We present a two-photon microscope that is approximately in mass and 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 microprism. 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.
© 2009 Optical Society of America
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 , minimally invasive diagnostics , and image-guided therapeutic intervention . Promising nonlinear contrast modalities for use in miniaturized microscopes include two-photon fluorescence [1, 4, 5], second-harmonic generation [2, 6], coherent anti-Stokes Raman scattering , and stimulated Raman scattering .
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, 12, 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 reflection off the MEMS mirror , which aids miniaturization but reduces robustness to light scattering when imaging deep in optically dense tissue. The report on the other endoscope included only spatially filtered images . Neither device was shown capable of imaging in live subjects or to have sufficient sensitivity for fast physiological measurements. Here, we describe a microscope based on a two-dimensional MEMS scanner and the use of this instrument for tracking erythrocyte flow in live mice.
We constructed a portable two-photon microscope that is in mass and ∼2.0×1.9×1.1 cm3 in volume. A hollow-core bandgap fiber (Blaze Photonics) delivers ultrashort excitation pulses ( FWHM) from a tunable Ti:sapphire laser (Tsunami, Spectra-Physics) to the microscope. In this fiber the pulses incur negligible self-phase modulation , which arises in conventional single-mode or double-clad fibers with the pulse energies commonly used for two-photon excitation. Group-velocity dispersion in the bandgap fiber vanishes at , so we tuned the laser emission to to obviate dispersion precompensation .
The light delivered to the microscope passes through an aspherical collimating lens (LightPath) and reflects off the MEMS scanner Figs. 1 a–d. The scanner is 1 mm×1 mm, has a gimbal design, and contains six banks of electrostatic vertical comb actuators for steering the laser beam in two angular dimensions (Fig. 1b). We batch fabricated scanners of this type on a double-side polished, double-silicon-on-insulator wafer using four deep reactive ion-etching steps . A double-sided printed circuit board (Figs. 1b and 1c) holds the scanner die and attaches to its voltage control wires.
After reflecting off the scanner, the illumination enters a micro-optical assembly (made to our design by GRINTECH, GmbH) of four gradient refractive index (GRIN) lenses and a -wide dichroic microprism (short pass, ) that separates the IR excitation and visible emission (Fig. 1d). Two of the GRIN lenses, a diameter scan lens (, 0.14 pitch, Ag doped) and a diameter tube lens (, 0.22 pitch, Li doped), serve together as a beam expander. The illumination under fills the back aperture of the objective, a diameter GRIN lens (, 0.23 pitch, Th doped), and is focused to the specimen with a lower NA than that for fluorescence collection. The working distance from the tip of the objective to the specimen plane is in air.
The full aperture of fluorescence emissions collected by the objective returns through the tube lens, the microprism, and a GRIN collection lens (, 0.27 pitch, Ag doped) that projects the emissions onto a multimode polymer fiber ( diameter core, , Edmund Optics). This fiber guides the light to a remotely placed photomultiplier tube. Our detection configuration, in which emissions are not routed back though the scanner, differs from that of several prior fiber-optic two-photon microscopes that used a double-clad fiber for both illumination delivery and fluorescence collection and thus required emission descanning [6, 10, 13, 16].
The microscope’s housing, base plate, and internal imaging head are composed of carbon-fiber filled polyetheretherketone (PEEK), a conductive plastic chosen to minimize static charge accumulation that could damage the scanner. The base plate permits coarse lateral adjustments in the microscope’s mounting position. The housing allows axial adjustments in the position of the imaging head, for coarse focusing. For fine focusing, a dc micromotor (Faulhaber) drives a shuttle, made of PEEK, that adjusts the axial position of the bandgap fiber. The shuttle’s range of movement is , yielding of focal adjustment in the specimen.
We generally used a raster-scanning pattern, with one axis of the scanner oscillating at resonance and the other at the frame rate of . In dc operation, the maximum optical angular ranges for the inner and outer axes of the scanner are about and , respectively (Fig. 2a ). In ac operation, scan rates can be adjusted from near dc to over the mechanical resonant frequencies of and for the inner and outer axes, respectively (Fig. 2b).
To characterize the microscope’s resolution, we imaged diameter fluorescent beads. The FWHM of bead images in cross section yielded transverse and axial resolutions of and (; beads), respectively (Figs. 2c and 2d). These values are comparable to those we reported for a resonant fiber-scanning two-photon microendoscope . Images had , and the maximum field of view was .
To demonstrate in vivo usage, we studied neocortical microvasculature in adult mice anesthetized during imaging by intraperitoneal injection of ketamine and xylazine (80–100 and body mass, respectively). We made a diameter cranioto my (stereotactic coordinates: relative to lambda, lateral) and injected into a tail vein of fluorescein isothiocyanate-dextran dye (MW 2,000,000) diluted in saline solution to brightly label the blood plasma. After placing the microscope atop the mouse’s head, we imaged vessels near the neocortical surface (Figs. 3a–3c ).
To observe microcirculation, we switched to a line-scanning mode in which the outer axis was scanned at resonance but the inner axis was held fixed. This allowed tracking of individual erythrocytes in vessels in which flow was parallel to the line scanning. Individual erythrocytes appeared as dark diagonal streaks against the labeled blood plasma in the resulting plots of space versus time (Figs. 3d–3f). The streaks’ slopes yielded velocity measurements for individual erythrocytes, with steeper streaks indicating faster velocities (Fig. 3d). In the examples of Figs. 3d–3f, flow speeds were , , and (), respectively, within ranges reported previously for mouse  and rat  neocortical capillaries.
In summary, we created a portable, two-photon microscope based on a two-dimensional MEMS scanner and made the initial demonstration of physiological measurements using a MEMS-based two-photon imaging device. Future applications of miniaturized two-photon microscopes based on MEMS scanners likely include physiological studies in alert animals or human subjects, as well as cellular level diagnostics. Our approach of using a MEMS mirror for laser scanning should also be applicable to other nonlinear optical contrast modalities.
This work was supported by grants to M. J. S. from the Office of Naval Research (ONR), the National Science Foundation (NSF), the NSF Center for Biophotonics, the National Institutes of Health (NIH) Nanomedicine Development Center for Optical Control of Biological Function, and the Packard and Beckman Foundations, NSF and Stanford Graduate Research fellowships (L. D. B.), training grants from NIH (E. C. and R. P. J. B.), and NECTEC (W. P.).
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