Simultaneous multiplane confocal microscopy using acoustic tunable lenses.

Maximizing the amount of spatiotemporal information retrieved in confocal laser scanning microscopy is crucial to understand fundamental three-dimensional (3D) dynamic processes in life sciences. However, current 3D confocal microscopy is based on an inherently slow stepwise process that consists of acquiring multiple 2D sections at different focal planes by mechanical or optical z-focus translation. Here, we show that by using an acoustically-driven optofluidic lens integrated in a commercial confocal system we can capture an entire 3D image in a single step. Our method is based on continuous axial scanning at speeds as high as 140 kHz combined with fast readout. In this way, one or more focus sweeps are produced on a pixel by pixel basis and the detected photons can be assigned to their corresponding focal plane enabling simultaneous multiplane imaging. We exemplify this method by imaging calibration and biological fluorescence samples. These results open the door to exploring new fundamental processes in science with an unprecedented time resolution.

[1]  Masatoshi Ishikawa,et al.  A variable-focus lens with 1 kHz bandwidth applied to axial-scan of a confocal scanning microscope , 2003, The 16th Annual Meeting of the IEEE Lasers and Electro-Optics Society, 2003. LEOS 2003..

[2]  Alberto Diaspro,et al.  Characterizing biostructures and cellular events in 2D/3D [using wide-field and confocal optical sectioning microscopy] , 1996 .

[3]  Nam-Trung Nguyen,et al.  Micro-optofluidic Lenses: A review. , 2010, Biomicrofluidics.

[4]  Benjamin Schmid,et al.  A high-level 3D visualization API for Java and ImageJ , 2010, BMC Bioinformatics.

[5]  J. Squier,et al.  Simultaneous imaging of multiple focal planes using a two-photon scanning microscope. , 2007, Optics letters.

[6]  Franck P. Martial,et al.  Programmable Illumination and High-Speed, Multi-Wavelength, Confocal Microscopy Using a Digital Micromirror , 2012, PloS one.

[7]  Craig B. Arnold,et al.  Enhanced depth of field laser processing using an ultra-high-speed axial scanner , 2013 .

[8]  J. Tiago Gonçalves,et al.  Simultaneous 2-photon calcium imaging at different cortical depths in vivo with spatiotemporal multiplexing , 2010, Nature Methods.

[9]  M. Hofmann,et al.  Axial scanning in confocal microscopy employing adaptive lenses (CAL). , 2014, Optics express.

[10]  Rodrigo Cuenca,et al.  Optical axial scanning in confocal microscopy using an electrically tunable lens. , 2014, Biomedical optics express.

[11]  Vidya Venkatachalam,et al.  Extended depth of field imaging for high speed cell analysis , 2007, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[12]  Yi-Hsin Lin,et al.  An endoscopic system adopting a liquid crystal lens with an electrically tunable depth-of-field. , 2013, Optics express.

[13]  Demetri Psaltis,et al.  Elastomer based tunable optofluidic devices. , 2012, Lab on a chip.

[14]  A. Cheng,et al.  simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing , 2011 .

[15]  Rimas Juskaitis,et al.  Real-time extended depth of field microscopy. , 2008, Optics express.

[16]  Benjamin Schmid,et al.  Rapid 3D light-sheet microscopy with a tunable lens. , 2013, Optics express.

[17]  Euan McLeod,et al.  High-speed varifocal imaging with a tunable acoustic gradient index of refraction lens. , 2008, Optics letters.

[18]  I. Parker,et al.  Construction of a confocal microscope for real-time x-y and x-z imaging. , 1999, Cell calcium.

[19]  Keith J. Kelleher,et al.  Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity , 2008, Nature Neuroscience.

[20]  Hiromasa Oku,et al.  High-speed liquid lens with 2-ms response and 80.3-nm root-mean-square wavefront error , 2010, MOEMS-MEMS.

[21]  Alexandre Mermillod-Blondin,et al.  Two-photon microscopy with simultaneous standard and extended depth of field using a tunable acoustic gradient-index lens. , 2009, Optics letters.

[22]  Martí Duocastella,et al.  Simultaneous imaging of multiple focal planes for three-dimensional microscopy using ultra-high-speed adaptive optics. , 2012, Journal of biomedical optics.

[23]  W. Cathey,et al.  Extended depth of field through wave-front coding. , 1995, Applied optics.

[24]  Uriel Levy,et al.  Tunable optofluidic devices , 2008 .

[25]  Hiromasa Oku,et al.  High-speed liquid lens with 2 ms response and 80.3 nm root-mean-square wavefront error , 2009 .

[26]  Benjamin F. Grewe,et al.  Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens , 2011, Biomedical optics express.