Advertisement

Longitudinal, 3D In Vivo Imaging of Sebaceous Glands by Coherent Anti-Stokes Raman Scattering Microscopy: Normal Function and Response to Cryotherapy

  • Yookyung Jung
    Affiliations
    Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA

    These authors contributed equally to this work
    Search for articles by this author
  • Joshua Tam
    Affiliations
    Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA

    These authors contributed equally to this work
    Search for articles by this author
  • H. Ray Jalian
    Affiliations
    Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Division of Dermatology, University of California, Los Angeles, Los Angeles, California, USA
    Search for articles by this author
  • R. Rox Anderson
    Affiliations
    Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA
    Search for articles by this author
  • Conor L. Evans
    Correspondence
    Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA
    Affiliations
    Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, USA

    Department of Dermatology, Harvard Medical School, Boston, Massachusetts, USA
    Search for articles by this author
      Sebaceous glands perform complex functions, and they are centrally involved in the pathogenesis of acne vulgaris. Current techniques for studying sebaceous glands are mostly static in nature, whereas the gland’s main function—excretion of sebum via the holocrine mechanism—can only be evaluated over time. We present a longitudinal, real-time alternative—the in vivo, label-free imaging of sebaceous glands using Coherent Anti-Stokes Raman Scattering (CARS) microscopy, which is used to selectively visualize lipids. In mouse ears, CARS microscopy revealed dynamic changes in sebaceous glands during the holocrine secretion process, as well as in response to damage to the glands caused by cooling. Detailed gland structure, plus the active migration of individual sebocytes and cohorts of sebocytes, were measured. Cooling produced characteristic changes in sebocyte structure and migration. This study demonstrates that CARS microscopy is a promising tool for studying the sebaceous gland and its associated disorders in three dimensions in vivo.

      Abbreviations

      CARS
      Coherent Anti-Stokes Raman Scattering

      Introduction

      Sebaceous glands have a predominant role in the etiology and pathology of acne vulgaris, the most prevalent skin disorder affecting over 85% of adolescents and many adults (
      • Bhate K.
      • Williams H.C.
      Epidemiology of acne vulgaris.
      ). Many current therapies for acne e.g. isotretinoin (
      • Rigopoulos D.
      • Larios G.
      • Katsambas A.D.
      The role of isotretinoin in acne therapy: why not as first-line therapy? facts and controversies.
      ), anti-androgens (
      • Katsambas A.D.
      • Dessinioti C.
      Hormonal therapy for acne: why not as first line therapy? facts and controversies.
      ), and photodynamic therapy (
      • Sakamoto F.H.
      • Lopes J.D.
      • Anderson R.R.
      Photodynamic therapy for acne vulgaris: a critical review from basics to clinical practice: part I. Acne vulgaris: when and why consider photodynamic therapy?.
      ) assert their effects, at least in part, by damaging sebaceous glands and/or suppressing their secretory function. This central involvement of sebaceous glands in acne, as well as the increasing appreciation for the complex neuro-immuno-endocrine functions performed by sebaceous glands, has led to a growing interest in the study of sebaceous gland physiology (
      • Nejati R.
      • Skobowiat C.
      • Slominski A.T.
      Commentary on the practical guide for the study of sebaceous glands.
      ). Of the various currently available investigative techniques, three-dimensional morphometric analysis of sebaceous glands has been found to be especially informative (
      • Hinde E.
      • Haslam I.S.
      • Schneider M.R.
      • et al.
      A practical guide for the study of human and murine sebaceous glands in situ.
      ). This is typically achieved by confocal microscopy in epidermal whole mounts, which requires ex vivo labeling of the glands (e.g., by immunofluorescence;
      • Hinde E.
      • Haslam I.S.
      • Schneider M.R.
      • et al.
      A practical guide for the study of human and murine sebaceous glands in situ.
      ), and thus, by necessity, limits the data to a static “snapshot” in time. This limitation is particularly significant as the gland’s main function—the excretion of sebum via the holocrine mechanism—is a dynamic process that can only be evaluated over time. In this study, we carried out in vivo, label-free longitudinal imaging of individual sebaceous glands using Coherent Anti-Stokes Raman Scattering (CARS) microscopy. CARS microscopy is a nonlinear imaging technology that can selectively visualize lipids based on their chemical structure to reveal dynamic changes in sebaceous glands, both during their normal holocrine secretion process and in response to damage caused by cryotherapy. No stains or genetic manipulations are needed for in vivo CARS microscopy, making it a promising tool for investigating sebaceous gland biology.
      CARS microscopy is a highly sensitive, chemically selective imaging technique that is capable of real-time, nonperturbative imaging in vivo (
      • Evans C.L.
      • Potma E.O.
      • Puoris'haag M.
      • et al.
      Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.
      ). The image contrast in CARS microscopy arises from molecular vibrational modes, such as the CH2 bonds in lipids. A nonlinear Raman technique CARS uses a pair of laser pulse trains, called “pump” (at a frequency ωp) and “Stokes” (at a frequency ωs), whose energy difference is set to correspond to a molecular vibration of interest (
      • Evans C.L.
      • Xie X.S.
      Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine.
      ). The combined pulses generate a beat frequency at ωpωs that can coherently drive specific molecular vibrations. When molecules that contain such vibrations are present in the microscope focal volume, strong emission at a new wavelength, called “anti-Stokes”, is generated at ωas=2ωp-ωs that can be readily collected using standard multiphoton filters and detection schemes (
      • Evans C.L.
      • Xie X.S.
      Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine.
      ). CARS is a multiphoton process, and as such it only generates emission at the objective focal point, enabling three-dimensional molecular imaging hundreds of microns deep in tissue (
      • Wright A.J.
      • Poland S.P.
      • Girkin J.M.
      • et al.
      Adaptive optics for enhanced signal in CARS microscopy.
      ). The turbid tissue environment strongly backscatters the anti-Stokes signal, allowing for highly sensitive imaging of tissue lipids in the cell membrane, cytoplasm, and other structures in vivo (
      • Evans C.L.
      • Potma E.O.
      • Puoris'haag M.
      • et al.
      Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.
      ).
      CARS is a spectroscopic technique that can be used to quantitatively measure chemical species present in intact tissue (
      • Evans C.L.
      • Xie X.S.
      Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine.
      ). In providing three-dimensional images with different chemical “weightings”, CARS and other coherent Raman imaging tools (
      • Freudiger C.W.
      • Min W.
      • Saar B.G.
      • et al.
      Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy.
      ;
      • Saar B.G.
      • Freudiger C.W.
      • Reichman J.
      • et al.
      Video-rate molecular imaging in vivo with stimulated Raman scattering.
      ) can be considered microscopic analogs to spectroscopic MRI, which have submicron spatial and video-rate temporal resolution. CARS microscopy is particularly sensitive to lipids, as their long hydrocarbon chains contain a multitude of CH2 moieties that have strong Raman vibrational modes. These properties have made CARS microscopy an attractive technology for biomedical imaging, with applications including the skin (
      • Evans C.L.
      • Potma E.O.
      • Puoris'haag M.
      • et al.
      Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.
      ), the peripheral nervous system (
      • Huff T.B.
      • Cheng J.X.
      In vivo coherent anti-Stokes Raman scattering imaging of sciatic nerve tissue.
      ;
      • Jung Y.K.
      • Ng J.H.
      • Keating C.P.
      • et al.
      Comprehensive evaluation of peripheral nerve regeneration in the acute healing phase using tissue clearing and optical microscopy in a rodent model.
      ), and brain (
      • Evans C.L.
      • Xu X.
      • Kesari S.
      • et al.
      Chemically-selective imaging of brain structures with CARS microscopy.
      ;
      • Fu Y.
      • Huff T.B.
      • Wang H-W.
      • et al.
      Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy.
      ).
      In this study, we used CARS microscopy to image normal sebaceous glands in mouse ears, before and after cryotherapy. Cryotherapy was an early (mid-1900s) treatment for acne, traditionally performed by applying liquid nitrogen (-196 °C) or a “slush” of frozen carbon dioxide and acetone (-78 °C) onto an area with acne until “superficial freezing” was achieved (
      • Dobes W.L.
      • Keil H.
      Treatment of acne vulgaris by cryotherapy (slush method).
      ;
      • Graham G.F.
      Cryosurgical treatment of acne.
      ). It has also been reported previously that sebaceous glands are especially sensitive to cooling-induced injury (
      • Gage A.A.
      • Meenaghan M.A.
      • Natiella J.R.
      • et al.
      Sensitivity of pigmented mucosa and skin to freezing injury.
      ). For the cryotherapy group in this study, we have applied cooling parameters that are found to be effective at damaging sebaceous glands, without causing gross cryogenic injury to the surrounding tissue (
      • Gage A.A.
      • Meenaghan M.A.
      • Natiella J.R.
      • et al.
      Sensitivity of pigmented mucosa and skin to freezing injury.
      , Supplementary Figures S1–S3 online).

      Results and Discussion

      Normal sebaceous glands

      Using CARS microscopy, sebaceous glands were easily seen because of their high contrast, and they were imaged in three dimensions with subcellular resolution. Intracellular lipid lobules were visible as bright granules within each sebocyte, whereas nuclei and cell membranes were visible in dark contrast because of their lower lipid content. The CARS images are consistent with the established characteristics of holocrine secretion. Sebocyte progenitor cells residing at the periphery of the gland develop into mature sebocytes, while migrating from the periphery to the central, ductal portion of the gland. These fully mature sebocytes then rupture their cell membranes and release their lipid contents, via the infundibulum (which also contains the hair shaft), ultimately onto the skin surface (
      • Thody A.J.
      • Shuster S.
      Control and function of sebaceous glands.
      ). In this study, the CARS signal was weakest in the lipid-poor progenitor sebocytes located along the periphery, becoming stronger in sebocytes in the interior portions of the gland, and strongest at the ductal outlet (Figure 1a, marked “1”). This signal pattern corresponds to the accumulation of lipids in maturing sebocytes as they migrate from the periphery toward the gland duct. Characteristic subcellular structures, including lipid granules, nuclei, and cell membranes, were observed to gradually degrade, and were eventually lost in sebocytes immediately adjacent to gland outlets. This corresponds to mature sebocytes undergoing cell death and releasing their lipid contents (Figure 1a, marked “2”). In addition, there was a strong CARS signal along the hair shaft, consistent with a lipid coating (Figure 1a, marked “3”, verified with a lipophilic stain in Supplementary Figure S4 online).
      Figure thumbnail gr1
      Figure 1Coherent Anti-Stokes Raman Scattering (CARS) imaging of normal sebaceous glands. (a) A sebaceous gland showing intracellular lipid granules (blue arrow), nuclei (red arrow), and cell membranes (green arrow). (1) CARS signal intensity increases as sebocytes approach the gland duct, corresponding to lipid accumulation as sebocytes mature. (2) Sebocytes near the duct show the highest CARS signal and lose cellular structures corresponding to cell death and lipid content release. (3) Hair shafts are coated with secreted lipid. (bd) Sebocyte migration in the same sebaceous gland shown in a over 3 consecutive days. Five sebocytes are marked in separate colors to facilitate identification. As the sebocytes migrated to the gland duct (*), there is a loss in intracellular structures and a concomitant increase in CARS signal. Scale bar = 50 μm.
      Using small tattoos as landmarks, we were able to repeatedly locate individual sebaceous glands over the course of serial imaging sessions carried out for up to 2 weeks. With this method, we were able to track individual sebocytes within the glands as they migrated toward the gland duct during holocrine secretion (Figures 1b–d). Individual sebocytes and cohorts of sebocytes were identified in glands over time by their connectivity to adjacent cells, which was maintained throughout the process of sebocyte migration. This connective pattern was maintained even as the sebocytes’ shape and size progressively changed during migration. The sebocytes migrated at a rate of approximately one cell layer (concentric about the gland outlet) per day. As each gland consists of roughly 6–8 lipid-containing cell layers, the migration rate observed by CARS microscopy is consistent with previous findings that sebocyte turnover occurs over ∼7–14 days (
      • Bertalanffy F.D.
      Mitotic activity and renewal rate of sebaceous gland cells in the rat.
      ;
      • Epstein E.H.
      • Epstein W.L.
      New cell formation in human sebaceous glands.
      ;
      • Plewig G.
      • Luderschmidt C.
      Hamster ear model for sebaceous glands.
      ). The data presented here mark, to our knowledge, previously unreported sebocyte migration visualization directly observed as it occurs in vivo. The ability to monitor holocrine secretion over time—both by detecting deviations from the normal patterning of CARS signal in sebaceous glands and by tracking the rate of sebocyte migration—should be a powerful tool for studying the myriad physiologic (e.g., androgen levels) and exogenous (e.g., therapeutic agents) factors that can affect sebaceous gland function. CARS microscopy could be used to investigate many currently unknown aspects of sebaceous gland physiology, e.g., sebocyte migration patterns, spatial and temporal relationships between sebocyte migration and lipid accumulation, and (in combination with other imaging modalities) the real-time interactions between sebocytes and other cells/organisms such as leukocytes or bacteria. In addition to its utility as a research tool, CARS microscopy could also potentially be used in clinical settings to noninvasively monitor sebaceous gland–related disease progression and response to therapy. Adapting CARS microscopy to clinical use will require adjustments in instrumentation (such as miniaturization of device components, and including tissue-stabilizing fixtures), which have been successfully achieved before for other optical imaging systems, e.g., confocal microscopy (
      • Rajadhyaksha M.
      • Anderson R.R.
      • Webb R.H.
      Video-rate confocal scanning laser microscope for imaging human tissues in vivo.
      ;
      • Nehal K.S.
      • Gareau D.
      • Rajadhyaksha M.
      Skin imaging with reflectance confocal microscopy.
      ).

      Response to cryotherapy

      Individual sebaceous glands were monitored over time to examine their response to cryotherapy (Figure 2). After a single cold exposure at -8 °C, there was a gradual loss of subcellular structures in sebocytes (including nuclei, intracellular lipid granules, and cell membranes), alongside a reduction in lipid content in each gland (Figure 3). These findings are consistent with previous findings (
      • Gage A.A.
      • Meenaghan M.A.
      • Natiella J.R.
      • et al.
      Sensitivity of pigmented mucosa and skin to freezing injury.
      ), as well as our own histologic results (Supplementary Figure S3 online). The cooling treatment did not substantially change the lipid composition, as confirmed by Raman spectroscopy (Supplementary Figure S5 online). CARS signal along the hair shaft was substantially reduced, and in some cases completely abolished, after cooling treatment (Figure 3, Supplementary Figure S4 online). This indicates that lipid excretion into the infundibulum may be halted after cooling.
      Figure thumbnail gr2
      Figure 2Transmission microscopy images of sebaceous glands before and after cryotherapy. A tattoo (T) was used as a landmark so that the same sebaceous glands could be identified across different imaging sessions. Four sebaceous glands (green arrows) were randomly chosen for longitudinal monitoring (af). Changes in the overall gland structure can be seen after cooling. Most glands appeared to recover eventually, but a few were unable to recover within the study period (one such gland marked by red arrow). The “grainy” background in the images taken before treatment and at later time points is caused by light transmitting through the subcutaneous fat. The clearing of this background in early post-treatment days is likely owing to changes in tissue refractive index caused by transient edema. Scale bar = 100 μm.
      Figure thumbnail gr3
      Figure 3Coherent Anti-Stokes Raman Scattering (CARS) images of four individual sebaceous glands (marked by green arrows in ) at different times post cooling, showing both damage to gland structures and subsequent recovery. Green background denotes glands with normal morphology. Blue background denotes glands showing damage to intracellular structures, whereas the general gland structure remained relatively intact. Yellow background denotes glands showing severe damage, with major disruption in the overall gland structure. Red background denotes time points where CARS signal could not be detected. Scale bars = 50 μm. Magnification at time 0 was maintained for all subsequent images.
      Sebocyte loss appears to occur first along the periphery of the gland, whereas the cells in the central portions of the gland persisted the longest. This finding is surprising because lipid-rich cells are thought to be especially susceptible to cryotherapy, owing to the propensity of lipids to crystallize at temperatures higher than the water ice point (
      • Manstein D.
      • Laubach H.
      • Watanabe K.
      • et al.
      Selective cryolysis: a novel method of non-invasive fat removal.
      ;
      • Quesada-Cortes A.
      • Campos-Munoz L.
      • Diaz-Diaz R.M.
      • et al.
      Cold panniculitis.
      ). This observation would lead one to expect that more mature sebocytes near the gland outlet should be more vulnerable to damage caused by lipid crystallization. The unexpected pattern of cell loss, as well as the low temperature required to induce discernable damage to the sebaceous glands, suggests that other mechanisms besides lipid crystallization may contribute to the damage to sebaceous glands caused by cryotherapy.
      CARS microscopy showed that the treated sebaceous glands began showing signs of recovery 1–2 weeks after treatment, which correlates with histology (Supplementary Figure S3 online). Post-treatment recovery appeared to occur in a somewhat disorganized manner, with gland features that were observed to deviate from normal. Distribution of sebocytes was discontinuous in some cases, with sebocytes present seemingly at random over different parts of the gland. Unilocular lipid-rich structures, devoid of nuclei or intracellular lipid granules, were often found mid-gland, instead of near the gland duct (Figure 4).
      Figure thumbnail gr4
      Figure 4Sebaceous glands at various stages of recovery, showing abnormal gland features such as discontinuous distribution of sebocytes, with sebocytes present seemingly at random over different parts of the gland. (a) The presence of unilocular lipid-rich structures, devoid of nuclei or intracellular lipid granules in middle-peripheral portions of the gland, instead of near the gland duct (ac). Other glands were able to recover normal gland features at the same time point (d). All glands were imaged at 8 days post cooling. Scale bars = 50 μm.
      Although most of the sebaceous glands were able to recover after cooling treatment, some of the glands never recovered within the course of the experiment (Figure 2), which suggests that some sebaceous glands may be permanently damaged by cooling. Future studies to characterize the glands that do and do not recover following cryotherapy, as well as the post-cooling recovery process, should be helpful for optimizing treatment parameters in our practical aim of improving cryotherapy for acne vulgaris.
      In this study, we have demonstrated, to our knowledge previously unreported, the ability to precisely monitor the dynamic behavior of sebaceous glands in vivo longitudinally, with subcellular resolution, and without the need for exogenous labeling or genetic manipulation. This capability of CARS microscopy makes it a promising tool for studying the sebaceous gland and its associated disorders. We have directly measured sebocyte migration, and the effects of a cold cycle on sebaceous glands in vivo using CARS. Surprisingly, this study revealed sensitivity of sebocytes at the gland periphery to cold injury. We also determined the recovery of a population of glands.
      In addition to monitoring sebaceous glands with CARS microscopy alone, as we demonstrated in this study, additional imaging modalities could be combined with CARS for even more comprehensive studies, especially for disease models. Two of the most prevalent disorders involving sebaceous glands—acne vulgaris and rosacea—both involve additional factors besides sebocytes themselves: colonization by Propionibacterium acnes in acne and dysfunction of the local vascular system in rosacea. Combining the monitoring of sebocytes by CARS microscopy with quantitative imaging of these related factors is likely to yield additional mechanistic insights into these disorders and contribute significantly to the development of effective therapies.

      Materials and Methods

      Animals

      All animal procedures were performed in compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee. The mouse ear model was chosen for this study because of its high density of sebaceous glands, as well as ease of access. Adult female BALB/c mice (12–16 weeks old) purchased from Jackson Lab (Bar Harbor, ME) were used for all experiments. During cooling and microscopy procedures the animals were anesthetized by inhaled isoflurane (1–3%). For cooling treatment, the ear was placed upon a conductive cooling plate and held in place by an insulation block weighing 100 g. Temperature in the cooling plate was maintained by computer-controlled thermoelectric coolers. The treated ears were held against the cooling plate at -8 °C for 10 minutes each. Untreated ears were used as controls. Small tattoos were placed in the ears to serve as landmarks, so that the same sebaceous glands can be identified and imaged repeatedly over the course of the study. Five treated and four control animals were imaged in this manner, with three to four glands in each animal imaged at various intervals. For microscopy, the ear being examined was flattened and secured to a glass coverslip using a thin layer of methyl cellulose solution (Fisher Scientific, Pittsburgh, PA).

      CARS microscopy

      The excitation light for CARS microscopy was generated using a pair of high repetition rate, low pulse power infrared lasers for deep, nonperturbative imaging in skin in vivo (
      • Evans C.L.
      • Potma E.O.
      • Puoris'haag M.
      • et al.
      Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.
      ). Briefly, a 7-ps, 1064-nm, mode-locked Nd:Vanadate laser (PicoTRAIN, High-Q Laser, Rankweil, Austria) was used to synchronously pump an optical parametric oscillator (Levante, APE, Berlin, Germany) that produced wavelength-tunable 3-ps-duration pulses. A portion (500 mW) of the 1064-nm output was used as the Stokes beam. For imaging of the lipid-rich sebum, the pump beam from the optical parametric oscillator was tuned between 814 and 816 nm to be in resonance with methylene stretching vibrations. The pump and Stokes pulse trains were combined using a 950 short-pass dichroic mirror (Chroma Technology, Bellows Fall, VT) and overlapped in time using a delay line.
      The combined pump and Stokes beam were steered into the infrared side port of a modified Olympus FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). The beam was aligned through the microscope as described elsewhere (
      • Jung Y.
      • Nichols A.J.
      • Klein O.J.
      • et al.
      Label-free, longitudinal visualization of pdt response in vitro with optical coherence tomography.
      ). A UPlanSApo 20 × 0.75NA (Olympus) objective was used as the objective lens for all experiments. The epi-collected anti-Stokes emission was passed through a 650-nm, 25-nm band-pass filter (Chroma Technology) and focused onto a Hamamatsu H7422PA-40 photomultiplier tube (Hamamatsu Photonics, Hamamatsu City, Japan). The current output of the photomultiplier tube passed through a high-speed current amplifier (Femto, Berlin, Germany, HCA-4 M-500 K) and was detected using Olympus input/output hardware (Olympus). Images were acquired using the built-in Olympus FV10 software (Olympus).
      Images were collected at standard acquisition rates (2 seconds per frames, 2 microseconds per pixel, Kalman averaging of 3) using ∼30 mW of pump and 35 mW of Stokes power at the focus. Animals were examined following imaging sessions to look for evidence of tissue perturbation, including redness or swelling. No signs or symptoms of tissue damage or perturbation were observed. Animals were mounted on a motorized state (Prior Scientific, Rockland, MA, H117 stage) with 0.01-μm resolution, which enabled precise and repeatable imaging of sebaceous glands over the course of days and weeks. Near-infrared light that passed through the thin mouse ear was collected on the microscope's transmission detector to create transmission images.

      Acknowledgments

      We thank Sunny Xie and Dan Fu of Harvard University for allowing the use of the Raman microscope. This study was funded, in part, by R21CA53335 (CLE) and a New Innovator Award (DP2 OD007096, CLE). Information on the New Innovator Award Program is at http://nihroadmap.nih.gov/newinnovator/.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

      References

        • Bertalanffy F.D.
        Mitotic activity and renewal rate of sebaceous gland cells in the rat.
        Anat Rec. 1957; 129: 231-241
        • Bhate K.
        • Williams H.C.
        Epidemiology of acne vulgaris.
        Br J Dermatol. 2013; 168: 474-485
        • Dobes W.L.
        • Keil H.
        Treatment of acne vulgaris by cryotherapy (slush method).
        Arch Derm Syphilol. 1940; 42: 547-558
        • Epstein E.H.
        • Epstein W.L.
        New cell formation in human sebaceous glands.
        J Investig Dermatol Symp Proc. 1966; 46: 453-458
        • Evans C.L.
        • Potma E.O.
        • Puoris'haag M.
        • et al.
        Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.
        Proc Natl Acad Sci USA. 2005; 102: 16807-16812
        • Evans C.L.
        • Xie X.S.
        Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine.
        Annu Rev Anal Chem. 2008; 1: 883-909
        • Evans C.L.
        • Xu X.
        • Kesari S.
        • et al.
        Chemically-selective imaging of brain structures with CARS microscopy.
        Opt Express. 2007; 15: 12076-12087
        • Freudiger C.W.
        • Min W.
        • Saar B.G.
        • et al.
        Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy.
        Science. 2008; 322: 1857-1861
        • Fu Y.
        • Huff T.B.
        • Wang H-W.
        • et al.
        Ex vivo and in vivo imaging of myelin fibers in mouse brain by coherent anti-Stokes Raman scattering microscopy.
        Opt Express. 2008; 16: 19396-19409
        • Gage A.A.
        • Meenaghan M.A.
        • Natiella J.R.
        • et al.
        Sensitivity of pigmented mucosa and skin to freezing injury.
        Cryobiology. 1979; 16: 348-361
        • Graham G.F.
        Cryosurgical treatment of acne.
        Cutis. 1975; 16: 509-513
        • Hinde E.
        • Haslam I.S.
        • Schneider M.R.
        • et al.
        A practical guide for the study of human and murine sebaceous glands in situ.
        Exp Dermatol. 2013; 22: 631-637
        • Huff T.B.
        • Cheng J.X.
        In vivo coherent anti-Stokes Raman scattering imaging of sciatic nerve tissue.
        J Microsc. 2007; 225: 175-182
        • Jung Y.
        • Nichols A.J.
        • Klein O.J.
        • et al.
        Label-free, longitudinal visualization of pdt response in vitro with optical coherence tomography.
        Isr J Chem. 2012; 52: 728-744
        • Jung Y.K.
        • Ng J.H.
        • Keating C.P.
        • et al.
        Comprehensive evaluation of peripheral nerve regeneration in the acute healing phase using tissue clearing and optical microscopy in a rodent model.
        PLoS One. 2014; 9: e94054
        • Katsambas A.D.
        • Dessinioti C.
        Hormonal therapy for acne: why not as first line therapy? facts and controversies.
        Clin Dermatol. 2010; 28: 17-23
        • Manstein D.
        • Laubach H.
        • Watanabe K.
        • et al.
        Selective cryolysis: a novel method of non-invasive fat removal.
        Lasers Surg Med. 2008; 40: 595-604
        • Nehal K.S.
        • Gareau D.
        • Rajadhyaksha M.
        Skin imaging with reflectance confocal microscopy.
        Semin Cutan Med Surg. 2008; 27: 37-43
        • Nejati R.
        • Skobowiat C.
        • Slominski A.T.
        Commentary on the practical guide for the study of sebaceous glands.
        Exp Dermatol. 2013; 22: 629-630
        • Plewig G.
        • Luderschmidt C.
        Hamster ear model for sebaceous glands.
        J Investig Dermatol Symp Proc. 1977; 68: 171-176
        • Quesada-Cortes A.
        • Campos-Munoz L.
        • Diaz-Diaz R.M.
        • et al.
        Cold panniculitis.
        Dermatol Clin. 2008; 26: 485-489
        • Rajadhyaksha M.
        • Anderson R.R.
        • Webb R.H.
        Video-rate confocal scanning laser microscope for imaging human tissues in vivo.
        Appl Opt. 1999; 38: 2105-2115
        • Rigopoulos D.
        • Larios G.
        • Katsambas A.D.
        The role of isotretinoin in acne therapy: why not as first-line therapy? facts and controversies.
        Clin Dermatol. 2010; 28: 24-30
        • Saar B.G.
        • Freudiger C.W.
        • Reichman J.
        • et al.
        Video-rate molecular imaging in vivo with stimulated Raman scattering.
        Science. 2010; 330: 1368-1370
        • Sakamoto F.H.
        • Lopes J.D.
        • Anderson R.R.
        Photodynamic therapy for acne vulgaris: a critical review from basics to clinical practice: part I. Acne vulgaris: when and why consider photodynamic therapy?.
        J Am Acad Dermatol. 2010; 63: 183-193
        • Thody A.J.
        • Shuster S.
        Control and function of sebaceous glands.
        Physiol Rev. 1989; 69: 383-416
        • Wright A.J.
        • Poland S.P.
        • Girkin J.M.
        • et al.
        Adaptive optics for enhanced signal in CARS microscopy.
        Opt Express. 2007; 15: 18209-18219