Zhenhua Lai

Stepwise multiphoton activation fluorescence reveals a new method of melanin detection

Abstract. The stepwise multiphoton activated fluorescence (SMPAF) of melanin, activated by a continuous-wave mode near infrared (NIR) laser, reveals a broad spectrum extending from the visible spectra to the NIR and has potential application for a low-cost, reliable method of detecting melanin. SMPAF images of melanin in mouse hair and skin are compared with conventional multiphoton fluorescence microscopy and confocal reflectance microscopy (CRM). By combining CRM with SMPAF, we can locate melanin reliably. However, we have the added benefit of eliminating background interference from other components inside mouse hair and skin. The melanin SMPAF signal from the mouse hair is a mixture of a two-photon process and a third-order process. The melanin SMPAF emission spectrum is activated by a 1505.9-nm laser light, and the resulting spectrum has a peak at 960 nm. The discovery of the emission peak may lead to a more energy-efficient method of background-free melanin detection with less photo-bleaching.

Photon absorption in step-wise multi-photon activation fluorescence (SMPAF) of Sepia melanin

Previous research has shown that melanin goes through a step-wise three-photon absorption process when the fluorescence is activated with high laser intensity. We have conducted further research using even higher laser intensity for the activation, and have shown the possibility of observing power dependence other than third-order. This article discusses the possible energy states of Sepia melanin by studying the power dependence curves of the step-wise multi-photon activated fluorescence signal. Three different excitation channels are activated. Possible reasons causing the three channels are discussed.

Melanin fluorescence spectra by step-wise three photon excitation

Melanin is the characteristic chromophore of human skin with various potential biological functions. Kerimo discovered enhanced melanin fluorescence by stepwise three-photon excitation in 2011. In this article, step-wise three-photon excited fluorescence (STPEF) spectrum between 450 nm -700 nm of melanin is reported. The melanin STPEF spectrum exhibited an exponential increase with wavelength. However, there was a probability of about 33% that another kind of step-wise multi-photon excited fluorescence (SMPEF) that peaks at 525 nm, shown by previous research, could also be generated using the same process. Using an excitation source at 920 nm as opposed to 830 nm increased the potential for generating SMPEF peaks at 525 nm. The SMPEF spectrum peaks at 525 nm photo-bleached faster than STPEF spectrum.

Stepwise multi-photon activation fluorescence reveals a new method of melanoma imaging for dermatologists

Previous research has shown that the stepwise multi-photon activated fluorescence (SMPAF) of melanin, activated by a continuous-wave (CW) mode near infrared (NIR) laser, is a low cost and reliable method of detecting melanin. SMPAF images of melanin in a mouse hair and a formalin fixed mouse melanoma were compared with conventional multiphoton fluorescence microscopy (MPFM) images and confocal reflectance microscopy (CRM) images, all of which were acquired at an excitation wavelength of 920 nm, to further prove the effectiveness of SMPAF in detecting melanin. SMPAF images add specificity for melanin detection to MPFM images and CRM images. Melanin SMPAF can be a promising technology to enable melanoma imaging for dermatologists.

Customized profile lens based linear response fast spinning on-axis optical scanners for 3D laser scanning microscopy

Previously, we presented an on-axis linear-response linear-motion optical scanner. While the linear design is highly desired for engineering consideration, it was still lacking the scanning speed required for imaging applications. We here present a customized profile lens (CPL), tailored for high speed performance while maintaining the advantages of a linear response on-axis optical scanner. The device was built and tested experimentally on an optical bench. The test results demonstrate precise linear response and fast scanning speed, and revealed video frame rate scanning ability. The implementation of the CPLs in laser scanning systems is promising in improving the current 3D laser scanning microscopy systems by reducing the size, error, and complexity of the system, as well as other systems unitizing high speed laser scanning technique.

In vivo stepwise multi-photon activation fluorescence imaging of melanin in human skin

The stepwise multi-photon activated fluorescence (SMPAF) of melanin is a low cost and reliable method of detecting melanin because the activation and excitation can be a continuous-wave (CW) mode near infrared (NIR) laser. Our previous work has demonstrated the melanin SMPAF images in sepia melanin, mouse hair, and mouse skin. In this study, we show the feasibility of using SMPAF to detect melanin in vivo. in vivo melanin SMPAF images of normal skin and benign nevus are demonstrated. SMPAF images add specificity for melanin detection than MPFM images and CRM images. Melanin SMPAF is a promising technology to enable early detection of melanoma for dermatologists.

A novel multimodal laser scanning microscope control system

Traditional laser scanning microscopes require complex control systems to synchronize and control image acquisition. The control system is especially cumbersome in the multimodal laser scanning microscope. We have developed a novel multimodal laser scanning microscope control system based on a National Instruments multifunction data acquisition device (DAQ), which serves as both a data acquisition device and a programmable signal generator. The novel control system is low-cost and easy-to-build, with all components off-the-shelf. We have applied the control system in a multimodal laser scanning microscope. The control system has not only significantly decreased the complexity of the microscope, but also increased the system flexibility. We have demonstrated that the system can be easily customized for various applications.

The stepwise multi-photon activation fluorescence guided ablation of melanin

Previous research has shown that the stepwise multi-photon activation fluorescence (SMPAF) of melanin, activated and excited by a continuous-wave (CW) mode near infrared (NIR) laser, is a low-cost and reliable method for detecting melanin. We have developed a device utilizing the melanin SMPAF to guide the ablation of melanin with a 975 nm CW laser. This method provides the ability of targeting individual melanin particles with micrometer resolution, and enables localized melanin ablation to be performed without collateral damage. Compared to the traditional selective photothermolysis, which uses pulsed lasers for melanin ablation, this method demonstrates higher precision and lower cost. Therefore, the SMPAF guided selective ablation of melanin is a promising tool of melanin ablation for both medical and cosmetic purposes.

Statistical analysis on activation and photo-bleaching of step-wise multi-photon activation fluorescence of melanin

Melanin is regarded as the most enigmatic pigments/biopolymers found in most organisms. We have shown previously that melanin goes through a step-wise multi-photon absorption process after the fluorescence has been activated with high laser intensity. No melanin step-wise multi-photon activation fluorescence (SMPAF) can be obtained without the activation process. The step-wise multi-photon activation fluorescence has been observed to require less laser power than what would be expected from a non–linear optical process. In this paper, we examined the power dependence of the activation process of melanin SMPAF at 830nm and 920nm wavelengths. We have conducted research using varying the laser power to activate the melanin in a point-scanning mode for multi-photon microscopy. We recorded the fluorescence signals and position. A sequence of experiments indicates the relationship of activation to power, energy and time so that we can optimize the power level. Also we explored regional analysis of melanin to study the spatial relationship in SMPAF and define three types of regions which exhibit differences in the activation process.

The arrangement of fibrous collagen in cornea using second harmonic generation (SHG) microscopy

In recent years, interest in studying the components of the cornea and their arrangement, with emphasis on the corneal stroma, has expanded rapidly. By determining the corneal stroma’s organization in detail, we will understand better the relationship between its structure and functionality. Here, the cornea’s collagen lamellae were scanned using second harmonic generation (SHG) microscopy, in order to determine the orientation of fibers in different directions within a two-dimensional cross section. A unique algorithm was used to quantitatively measure the directions. Cross sections were obtained at several different depths in each sample. This work offers supplemental sectioning revelations to the methods historically used to scan at the lamella level, such as X-ray diffraction.