A confocal reflectance theta line scanning microscope demonstrates imaging of nuclear and cellular detail in human epidermis in vivo. Experimentally measured line-spread functions determine the instrumental optical section thickness to be 1.7 +/- 0.1 microm and the lateral resolution to be 1.0 +/- 0.1 microm. Within human dermis (through full-thickness epidermis), the measured section thickness is 9.2 +/- 1.7 microm and the lateral resolution is 1.7 +/- 0.1 microm. An illumination line is scanned directly in the pupil of the objective lens, and the backscattered descanned light is detected with a linear array, such that the theta line scanner consists of only seven optical components.
"It has been established that although Intralipid models the dominant scattering properties of tissue, it lacks the micro-architectural heterogeneities to serve as a realistic scattering phantom for confocal microscopy. Structural heterogeneities such as cellular nuclei, glands, and vasculature, can cause significant beam steering and lensing (refractive effects) in diffraction-limited optical beams, thus leading to misalignments and degraded image resolution and contrast [18–20]. Therefore, we have formulated a tissue phantom that more closely mimics the confocal-imaging properties of human epithelium, our target of interest, both in terms of scattering magnitude and the heterogeneity-induced degradation in resolution. "
[Show abstract][Hide abstract] ABSTRACT: Phantoms play an important role in the development, standardization, and calibration of biomedical imaging devices in laboratory and clinical settings, serving as standards to assess the performance of such devices. Here we present the design of a liquid optical phantom to facilitate the assessment of optical-sectioning microscopes that are being developed to enable point-of-care pathology. This phantom, composed of silica microbeads in an Intralipid base, is specifically designed to characterize a reflectance-based dual-axis confocal (DAC) microscope for skin imaging. The phantom mimics the scattering properties of normal human epithelial tissue in terms of an effective scattering coefficient and a depth-dependent degradation in spatial resolution due to beam steering caused by tissue micro-architectural heterogeneities.
"The results to date indicate that the divided-pupil approach may offer improved imaging performance in scattering tissues, compared to the full-pupil, with either point-scanning or line-scanning. Moreover, as part of ongoing efforts to translate confocal microscopy for detection of skin cancer, we are exploring a simpler and lower-cost line-scanning configuration [7–9] that may offer a practical alternative to currently available point-scanning technology. Thus, to determine the best possible approach for clinical applications, we are now developing a quantitative understanding of imaging performance for a set of scanning and pupil conditions. "
[Show abstract][Hide abstract] ABSTRACT: Both point-scanning and line-scanning confocal microscopes provide resolution and optical sectioning to observe nuclear and cellular detail in human tissues, and are being translated for clinical applications. While traditional point-scanning is truly confocal and offers the best possible optical sectioning and resolution, line-scanning is partially confocal but may offer a relatively simpler and lower-cost alternative for more widespread dissemination into clinical settings. The loss of sectioning and loss of contrast due to scattering in tissue is more rapid and more severe with a line-scan than with a point-scan. However, the sectioning and contrast may be recovered with the use of a divided-pupil. Thus, as part of our efforts to translate confocal microscopy for detection of skin cancer, and to determine the best possible approach for clinical applications, we are now developing a quantitative understanding of imaging performance for a set of scanning and pupil conditions. We report a Fourier-analysis-based computational model of confocal microscopy for six configurations. The six configurations are point-scanning and line-scanning, with full-pupil, half-pupil and divided-pupils. The performance, in terms of on-axis irradiance (signal), resolution and sectioning capabilities, is quantified and compared among these six configurations.
"Optical coherence microscopy (OCM) combines the high-sensitivity coherence gate of OCT and high transverse resolution of confocal microscopy to achieve cellular level imaging of tissue microstructures in 3-D , , , . Confocal microscopy can detect either the reflectance (scattered) light – or the fluorescence light –. Multiphoton microscopy (MPM) utilizes the nonlinear generation of fluorescence from endogenous and exogenous markers in the tissue , , . "
[Show abstract][Hide abstract] ABSTRACT: We developed a combined optical coherence tomography (OCT) and fluorescence laminar optical tomography (FLOT) system for co-registered depth-resolved structural and molecular imaging. Experimental results using a mouse model with human breast cancer xenograft are presented.
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