Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce two-photon imaging based on microelectromechanical systems (MEMS) scanners. Single crystalline silicon scanning mirrors that are 0.75 mm x 0.75 mm in size and driven in two dimensions by microfabricated vertical comb electrostatic actuators can provide optical deflection angles through a range of approximately16 degrees . Using such scanners we demonstrated two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.52 kHz.
We present a two-photon microscope that is approximately 2.9 g in mass and 2.0×1.9×1.1 cm 3 in size and based on a microelectromechanical systems (MEMS) laser-scanning mirror. The microscope has a focusing motor and a micro-optical assembly composed of four gradient refractive index lenses and a dichroic micro-prism. Fluorescence is captured without the detected emissions reflecting off the MEMS mirror, by use of separate optical fibers for fluorescence collection and delivery of ultrashort excitation pulses. Using this microscope we imaged neocortical microvasculature and tracked the flow of erythrocytes in live mice.An important aim of recent research on nonlinear optical microscopy has been to create miniaturized laser-scanning microscopes and endoscopes for biomedical investigation and clinical purposes. Such devices would propel a range of applications benefiting from portable instrumentation and should ultimately be more economical than tabletop instrumentation. Among key potential applications are imaging microvascular function or cellular dynamics in live or behaving animals [1], minimally invasive diagnostics [2], and image-guided therapeutic intervention [3]. Promising nonlinear contrast modalities for use in miniaturized microscopes include two-photon fluorescence [1,4,5], second-harmonic generation [2,6], coherent antiStokes Raman scattering [7], and stimulated Raman scattering [8].Crucial design issues with miniaturized nonlinear imaging devices concern laser-scanning mechanisms. Several mechanisms have been explored for two-photon imaging, including cantilever fiber-scanners operating at resonant [9,10] or non-resonant [11-13] frequencies, as well as microelectromechanical systems (MEMS) scanning mirrors [3,6,14,15]. Cantilever scanners generally have longer lengths than MEMS scanners and if operated at resonance also restrict the choice of scanning frequencies. MEMS scanners allow batch fabrication, generally at least modest abilities to zoom and pan across the imaging field, and flexibility in the adjustment of scan rates. To date, two endoscopes for two-photon imaging based on MEMS scanners have been described [3,16], both of which are larger than the microscope we present below. One endoscope involved a double-clad optical fiber and routed fluorescence signals in *Corresponding author: mschnitz@stanford.edu. We constructed a portable two-photon microscope that is 2.9 g in mass and ~2.0×1.9×1.1 cm 3 in volume. A hollow-core bandgap fiber (Blaze Photonics) delivers ultrashort excitation pulses (~110 fs FWHM) from a tunable Ti:sapphire laser (Tsunami, Spectra-Physics) to the microscope. In this fiber the pulses incur negligible self-phase modulation [17], which arises in conventional single-mode or double-clad fibers with the 0.01-1 nJ pulse energies commonly used for two-photon excitation. Group-velocity dispersion in the bandgap fiber vanishes at 800 nm, so we tuned the laser emission to ~790-810 nm to obviate dispersion precompensation [17]. NIH Public AccessThe light delivered to the m...
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.
The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5-7 microm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 x 100 microm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy.
Small interfering RNAs (siRNAs) can be designed to specifically and potently target and silence a mutant allele, with little or no effect on the corresponding wild-type allele expression, presenting an opportunity for therapeutic intervention. Although several siRNAs have entered clinical trials, the development of siRNA therapeutics as a new drug class will require the development of improved delivery technologies. In this study, a reporter mouse model (transgenic click beetle luciferase/ humanized monster green fluorescent protein) was developed to enable the study of siRNA delivery to skin; in this transgenic mouse, green fluorescent protein reporter gene expression is confined to the epidermis. Intradermal injection of siRNAs targeting the reporter gene resulted in marked reduction of green fluorescent protein expression in the localized treatment areas as measured by histology, real-time quantitative polymerase chain reaction and intravital imaging using a dual-axes confocal fluorescence microscope. These results indicate that this transgenic mouse skin model, coupled with in vivo imaging, will be useful for development of efficient and 'patient-friendly' siRNA delivery techniques and should facilitate the translation of siRNA-based therapeutics to the clinic for treatment of skin disorders.
We designed and constructed a single-fiber-optic confocal microscope (SFCM) with a microelectromechanical system (MEMS) scanner and a miniature objective lens. Axial and lateral resolution values for the system were experimentally measured to be 9.55 mum and 0.83 mum respectively, in good agreement with theoretical predictions. Reflectance images were acquired at a rate of 8 frames per second, over a 140 mum x 70 mum field-of-view. In anticipation of future applications in oral cancer detection, we imaged ex vivo and in vivo human oral tissue with the SFCM, demonstrating the ability of the system to resolve cellular detail.
Abstract.A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-offocus light for high-contrast optical sectioning. A biaxial microelectromechanical system ͑MEMS͒ scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500ϫ 500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections.
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