3‐photon microscopy of myelin in mouse digital skin excited at the 1700‐nm window

Journal of Biophotonics

Tong

et al. 2021

Self Cite

Multiphoton microscopy (MPM) excited at the 1700-nm window has enabled deep-tissue penetration in biological tissue, especially brain. MPM of skin may also benefit from this deep-penetration capability. Skin is a layered structure with varying refractive index (from 1.34 to 1.5). Consequently, proper immersion medium should be selected when imaging with high numerical aperture objective lens. To provide guidelines for immersion medium selection for skin MPM, here we demonstrate comparative experimental investigation of deep-skin MPM excited at 1600 nm in vivo, using both silicone oil and deuterium dioxide (D 2 O) immersion. We specifically characterize imaging depths, signal levels and spatial resolution. Our results show that both immersion media give similar performance in imaging depth and spatial resolution, while signal levels are slightly better with silicone oil immersion. We also demonstrate that local injection of fluorescent beads into the skin is a viable technique for spatial resolution characterization in vivo.Skin is the outside layer of tissue covering the body of many animals including human beings, which plays important roles in protection, regulation and sensation. Research on skin structures and even functions leverage on various imaging modalities. Among them, multiphoton microscopy (MPM) finds a unique niche by combining reasonable penetration depth (>100 μm) and subcellular resolution. Consequently, MPM has found successful applications in skin imaging of both animals and more importantly, human subjects in clinical environments [1][2][3][4][5][6][7][8][9][10][11][12][13].Even MPM itself comprises a variety of modalities, examples include 2-/3-/4-photon fluorescence (i.e., 2PF/3PF/4PF)

mentioning

confidence: 99%

“…Excitation at the 1700-nm window has shown potential for deep-tissue penetration, enabling the largest brain 3PF [14] and 2PF [15] imaging depths so far. In terms of skin imaging, excitation at the 1700-nm window has been demonstrated to image dye-labeled elastic fibers [10] and myelin sheaths [11] deep in the mouse skin.…”

mentioning

confidence: 99%

Deep‐skin multiphoton microscopy in vivo excited at 1600 nm: A comparative investigation with silicone oil and deuterium dioxide immersion

Wang

Journal of Biophotonics

Tong

et al. 2021

Self Cite

Deep-skin third-harmonic generation (THG) imagingin vivoexcited at the 2200 nm window

Chen

J. Innov. Opt. Health Sci.

Qiu

et al. 2022

The skin is heterogeneous and exerts strong scattering and aberration onto excitation light in multiphoton microscopy (MPM). Shifting to longer excitation wavelengths may help reduce skin scattering and aberration, potentially enabling larger imaging depths. However, previous demonstrations of skin MPM employ excitation wavelengths only up to the 1700[Formula: see text]nm window, leaving an open question as to whether longer excitation wavelengths are suitable for deep-skin MPM. Here, in order to explore the longer-wavelength territory, first, we demonstrate characterization of the broadband transmittance of excised mouse skin, revealing a high transmittance window at 2200[Formula: see text]nm. Then, we demonstrate third-harmonic generation (THG) imaging in mouse skin in vivo excited at this window. With 9[Formula: see text]mW optical power on the skin surface operating at 1[Formula: see text]MHz repetition rate, we can get THG signals of 250[Formula: see text][Formula: see text]m below the skin surface. Comparative THG imaging excited at the 1700[Formula: see text]nm window shows that as imaging depth increases, THG signals decay even faster than those excited at 2200[Formula: see text]nm. Our results thus uncover the 2200[Formula: see text]nm window as a new, promising excitation window potential for deep-skin MPM.

3-photon fluorescence and third-harmonic generation imaging of myelin sheaths in mouse digital skin in vivo: A comparative study

Wang

J. Innov. Opt. Health Sci.

Chen

et al. 2021

Myelin sheaths wrapping axons are key structures that help maintain the propagation speed of action potentials in both central and peripheral nervous systems (CNS and PNS). However, noninvasive, deep imaging technologies visualizing myelin sheaths in the digital skin in vivo are lacking in animal models. 3-photon fluorescence (3PF) imaging excited at the 1700-nm window enables deep imaging of myelin sheaths, but necessitates labeling by exogenous fluorescent dyes. Since myelin sheaths are lipid-rich structures which generate strong third-harmonic signals, in this paper, we perform a detailed comparative experimental study of both third-harmonic generation (THG) and 3PF imaging in the mouse digital skin in vivo. Our results show that THG imaging also enables visualization of myelin sheaths deep in the mouse digital skin, which shows colocalization with 3PF signals from labeled myelin sheaths. Besides its superior label-free advantage, THG does not suffer from photobleaching due to its 3PF property.