Second-harmonic generation (SHG) microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Because of its underlying physical origin, it is highly sensitive to the collagen fibril/fiber structure, and, importantly, to changes that occur in diseases such as cancer, fibrosis and connective tissue disorders. We discuss how SHG can be used to obtain more structural information on the assembly of collagen in tissues than is possible by other microscopy techniques. We first provide an overview of the state of the art and the physical background of SHG microscopy, and then describe the optical modifications that need to be made to a laser-scanning microscope to enable the measurements. Crucial aspects for biomedical applications are the capabilities and limitations of the different experimental configurations. We estimate that the setup and calibration of the SHG instrument from its component parts will require 2-4 weeks, depending on the level of the user's experience.

Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure

Second Harmonic Generation microscopy has emerged as a powerful new optical imaging modality. This Feature describes its chemical and physical principles and highlights current applications in disease diagnostics.

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Second Harmonic Generation Imaging Microscopy: Applications to Diseases Diagnostics

Background
Remodeling of the extracellular matrix (ECM) has been implicated in ovarian cancer, and we hypothesize that these alterations may provide a better optical marker of early disease than currently available imaging/screening methods and that understanding their physical manifestations will provide insight into invasion.

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Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy

In this review, the authors discuss the underlying principles of SHG and specific factors that affect the generation properties and describe the essential components of a SHG instrument. In addition, results on the recent progress and impact SHG microscopy has made in different areas of biology and medicine are presented. In particular, the authors focus on disease diagnosis and basic research associated with connected tissues, musculo-skeletal disorders, and epithelial cancers. The presentation is concluded by offering a perspective on the future technical development of SHG microscopy and additional forefronts to be addressed.

Second harmonic generation microscopy: principles and applications to disease diagnosis

Automated quantification of aligned collagen for human breast carcinoma prognosis.

Phase Matching considerations in Second Harmonic Generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology.

A continuous wave photolytic iodine laser has a gain cell for receiving a continuous supply of gaseous fuel. The gain cell is connected to laser beam transfer optics, a laser resonator for shaping a laser beam, and a lamp. The lamp is driven by a microwave subsystem such that a laser gain medium is pumped through the gain cell. The continuous wave photolytic iodine laser of the present invention incorporates a closed loop fuel system for presenting gaseous fuel to the gain cell at a rate sufficient to sweep any lasing by-products out of the gain cell, thereby preventing quenching of the lasing process. The fuel system also includes a condenser for converting the gaseous fuel to a liquid after it has passed through the gain cell, a scrubber for removing the by-products of the lasing process from the fuel, and an evaporator for converting the recycled liquefied fuel back to a gas. The closed loop fuel system also includes a pump for pressurizing and transporting the liquefied fuel. Provision of pulsed, high energy ultra-violet radiation to the interior gain modules provides a pulsed photolytic iodine laser with a plurality of gain modules, generating very high energy/high power laser output radiation pulses. These output pulses from a laser with a plurality of gain cells can be a frequency greater than 1 Hz.

High power photolytic iodine laser

A broadband optical limiter for use in combination with a sensor system is operative to prevent irradiation of the sensor system by laser radiation of unknown wavelengths having intensity levels sufficient to damage or disable the sensor system. The broadband optical limiter is further operative to throughput, with minimal optical distortion at wide angle fields of view, electromagnetic radiation in the operating spectral band(s) of the sensor system. The broadband optical limiter includes a flat or spherically shaped sacrificial mirror that is operative to reflect electromagnetic radiation in the operating spectral band(s) of the sensor system and to be optically machined, i.e., vaporized, by focused laser radiation of unknown wavelengths having intensity levels sufficient to damage or disable the sensor system to create a reflective dead spot. The reflective dead spot prevents the focused laser radiation from being throughputted to the sensor system. The broadband optical limiter further includes optical components to focus incident electromagnetic and laser raidation onto the sacrificial mirror, to turn incident electromagnetic and laser radiation out of the field of view of the sensor system, and to turn electromagnetic radiation reflected by the sacrificial mirror back into the field of view of the sensor system.

Broadband optical limiter with sacrificial mirror to prevent irradiation of a sensor system by high intensity laser radiation

A continuous wave photolytic iodine laser has a gain cell for receiving a continuous supply of gaseous fuel. The gain cell is connected to laser beam transfer optics, a laser resonator for shaping a laser beam, and a lamp. The lamp is driven by a microwave subsystem such that a laser gain medium is pumped through the gain cell. The continuous wave photolytic iodine laser of the present invention incorporates a closed loop fuel system for presenting gaseous fuel to the gain cell at a rate sufficient to sweep any lasing by-products out of the gain cell, thereby preventing quenching of the lasing process. The fuel system also includes a condenser for converting the gaseous fuel to a liquid after it has passed through the gain cell, a scrubber for removing the by-products of the lasing process from the fuel, and an evaporator for converting the recycled liquefied fuel back to a gas. The closed loop fuel system also includes a pump for pressurizing and transporting the liquefied fuel.

Continuous wave photolytic iodine laser

A laser with an unstable resonator configuration is described with a second, lower power auxiliary laser to provide a feedback beam to pulse high power CW lasers without exceeding the power handling capability limitation of currently available modulators. The present laser relies on adjoint mode feedback provided by the auxiliary laser to accomplish pulsed operation with a modulator in the low power feedback beam path. Higher power output beams can be generated with multiple stages of auxiliary laser providing adjoint mode feedback, thereby reducing incident power on modulation devices while providing adequate adjoint beam power to effectively pulse the high power laser. In certain embodiments a single gain generator is used to drive the main and auxiliary lasers.

Sallie S. Townsend

Papers

Phase Matching considerations in Second Harmonic Generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology.

High power photolytic iodine laser

Broadband optical limiter with sacrificial mirror to prevent irradiation of a sensor system by high intensity laser radiation

Continuous wave photolytic iodine laser

High power laser having staged laser adjoint pulsed feedback