et al.
      Cutaneous immunosurveillance by self-renewing dermal gammadelta T cells.
      ). Using CD11c-EYFP mice, dendritic cell function and migratory behavior were evaluated in sterile skin injury in which the chemoattractants to the site of injury were proposed to rise from the resident or recruited inflammatory cells (
      • Goh C.C.
      • Li J.L.
      • Devi S.
      • Bakocevic N.
      • See P.
      • Larbi A.
      • et al.
      Real-time imaging of dendritic cell responses to sterile tissue injury.
      ). TPM microscopy can also be enhanced by combining it with other optical techniques. Using a Cre-inducible E-cadherin–GFP transgenic mouse model,
      • Erami Z.
      • Herrmann D.
      • Warren S.C.
      • Nobis M.
      • McGhee E.J.
      • Lucas M.C.
      • et al.
      Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue.
      combined TPM and fluorescence recovery after photobleaching to assess alteration in cadherin-based cell-cell junction integrity in the setting of tumor progression. Although this is not a comprehensive review of all the advances made through multiphoton imaging of the skin, these examples serve to show that this technology has significantly advanced our understanding of the spatiotemporal interactions of immune cell subsets in lymphoid organs and peripheral tissues, including the skin (
      • Germain R.N.
      • Robey E.A.
      • Cahalan M.D.
      A decade of imaging cellular motility and interaction dynamics in the immune system.
      ,
      • Jain R.
      • Weninger W.
      Shedding light on cutaneous innate immune responses: the intravital microscopy approach.
      ). Moreover, although animal models can be used to elucidate the basic cellular and molecular mechanisms to obtain real-time, quantifiable details of complex biological mechanisms in intact tissues, the ultimate goal is to translate our understanding into clinical applications.

      Clinical Dermatology and TPM

      Multiphoton microscopy is now being considered as a potential noninvasive diagnostic tool in dermatology, because the skin is very accessible for imaging. Although currently it is only possible to derive structural information from autofluorescent signals within human tissue in vivo, TPM is able to provide near-histological grade images without the need for a skin biopsy or tissue processing. The use of autofluorescence as a source of natural contrast has been shown in reflectance confocal microscopy. However, the multiphoton microscope has multiple advantages, allowing “optical biopsies” of human skin in vivo (
      • Luo Y.
      • Singh V.R.
      • Bhattacharya D.
      • Yew E.Y.S.
      • Tsai J.C.
      • Yu S.L.
      • et al.
      Talbot holographic illumination nonscanning (THIN) fluorescence microscopy.
      ). Nevertheless, there are limited human safety data for multiphoton imaging in the skin, and thus its use currently is restricted to the experimental setting. Intravital imaging studies using TPM in mouse skin have not shown signs of acute phototoxicity (
      • Li J.L.
      • Goh C.C.
      • Keeble J.L.
      • Qin J.S.
      • Roediger B.
      • Jain R.
      • et al.
      Intravital multiphoton imaging of immune responses in the mouse ear skin.
      ,
      • Roediger B.
      • Ng L.G.
      • Smith A.L.
      • Fazekas de St Groth B.
      • Weninger W.
      Visualizing dendritic cell migration within the skin.
      ), and clinical studies indicate minimal erythema and cellular damage from two-photon excitation (
      • Fischer F.
      • Volkmer B.
      • Puschmann S.
      • Greinert R.
      • Breitbart E.
      • Kiefer J.
      • et al.
      Assessing the risk of skin damage due to femtosecond laser irradiation.
      ).
      Commercial multiphoton microscope systems are now available and have been used experimentally in the clinical setting to investigate the structural composition of human skin with the promise of future application in disease processes in vivo (
      • Shirshin E.A.
      • Gurfinkel Y.I.
      • Priezzhev A.V.
      • Fadeev V.V.
      • Lademann J.
      • Darvin M.E.
      Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo: assessment of blood capillaries and structural proteins localization.
      ). These studies have mainly focused on the utility of this technology in skin aging studies (
      • Koehler M.J.
      • Preller A.
      • Kindler N.
      • Elsner P.
      • Konig K.
      • Buckle R.
      • et al.
      Intrinsic, solar and sunbed-induced skin ageing measured in vivo by multiphoton laser tomography and biophysical methods.
      ), inflammatory dermatoses (
      • Koehler M.J.
      • Preller A.
      • Elsner P.
      • Konig K.
      • Hipler U.C.
      • Kaatz M.
      Non-invasive evaluation of dermal elastosis by in vivo multiphoton tomography with autofluorescence lifetime measurements.
      ,
      • Sugata K.
      • Osanai O.
      • Sano T.
      • Takema Y.
      Evaluation of photoaging in facial skin by multiphoton laser scanning microscopy.
      ), and role of skin cancer diagnosis (
      • Balu M.
      • Zachary C.B.
      • Harris R.M.
      • Krasieva T.B.
      • Konig K.
      • Tromberg B.J.
      • et al.
      In vivo multiphoton microscopy of basal cell carcinoma.
      ). In addition, in combination with fluorescence lifetime imaging, TPM has been used to study the metabolic state of keratinocytes in normal and inflamed human epidermis (
      • Huck V.
      • Gorzelanny C.
      • Thomas K.
      • Getova V.
      • Niemeyer V.
      • Zens K.
      • et al.
      From morphology to biochemical state - intravital multiphoton fluorescence lifetime imaging of inflamed human skin.
      ). Moreover, similar to the murine laser injury model (
      • Ng L.G.
      • Qin J.S.
      • Roediger B.
      • Wang Y.
      • Jain R.
      • Cavanagh L.L.
      • et al.
      Visualizing the neutrophil response to sterile tissue injury in mouse dermis reveals a three-phase cascade of events.
      ), it has been proposed that the same femtosecond laser used to excite and image endogenous fluorophores in human skin can be used as a dermal cutting tool. Because of the two-photon effect, the desired femtosecond laser ablation occurs at the focal point within the intact skin (
      • Garvie-Cook H.
      • Stone J.M.
      • Yu F.
      • Guy R.H.
      • Gordeev S.N.
      Femtosecond pulsed laser ablation to enhance drug delivery across the skin.
      ). Although such approaches are purely experimental, it does highlight the potential significance of in vivo imaging, with possible application in the simultaneous treatment of human skin conditions.

      Further Reading

      Two-photon excitation has been extensively reviewed in many recent articles (e.g.,
      • Secklehner J.
      • Lo Celso C.
      • Carlin L.M.
      Intravital microscopy in historic and contemporary immunology.
      ). In addition, the US National Institutes of Health Resource for Biophysical Imaging (http://www.drbio.cornell.edu/ and http://www.jenlab.de/) provides further information on fluorophores and equipment for multiphoton microscopy for the interested reader.

      Conclusions

      TPM is an advanced optical imaging technique that uses brief, intense, long-wavelength laser pulses with the capacity to penetrate into the deep layer of the skin. This technology has thus become popular in experimental and clinical dermatology research to investigate the mechanisms underlying skin pathologies. Commercial instruments are now available for real-time microscopy using endogenous autofluorescent components like melanin, elastin, collagen, and NAD(P)H. Among many advantages of TPM are penetration depth, minimal out-of-focus signals, minimal photobleaching, and reduced phototoxicity, as well as the ability to image structures label free. The increased availability of transgenic mice and fluorescent probes and instrumentational improvements like laser safety, together with enhanced analytical capability, make TPM an important part of the biomedical investigation.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We wish to thank Rohit Jain and Shweta Tikoo for critical reading of the manuscript and Kathy On for her technical expertise. This work was supported by National Health and Medical Research Council grants 1106439 , 1104876 , and 1085981 (to WW).

      Author Contributions

      PO and PLT were involved in figure development and writing the manuscript. WW contributed to concept and design, writing the manuscript, and final approval.

      Multiple Choice Questions

      • 1.
        Which of the following is true about the basic principles of two-photon microscopy (TPM)?
        • A.
          A two-photon microscope uses a pinhole.
        • B.
          TPM requires collision of two low-energy photons almost simultaneously.
        • C.
          UV spectra are used in TPM.
        • D.
          Generation of second harmonic generation (SHG) requires absorption of two low-energy photons.
      • 2.
        Which of the following is not an advantage of TPM?
        • A.
          The depth of light penetration
        • B.
          Less photobleaching outside the confocal volume
        • C.
          Excellent z-resolution
        • D.
          None of the above
      • 3.
        TPM has been used in which of the following applications?
        • A.
          Aging studies
        • B.
          Inflammatory dermatoses
        • C.
          Skin cancer diagnosis
        • D.
          All of the above
      • 4.
        Endogenous autofluorescent signals in the skin can be generated from which of the following?
        • A.
          Elastin and collagen
        • B.
          NAD(P)H
        • C.
          Melanin
        • D.
          All of the above
      • 5.
        Based on Figure 2b, using a mode-locked titanium-sapphire laser at 920 nm wavelength, which band-pass (BP) filter is most appropriate?
        • A.
          Evans Blue with a BP filter of 505/30
        • B.
          SHG with a BP filter of 641/25
        • C.
          EGFP with a BP filter of 505/30
        • D.
          None of the above

      Supplementary Material

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