2.1.

Samples

The eumelanin sample from Sepia for spectral measurement was purchased from the Sigma Chemical Company. The mouse hair and skin samples were from the skin of a black 6 strain laboratory mouse sacrificed in unrelated research. All mouse hair and skin samples were imaged with the Keck 3D fusion multimodal microscope. The samples were mounted inside culture dishes, so that the air could be purged with nitrogen in order to reduce photo-bleaching. To prevent dehydration of the sample, 100% relative humidity on the sample was maintained by bubbling the nitrogen through water.

2.2.

Keck 3D Fusion Multimodal Microscope

The Keck 3D fusion multimodal microscope (3DFM) in the Optical Science Laboratory (OSL) at Northeastern University allows us to image samples with multiple modalities on the same stage.²³ The modalities on the 3DFM include differential interference contrast microscopy (DIC), confocal reflectance microscopy (CRM), confocal fluorescence microscopy (CFM), multiphoton fluorescence microscopy (MPFM), optical quadrature microscopy (OQM), and others. An imaging spectrometer is integrated in the 3DFM for spectrum measurement. In this work, only CRM and MPFM modalities are used.

The layout of the 3DFM is shown in Fig. 1. A detailed description of the microscope system can be found in Warger et al.²³ Only the modalities and functions related to this work are shown in this figure. A Tsunami Ti:Sapphire Laser from Spectra-Physics is used as the light source. A small portion (less than 10%) of light is reflected by beam splitter 1 for real-time power monitoring. The power meter (Newport, 1830C) is connected to a computer to quantify the number of photons exciting the sample. Beam splitter 2, a polarizing beam splitter, reflects light that returns from the sample and is used for the CRM. The APD (Hamamatsu, C5460) is used for detection of the CRM signal.

Fig. 1

Layout of the KECK 3D fusion multimodal microscope. Only modalities and functions related to this work are shown in the graph.

The XY scanner consists of a polygon scanner and a galvanometer scanner. The polygon scanner performs fast scanning along a linear direction with line speeds of roughly 6 kHz and a pixel dwell time of 200 ns, while the galvanometer scans the line in the perpendicular direction, resulting in an imaging rate of 10 frames per second.²⁰ Five frames were averaged on each image presented in this article for noise reduction. In the configuration of fluorescence spectra measurement and fluorescence output power dependence measurement, the galvanometer was turned off, so that the polygon scanner performs continuous line scanning at a selected location on the sample.

The quarter wave plate is used for changing the polarization of input and output light to maximize the signal to the APD through the polarizing beam splitter. The dichroic mirror reflects the fluorescence signal while passing the input and reflected laser light. The filter box contains switchable optical filters. The filters used in this work are a 460-nm filter (bandwidth 50 nm), a 525-nm filter (bandwidth 40 nm), a 620-nm filter (bandwidth 60 nm) and a broadband filter (370 to 650 nm). The selection of filters corresponds to the broad melanin SMPAF spectra previously detected.²¹ The photo multiplier tube (PMT) (Hamamatsu, HC 124) is used for detection of the fluorescence signal. The linearity of the PMT was tested and corrected, as needed, by software. Brightfield images can also be taken by a camera (SPOT RT900). The imaging spectrometer consists of a 150-g/mm grating (Princeton Instruments SP2150) and the SPOT camera.

2.3.

Activation of Melanin SMPAF

A Tsunami Ti:Sapphire Laser was used to induce the enhanced emission from the melanin and to perform the multiphoton fluorescence imaging in either pulsed or CW mode. The enhanced emission could be generated equally well in pulsed or CW mode using the same average laser power level. The laser spot size in the system is $\sim {10}^{-18}{\mathrm{m}}^3$. The procedure used by Kerimo²⁰ was followed to activate and image the specimen. First, a large photon density ($\ge {10}^{29}$ $\text{photon}/{\mathrm{m}}^2$) was used to activate melanin SMPAF. Once SMPAF was activated, the laser power was turned down for measurements to avoid photon-bleaching.

2.4.

Fluorescence Spectrum Measurement

As the spectrometer integrated in the 3DFM only works at the range of 450 to 700 nm, a separate experimental setup is utilized for detection of the peak spectrum of melanin SMPAF (Fig. 2).

Fig. 2

Experimental setup for detection of the peak spectrum of melanin SMPAF.

A 1505.9-nm laser is used (Velocity 6300-LN) as the activation source. The monochromator (Princeton Instruments ACTON sp-2500i) consists of a 300-groove grating and a liquid-nitrogen-cooled CCD (Princeton Instruments Spec-10:400B/LN). The wavelength of the spectrometer is calibrated using the software provided by the manufacturer. The spectral intensity of the spectrometer is calibrated using a tungsten light source at a predetermined temperature (GE.Q6.6AT3).

3.1.

SMPAF and Photo-Bleaching

Figure 3 shows brightfield images of mouse hairs taken by the SPOT camera. The signal generated by SMPAF was strong enough that it was visible to eyesight alone. Figure 3(a) is a brightfield image of the mouse hairs before laser activation. Figure 3(b) is a brightfield image of the mouse hairs after laser exposure for 7 s. Within these two figures, the orange circular areas are places where melanin is activated. Figure 3(c) is an expanded subimage on the orange circular area in Fig. 3(a). Figure 3(d) is an expanded subimage on the orange circular area in Fig. 3(a) after laser exposure for 60 s. There is an obvious change to the melanin in the area of the red circle, which is consistent with our earlier report.²⁰ The activation source used for all images in Fig. 3 was an 830-nm pulsed laser.

Fig. 3

Brightfield images of mouse hairs taken by the SPOT camera. The signal generated by SMPAF was strong enough that it was visible to eyesight alone. (a) Mouse hairs before laser activation. (b) Mouse hairs after laser exposure for 7 s. Within these two figures, the orange circular areas are places where melanin is activated. (c) An expanded subimage on the orange circular area in (a). (d) An expanded subimage on the orange circular area in (a) after laser exposure for 60 s. There is an obvious change to the melanin in the area of the red circle, which is consistent with our earlier report. (See Ref. 20.) The activation source used for all images was an 830-nm pulsed laser.

Even though the laser power is uniform at the focal plane, the time required for melanin to be activated varies. In some places, the SMPAF signal is detected immediately after laser exposure, while in other places, no SMPAF signal is detected even after laser exposure for 20 min. There is no clear understanding of how the activation time is related to any physical features of melanin. The SMPAF signal is detected by excitation of much lower laser power ($\le {10}^{28}$ $\text{photon}/{\mathrm{m}}^2$) after laser activation. There is no SMPAF signal detected at the same power level without laser activation.

3.2.

Multimodal Microscopy

Multimodal images of the mouse hair and skin were taken using the MPFM and CRM mode of the 3DFM. Although the laser power was turned down after activation to reduce the photo-bleaching, in order to obtain a detectable signal under different filters, a relatively high laser input power ($\sim {10}^{29}$ $\text{photon}/{\mathrm{m}}^2$) was used and maintained. Therefore, the photo-bleaching was strong.

Figure 4 shows images of a mouse hair at the same position using different imaging modalities and filters. The first four images show SMPAF using a 460-nm filter (a), a 525-nm filter (b), a 620-nm filter (c), and a broadband filter (370 to 650 nm) (d). The activation source is a 920-nm CW laser. The broad distribution of the fluorescence signal activated by the CW laser matches the spectra of data we acquired previously,²¹ which confirms that the signals acquired are SMPAF images of melanin. Figure 4(e) is a CRM image. Figure 4(f) is a MPFM mode image using the broadband filter (370 to 650 nm) with activation from a 920-nm pulsed (100-fs pulse width) laser. Figure 4(g) is a composite image of Fig. 4(d) (red channel), Fig. 4(e) (blue channel), and Fig. 4(f) (green channel). The red channel shows the location of melanin SMPAF. The blue channel shows the physical structure of the mouse hair, and the green channel shows the auto-fluorescence of hair components as well as SMPAF of melanin. Figure 4(h) is the composite image of Fig. 4(d) and 4(e). By combining CRM with SMPAF, we can locate the melanin, with respect to other hair components.

Fig. 4

Images of a mouse hair at the same position using different imaging modalities and filters. The first four images show SMPAF using a 460-nm filter (a), a 525-nm filter (b), a 620-nm filter (c) and a broadband filter (370 to 650 nm) (d). The activation source is a 920-nm CW laser. The broad distribution of the fluorescence signal activated by the CW laser matches the spectra of data we acquired previously (see Ref. 21), which confirms that the signals acquired are SMPAF images of melanin. (e) CRM image. (f) MPFM mode image using the broadband filter with activation from a 920-nm pulsed (100-fs pulse width) laser. (g) A composite image of (d) (red channel), (e) (blue channel), and (f) (green channel). The red channel shows the location of melanin SMPAF. The blue channel shows the physical structure of mouse hair, and the green channel shows the auto-fluorescence of hair components as well as SMPAF of melanin. (h) The composite image of (d) and (e). By combining CRM with SMPAF, we can locate the melanin, with respect to other hair components.

The CW laser prevents the images in MPFM mode from being affected by the auto-fluorescence signal from other hair components. Therefore, the signal-noise-ratio (SNR) of the image is greatly increased, such that melanin is more easily detected and located. For example, the femtosecond pulsed laser generates background interference, due to the multiphoton absorption by keratin, as shown in Fig. 4(f). The CW laser image lacks this background, as can be seen in Fig. 4(a)–4(d).

The background interference was even greater when imaging mouse skin with the pulsed laser. Figure 5 shows images of mouse skin at the same position using different imaging modalities. The image in Fig. 5(a) was collected using the broadband filter (370 to 650 nm) and CW laser at 920 nm. Figure 5(b) is the corresponding CRM image. Figure 5(c) is a multiphoton fluorescence image using the broadband filter activated by the 920-nm pulsed laser. It is clear that the background auto-fluorescence is very high with the pulsed laser and is not present in the CW laser image. Figure 5(d) is a composite image of Fig. 5(a) (red channel) and Fig. 5(b) (blue channel). The red channel shows the location of melanin SMPAF, and the blue channel shows the physical structure of mouse skin.

Fig. 5

Images of mouse skin at the same position using different imaging modalities. (a) An image collected using the broadband filter (370 to 650 nm) and CW laser at 920 nm. (b) The corresponding CRM image. (c) A multiphoton fluorescence image using the broadband filter activated by the 920-nm pulsed laser. It is clear the background auto-fluorescence is very high with the pulsed laser and is not present in the CW laser image. (d) A composite image of (a) (red channel) and (b) (blue channel). The red channel shows the location of melanin SMPAF, and the blue channel shows the physical structure of mouse skin.

3.3.

SMPAF Spectra and Power Dependence

Figure 6 shows a SMPAF spectrum of mouse hair melanin. The spectrum matches the data we previously obtained on other types of melanin samples. Note that this spectrum has been corrected for the overall spectral response of the system.²¹ The continuous rising slope of the spectrum indicates that the SMPAF peak of melanin must be well past 700 nm, and that even larger signal levels are to be expected when it is used to image and detect melanin. As the PMT in our microscope can detect only wavelengths less than 650 nm, a small fraction of the total SMPAF signal is detected. The peak spectrum of melanin is measured in a separate experimental setup (Fig. 2). The new setup does not allow us to image the specimen, so it is hard to locate and activate the melanin at a specific point. Instead, we used a bulk sample of pure sepia melanin as the specimen. Figure 7 shows the corrected SMPAF spectrum of sepia melanin. The sample was excited with a CW laser at 1505.9 nm, and the peak of the fluorescence spectrum is around 960 nm.

Fig. 6

SMPAF spectrum of mouse hair melanin. ${\lambda }_{\mathrm{exc}}=920\mathrm{nm}$, CW.

Fig. 7

SMPAF spectrum of sepia melanin. ${\lambda }_{\mathrm{exc}}=1505.9\mathrm{nm}$, CW.

Figure 8 shows a typical power dependence of the melanin SMPAF in mouse hair. The red curve is the polynomial fit of fluorescence intensity versus incident power. The activation source is a 920-nm pulsed laser. The filter used is the broadband filter (370 to 650 nm). The polynomial fit equation is

Fig. 8

Power dependence of SMPAF of mouse hair melanin. The red curve is the polynomial fit of fluorescence intensity versus incident power. ${\lambda }_{\mathrm{exc}}=920\mathrm{nm}$, pulsed. The filter used is the broadband filter (370 to 650 nm).