In-vivo confocal microscope technology can be applied to the medical imaging diagnosis and new drug development.
We present an in-vivo confocal microscope that can acquire a reflection image and a fluorescence image simultaneously
and independently. To obtain reflection confocal images, we used a linearly polarized diode laser with the wavelength of
830 nm. To acquire fluorescence confocal images, we used two diode lasers with the wavelength of 488 nm and 660 nm,
respectively. Because of a broad wavelength bandwidth from visible (488 nm) to near-IR (830 nm), we designed and
optimized the optical system to reduce various optical aberrations. With the developed in-vivo confocal microscope, we
performed ex-vivo cell imaging and in-vivo imaging of the human skin.
Living skin for basic and clinical research can be evaluated by Confocal Laser Scanning Microscope (CLSM) non-invasively.
CLSM imaging system can achieve skin image its native state either "in vivo" or "fresh biopsy (ex
vivo)" without fixation, sectioning and staining that is necessary for routine histology. This study examines the potential
fluorescent CLSM with a various exogenous fluorescent contrast agent, to provide with more resolution images in skin.
In addition, in vivo fluorescent CLSM researchers will be extended a range of potential clinical application. The
prototype of our CLSM system has been developed by Prof. Gweon's group. The operating parameters are composed of
some units, such as illuminated wavelength 488 nm, argon illumination power up to 20mW on the skin, objective lens,
0.9NA oil immersion, axial resolution 1.0μm, field of view 200μm x 100μm (lateral resolution , 0.3μm). In human
volunteer, fluorescein sodium was administrated topically and intradermally. Animal studies were done in GFP
transgenic mouse, IRC mouse and pig skin. For imaging of animal skin, fluorescein sodium, acridine orange, and
curcumine were used for fluorescein contrast agent. We also used the GFP transgenic mouse for fluorescein CLSM
imaging. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated
the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. Curcumin is a yellow food dye that has
similar fluorescent properties to fluorescein sodium. Acridin Orange can be highlight nuclei in viable keratinocyte. In
vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals
are brightest in corneocyte, kertinocyte, hair and eccrine gland. In intact skin, absorption of fluorescein sodium by
individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell
border could be seen. In papillary dermis, fluorescein distribution is more homogeneous. Curcumin is a yellow food dye
that has similar fluorescent properties to fluorescein sodium. In vivo CLSM of transgenic GFP mouse enable on in vivo,
high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, skin appendage
and blood vessels. In conclusion, this study demonstrates the usefulness of CLSM as technique for imaging skin in vivo.
In addition, CLSM is non-invasive, the same tissue site may be imaged over a period of time to monitor the various
change such as wound healing, severity of skin diseases and effect of therapeutic management.
In this research, the method how to estimate the image quality for different scanning rate is suggested and experimentally shown with the laboratory-built confocal laser scanning microscope. The confocal microscope is designed for in vivo reflectance imaging of a biological tissue, which uses the refractive index mismatch at the boundaries of a tissue to generate an image without any additional staining process. The two-dimensional scanning mechanism is built up with a polygonal mirror and a galvanometric mirror that can be controlled to operate at a specific speed. To examine the effect of scanning rate on the image contrast, confocal scanning images of a biological specimen are acquired with various scanning rate while the other conditions are kept same. The contrast of confocal microscopic image is transformed into the numeric expression to describe the relation between image contrast and scanning rate quantitatively. Results suggest some useful methodology of how to determine the allowable maximum scanning rate for the specific application of confocal microscopy.