Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two- dimensional scanning mirror - PubMed (original) (raw)

Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two- dimensional scanning mirror

Wibool Piyawattanametha et al. Opt Lett. 2006.

Abstract

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.

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Figures

Fig. 1

Fig. 1

Electron micrographs of a two-dimensional MEMS scanner. a, 750 _μ_m × 750 _μ_m scanning mirror in a 3.2 mm × 3.0 mm die. Six banks of vertical comb actuators drive the mirror, which has a gimbal design. b, Inner axis torsional spring. c, Outer axis comb bank. Scale bars are 250 _μ_m.

Fig. 2

Fig. 2

(Color online) Response characteristics of a 750 _μ_m × 750 _μ_m MEMS scanner. For both a and b, the voltage signal was applied to only one of the two opposing comb banks for each rotational axis. a, Optical deflection angle as a function of dc voltage. The maximum deflection angles are ±7.6° and ±3.0° for the inner (blue solid curve) and outer (red dashed curve) axes, respectively. b, Frequency response functions for the inner (blue solid curve) and outer (red dashed curve) axes, obtained by applying voltage signals of peak-to-peak amplitudes 45 and 58 V to the inner and outer axes, respectively.

Fig. 3

Fig. 3

Two-photon fluorescence images of pollen grains acquired using instrumentation based on a MEMS scanner and a Ti:sapphire laser tuned to 850 nm. a–c, Images acquired using a 40× 0.8 NA water-immersion microscope objective. The fast-axis acquisition rate was 3.2 kHz for a and b and 3.0 kHz for c. Image b is a sum projection from a stack of 38 images acquired at 1 _μ_m increments. d and e, Images acquired using a doublet GRIN microendoscope probe of 0.47 NA and a 10× 0.25 NA microscope objective to couple light into the probe., The fast axis was driven at resonance, allowing a double-sided acquisition rate of 3.52 kHz. Image e is a maximum intensity projection from a stack of 46 images acquired at 1 _μ_m increments. Laser power at the sample was 20 mW for a and b, 28 mW for c, and 40 mW for d and e. These power levels were needed because of the fast acquisition rate. Scale bars are 5 _μ_m.

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