In Vivo Non-Invasive Observation of Dynamic Changes in Hair Shaft, Melanin, and Collagen During the Mice Hair Follicle Cycle

Summary

This study presents a non-invasive, in vivo method using two-photon and second-harmonic generation microscopy to longitudinally monitor hair shaft morphology, melanin dynamics, and dermal collagen density throughout the entire hair follicle cycle in mice, providing a robust methodological framework for hair-loss therapeutic research.

Abstract

Traditional evaluations of the hair follicle cycle rely on invasive histological methods, limiting continuous longitudinal observation. It was hypothesized that non-invasive optical imaging, particularly two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG), can accurately track and quantify in vivo dynamic changes in hair shafts, melanin, and collagen across multiple hair follicle cycles. The hair shafts of C57BL/6 mice were monitored in vivo during the anagen, catagen, and telogen phases using dermoscopy, photography, and electron microscopy. Specifically, TPEF under 800 nm excitation and imaging were used to quantify melanin dynamics, while SHG under 950 nm excitation was used to observe interfollicular collagen changes. To ensure accuracy, the in vivo optical measurements were cross-validated: melanin dynamics were confirmed via skin photography for overall tissue appearance and hematoxylin and eosin (HE) staining for hair bulb expression, while collagen changes were validated by assessing fiber density and orientation using a liquid crystal polarizing imaging system on Sirius Red-stained sections. The findings observations demonstrated that during the anagen phase, hair shaft diameter and melanin production progressively increased, accompanied by a significant reduction in interfollicular collagen density. During the catagen and telogen phases, melanin levels gradually decreased, and collagen density gradually recovered to baseline levels. This project establishes a validated, non-invasive in vivo methodology for evaluating the comprehensive dynamics of hair follicles, providing a robust experimental framework for the preclinical evaluation of active agents for hair loss treatments.

Introduction

The hair follicles of C57BL/6 mice exhibit synchronicity and regularity, offering experimental convenience and making them a common animal model for studying skin diseases^1,2. The hair follicles enter the second telogen phase in the seventh week after the birth of the C57BL/6 mouse³. In the absence of artificial intervention, this telogen phase will be maintained until the twelfth week after birth⁴. This extended period of telogen is a common time point used in research to synchronize the hair follicle growth cycle⁵. After artificial depilation of the mice, the hair follicles immediately begin to enter the anagen phase⁶.

After entering the growth phase, stem cells in the bulge region proliferate and differentiate into the outer root sheath, growing downward to increase follicle length⁷. The hair shaft's base extends upward as the follicle deepens⁸. Stem cells in the bulb continuously differentiate into the hair shaft and inner root sheath⁹. The secretion of collagen and melanin in the skin also changes rapidly during the anagen phase¹⁰, playing an important role in hair follicle development^11,12. In hair-loss-related studies, melanin secretion is generally assessed via skin color, which does not allow precise quantification¹³. To date, research on collagen has predominantly focused on development and aging rather than on the cyclical dynamics of quantification.

Currently, a major limitation in evaluating hair follicle cycles and regenerative therapies is the reliance on destructive histological endpoints, which necessitates euthanizing different cohorts of animals at specific time points¹⁴. This prevents continuous, longitudinal tracking of the exact same follicular microenvironment across multiple cycles. Furthermore, conventional non-invasive techniques like dermatoscopy lack sufficient tissue penetration to visualize deep follicle changes¹⁵, and macroscopic observation fails to precisely quantify melanin, making the melanin-deficient root of club hair difficult to detect during late regression and resting phases^16,17. While various optical technologies have advanced longitudinal in vivo observation^18,19, such as liquid crystal polarimetry for evaluating collagen fibers^20,21, skin confocal microscopy for observing pigmentation^22,24, and optical coherence tomography for measuring skin thickness^25,26,27, multiphoton microscopy offers a uniquely compelling solution. The combined use of two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) provides distinct advantages: it enables label-free, high-resolution optical sectioning deep into the dermis, allowing simultaneous in vivo quantification of melanin autofluorescence and collagen fiber architecture without exogenous dyes^28,29. In this study, the authors hypothesize that non-invasive TPEF/SHG imaging can dynamically and precisely quantify these structural changes with an accuracy comparable to invasive histological gold standards. By addressing this key scientific problem, this study aims to establish a robust, longitudinal in vivo imaging framework to support the non-invasive evaluation of therapeutic agents for alopecia³⁰.

Protocol

All methods that involve the use of vertebrate subjects were performed in compliance with institutional guidelines. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Experimental Research Center of China Academy of Chinese Medical Sciences (Protocol Code: ERCCACMS11-2210-03). All the materials used in this study are listed in the Table of Materials.

Animal preparation and macroscopic observation
Male C57BL/6 mice, aged 6–7 weeks and weighing 20 ± 2 g, were obtained from a commercial supplier. The mice were divided into a histological analysis cohort (n = 39) and a continuous in vivo imaging cohort (n = 3). The dorsal hair of all mice was depilated to synchronize the hair follicle cycle. Hair growth status was examined every other day from day 2 to day 26 post-depilation. The backs of the mice in the imaging cohort were photographed every other day until day 26 using a camera. A fixed focal distance of 15 cm was maintained, and consistent, standard ambient laboratory lighting conditions were ensured to accurately document macroscopic changes.

In vivo TPEF and SHG imaging
Imaging sessions were performed on days 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 post-depilation. The mice were anesthetized using isoflurane. Complete loss of consciousness was ensured by observing the breathing rate and testing the foot reflex with a gentle toe pinch. The anesthetized mouse was secured onto the stage of a two-photon microscope. For TPEF imaging of melanin, the microscope was equipped with a 25x water immersion objective (numerical aperture = 1.05), and the excitation wavelength was set to 800 nm. A z-stack scan of the melanin autofluorescence was performed, reaching a total scanning depth of 45 µm. The step size was set to 2 µm, the image resolution to 1,024 × 1,024 pixels, and the acquisition speed to 4 µs/pixel. For SHG imaging of collagen fibers, the excitation wavelength was adjusted to 950 nm using the same 25x water immersion objective. A z-stack scan from the upper surface of the dermis to a total scanning depth of 45 µm was initiated, maintaining a step size of 2 µm, an image resolution of 1,024 × 1,024 pixels, and an acquisition speed of 4 µs/pixel. Following the imaging session, the mouse was carefully removed from the microscope stage, isoflurane was discontinued, and the mouse was placed on a warming pad until it fully recovered consciousness before being returned to the home cage.

Tissue collection and histological processing
A distinct, separate cohort of three mice from the histological group was euthanized every other day from day 2 to day 26, according to institutional guidelines, ensuring that no mouse was reused for longitudinal histological sampling. A 2 cm × 2 cm full-thickness dorsal skin tissue sample centered precisely at the intersection of the horizontal and vertical midlines on the back of each mouse was surgically excised. The excised skin tissue was placed flatly onto a hard card to prevent edge curling and immediately immersed in a test tube containing 4% paraformaldehyde. The fixed tissues were dehydrated and embedded to prepare 6 µm thick paraffin sections. Prior to de-waxing, the paraffin sections were heated on a slide warmer for 120 min. The sections were stained with hematoxylin and eosin (HE) and Sirius Red using standard staining kits according to the manufacturer's instructions. The sections were mounted with neutral gum and observed using an inverted microscope. For liquid-crystal-polarized imaging, the de-waxed and Sirius Red-stained sections were photographed under a polarized light microscope. A unified polarizer angle was established by crossing the polarizer and analyzer at exactly 90° to achieve a maximally dark background (extinction state), and this specific angle was consistently locked for all subsequent image captures.

In vivo dermoscopy and electron microscopy of hair shafts
Prior to plucking, the dorsal hair growth area was evaluated in vivo using a clinical dermatoscope at 20x magnification. Hair regeneration was quantified using a standard 0–3 scoring system based on hair shaft emergence and density (0 = no visible hair; 1 = sparse hair emergence; 2 = moderate hair density; 3 = dense, full hair coverage). To ensure unbiased sampling, the dorsal depilated area of each mouse was visually divided into four equal quadrants. A random number generator was used to select two quadrants per mouse, and five newly grown hair shafts were firmly plucked from each selected quadrant using sterile forceps. The plucked hair shafts were immediately immersed and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 4 h at 4 °C to preserve the ultrastructure. The fixed hair shafts were washed three times in 0.1 M phosphate buffer and sequentially dehydrated through a graded ethanol series (50%, 70%, 80%, 90%, and 100%) for 15 min at each concentration, followed by critical point drying using liquid CO₂. The dried hair shafts were mounted horizontally onto conductive double-sided carbon tape attached to aluminum stubs. The samples were sputter-coated with a 10 nm gold layer using a vacuum sputter coater for 60 s to prevent electron charging. The coated samples were inserted into a scanning electron microscope, and the hair shafts were observed using an accelerating voltage of 5.0 kV. Ultramicroscopic morphological changes were systematically evaluated by capturing images at 500x and 1,000x magnifications, specifically documenting the hair shaft diameter and the structural integrity and arrangement of the cuticular scales.

Data and statistical analysis
Image parameters were measured and quantified using generic image analysis software. All statistical analyses and graphical representations were conducted using standard statistical analysis software. For continuous in vivo monitoring data obtained from the same animals across multiple time points, statistical significance was determined using repeated-measures analysis of variance (RM-ANOVA) followed by Bonferroni's post-hoc test. For histological data involving independent animal samples, an ordinary one-way ANOVA followed by Tukey's post-hoc test was used to evaluate statistical differences. All results were expressed as the mean ± standard deviation (SD). Differences were considered statistically significant at *p < 0.05, p < 0.01, and ***p < 0.001.

Results

Mouse hair observation
Photographic observations of murine hair growth are presented in Figure 1A. Hair emergence through the skin surface commenced on day 10, with continued development maintained between day 12 and day 16 (p < 0.05). Hair coverage subsequently plateaued from day 18 to day 26 (p > 0.05) (Figure 1B). Furthermore, dermoscopy facilitated the non-invasive in vivo observation of hair growth dynam...

Discussion

The C57BL/6 mouse model was selected because its second telogen phase stably lasts from week 7 to week 12. Since the 26-day observation window falls entirely within this static phase, the profound dynamic changes observed are explicitly driven by the depilation-induced cycle progression, entirely excluding natural age-related fluctuations. When depilation is performed during the telogen phase, the hair follicles immediately enter the anagen phase. To the best of the author’s knowledge, this study presents a novel n...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6 mice,	Beijing Vital River Laboratory Animal Technology	SCXK (Jing) 2021-0006	Used for animal models
Digital DSLR Camera	Canon	EOS 80D	Used to take pictures of the back of mice
H&E Staining Kit	LtdSolarbio Science & Technology Co.	G1120	Used for staining tissue sections
Inverted Microscope	Olympus	BX51	Used for taking photos
lsoflurane	RWD Life Science Co.	R510-22-16	Used for anesthetizing animals
Mai Tai DeepSee	SpectraPhysics	Mai Tai HP	Used for For TPEF and SHG in vivo imaging
Medical Electronic Dermatoscope	China Chuanghong Medical Technology Co.	CH-DSIS-2000	Used for in vivo imaging of skin color
Polarized Light Microscope	Olympus	BX51-P	Used for imaging Sirius Red staining
Scanning electron microscope (SEM)	Hitachi High-Tech Corporation	SU8010	For hair shaft ultrastructure observation
Sirius Red staining kit	Solarbio	G1472	For collagen fiber staining
Small Animal Anesthesia Machine	RWD Life Science Co.	R500	Used for anesthetizing animals