Vertical optical sectioning using a magnetically driven confocal microscanner aimed for in vivo clinical imaging.

This paper presents a confocal microscanner for direct vertical optical sectioning of biological samples. Confocal imaging is performed by transverse (X-axis) and axial (Z-axis) scanning of a focused laser beam using an optical fiber and a microlens respectively. The actuators are fabricated by laser micromachining techniques and are driven by electromagnetic forces. Optical and mechanical performance of the system is predicted by simulation software packages and characterized by experimental measurements. The scanner has lateral resolution of 3.87 µm and axial resolution of 10.68 µm with a field of view of 145 µm in X and 190 µm in Z directions. Confocal imaging of a polymer layer deposited on a silicon wafer and onion epidermal cells is demonstrated.

In vivo video rate multiphoton microscopy imaging of human skin.

We present a multiphoton microscopy instrument specially designed for in vivo dermatological use that is capable of imaging human skin at 27 frames per second with 256 pixels × 256 pixels resolution without the use of exogenous contrast agents. Imaging at fast frame rates is critical to reducing image blurring due to patient motion and to providing practically short clinical measurement times. Second harmonic generation and two-photon fluorescence images and videos acquired at optimized wavelengths are presented showing cellular and tissue structures from the skin surface down to the reticular dermis.

Imaging-guided two-photon excitation-emission-matrix measurements of human skin tissues.

There are increased interests on using multiphoton imaging and spectroscopy for skin tissue characterization and diagnosis. However, most studies have been done with just a few excitation wavelengths. Our objective is to perform a systematic study of the two-photon fluorescence (TPF) properties of skin fluorophores, normal skin, and diseased skin tissues. A nonlinear excitation-emission-matrix (EEM) spectroscopy system with multiphoton imaging guidance was constructed. A tunable femtosecond laser was used to vary excitation wavelengths from 730 to 920 nm for EEM data acquisition. EEM measurements were performed on excised fresh normal skin tissues, seborrheic keratosis tissue samples, and skin fluorophores including: NADH, FAD, keratin, melanin, collagen, and elastin. We found that in the stratum corneum and upper epidermis of normal skin, the cells have large sizes and the TPF originates from keratin. In the lower epidermis, cells are smaller and TPF is dominated by NADH contributions. In the dermis, TPF is dominated by elastin components. The depth resolved EEM measurements also demonstrated that keratin structure has intruded into the middle sublayers of the epidermal part of the seborrheic keratosis lesion. These results suggest that the imaging guided TPF EEM spectroscopy provides useful information for the development of multiphoton clinical devices for skin disease diagnosis.