Two-dimensional random access multiphoton spatial frequency modulated imaging.

Spatial frequency modulated imaging (SPIFI) enables the use of an extended excitation source for linear and nonlinear imaging with single element detection. To date, SPIFI has only been used with fixed excitation source geometries. Here, we explore the potential for the SPIFI method when a spatial light modulator (SLM) is used to program the excitation source, opening the door to a more versatile, random access imaging environment. In addition, an in-line, quantitative pulse compensation and measurement scheme is demonstrated using a new technique, spectral phase and amplitude retrieval and compensation (SPARC). This enables full characterization of the light exposure conditions at the focal plane of the random access imaging system, an important metric for optimizing, and reporting imaging conditions within specimens.

Sources of variability in the quantification of tissue optical properties by multidiameter single-fiber reflectance and fluorescence spectroscopy.

Recently, a multidiameter single-fiber reflectance and fluorescence spectroscopy device has been developed that enabled us to extract the autofluorescence of tissue that is corrected for the optical properties. Such a system has been incorporated in the population-based Rotterdam Study to investigate the autofluorescence of the skin. Since the device will be used by different operators over many years, it is essential that the results are comparable between users. It is, however, unclear how different methods of handling the probe might influence the outcome. Variability of blood oxygen saturation, blood volume fraction and vessel diameter, average gamma, reduced scattering coefficient at 800 nm, and integrated intrinsic fluorescence measured in three volunteers were assessed within and between eight untrained users. A variability of less than one standard deviation from the group mean was defined as an acceptable limit. Three mature volunteers were also included to assess the intrauser variability of mature skin. The variation in the measured parameters suggests that variation is dominated by tissue heterogeneity. Most users measured within one standard deviation of the group mean. Notably, corrected intrinsic fluorescence showed low intra- and interuser variability. These results strongly suggest that variability is mostly caused by tissue heterogeneity and is not user induced.

Estimating the risk of squamous cell cancer induction in skin following nonlinear optical imaging.

High power femto-second (fs) laser pulses used for in-vivo nonlinear optical (NLO) imaging can form cyclobutane pyrimidine dimers (CPD) in DNA, which may lead to carcinogenesis via subsequent mutations. Since UV radiation from routine sun exposure is the primary source of CPD lesions, we evaluated the risk of CPD-related squamous cell carcinoma (SCC) in human skin due to NLO imaging relative to that from sun exposure. We developed a unique cancer risk model expanding previously published estimation of risk from exposure to continuous wave (CW) laser. This new model showed that the increase in CPD-related SCC in skin from NLO imaging is negligible above that due to regular sun exposure.

Method for rapid multidiameter single-fiber reflectance and fluorescence spectroscopy through a fiber bundle.

We have recently demonstrated a means for quantifying the absorption and scattering properties of biological tissue through multidiameter single-fiber reflectance (MDSFR) spectroscopy. These measurements can be used to correct single-fiber fluorescence (SFF) spectra for the influence of optical properties, enabling quantification of intrinsic fluorescence. In our previous work, we have used a series of pinholes to show that selective illumination and light collection using a coherent fiber bundle can simulate a single solid-core optical fiber with variable diameter for the purposes of MDSFR spectroscopy. Here, we describe the construction and validation of a clinical MDSFR/SFF spectroscopy system that avoids the limitations encountered with pinholes and free-space optics. During one measurement, the new system acquires reflectance spectra at the effective diameters of 200, 600, and 1000 µm, and a fluorescence spectrum at an effective diameter of 1000 µm. From these spectra, we measure the absolute absorption coefficient, µ(a), reduced scattering coefficient, µ'(s'), phase function parameter, γ, and intrinsic fluorescence, Qµ(a,x)(f), across the measured spectrum. We validate the system using Intralipid- and polystyrene sphere-based scattering phantoms, with and without the addition of the absorber Evans Blue. Finally, we demonstrate the combined MDSFR/SFF of phantoms with varying concentrations of Intralipid and fluorescein, wherein the scattering properties are measured by MDSFR and used to correct the SFF spectrum for accurate quantification of Qµ(a,x)(f).

Towards endoscopic ultrafast laser microsurgery of vocal folds.

Vocal fold scarring is a predominant cause of voice disorders yet lacks a reliable treatment method. The injection of soft biomaterials to improve mechanical compliance of the vocal folds has emerged as a promising treatment. Here, we study the use of precise femtosecond laser microsurgery to ablate subsurface voids, with a goal of eventually creating a plane in dense subepithelial scar tissue into which biomaterials can be injected for their improved localization. Specifically, we demonstrate the ablation of small subepithelial voids in porcine vocal fold tissue up to 120 [micro sign]m below the surface such that larger voids in the active area of vocal fold mucosa (~3×10 mm(2)) can eventually be ablated in about 3 min. We use sub-µJ, 776-nm pulses from a compact femtosecond fiber laser system operating at a 500-kHz repetition rate. The use of relatively high repetition rates, with a small number of overlapping pulses, is critical to achieving ablation in a very short time while still avoiding significant heat deposition. Additionally, we use the same laser for nonlinear optical imaging to provide visual feedback of tissue structure and to confirm successful ablation. The ablation parameters, including pulse duration, pulse energy, spot size, and scanning speed, are comparable to the specifications in our recently developed miniaturized femtosecond laser surgery probes, illustrating the feasibility of developing an ultrafast laser surgical instrument.

Optical design and imaging performance testing of a 9.6-mm diameter femtosecond laser microsurgery probe.

We present the optical design of a 9.6-mm diameter fiber-coupled probe for combined femtosecond laser microsurgery and nonlinear optical imaging. Towards enabling clinical use, we successfully reduced the dimensions of our earlier 18-mm microsurgery probe by half, while improving optical performance. We use analytical and computational models to optimize the miniaturized lens system for off-axis scanning aberrations. The optimization reveals that the optical system can be aberration-corrected using simple aspheric relay lenses to achieve diffraction-limited imaging resolution over a large field of view. Before moving forward with custom lenses, we have constructed the 9.6-mm probe using off-the-shelf spherical relay lenses and a 0.55 NA aspheric objective lens. In addition to reducing the diameter by nearly 50% and the total volume by 5 times, we also demonstrate improved lateral and axial resolutions of 1.27 µm and 13.5 µm, respectively, compared to 1.64 µm and 16.4 µm in our previous work. Using this probe, we can successfully image various tissue samples, such as rat tail tendon that required 2-3 × lower laser power than the current state-of-the-art. With further development, image-guided, femtosecond laser microsurgical probes such as this one can enable physicians to achieve the highest level of surgical precision anywhere inside the body.

Fast-updating and nonrepeating Lissajous image reconstruction method for capturing increased dynamic information.

We present a fast-updating Lissajous image reconstruction methodology that uses an increased image frame rate beyond the pattern repeat rate generally used in conventional Lissajous image reconstruction methods. The fast display rate provides increased dynamic information and reduced motion blur, as compared to conventional Lissajous reconstruction, at the cost of single-frame pixel density. Importantly, this method does not discard any information from the conventional Lissajous image reconstruction, and frames from the complete Lissajous pattern can be displayed simultaneously. We present the theoretical background for this image reconstruction methodology along with images and video taken using the algorithm in a custom-built miniaturized multiphoton microscopy system.

Miniaturized probe for femtosecond laser microsurgery and two-photon imaging.

Combined two-photon fluorescence microscopy and femtosecond laser microsurgery has many potential biomedical applications as a powerful "seek-and-treat" tool. Towards developing such a tool, we demonstrate a miniaturized probe which combines these techniques in a compact housing. The device is 10 x 15 x 40 mm(3) in size and uses an aircore photonic crystal fiber to deliver femtosecond laser pulses at 80 MHz repetition rate for imaging and 1 kHz for microsurgery. A fast two-axis microelectromechanical system scanning mirror is driven at resonance to produce Lissajous beam scanning at 10 frames per second. Field of view is 310 microm in diameter and the lateral and axial resolutions are 1.64 microm and 16.4 microm, respectively. Combined imaging and microsurgery is demonstrated using live cancer cells.