Spatial light modulation for improved microscope stereo vision and 3D tracking Michael P. Lee a , Graham Gibson a , Manlio Tassieri b , Dave Phillips c , Stefan Bernet d , Monika Ritsch-Marte d and Miles J. Padgett a a School of Physics and Astronomy, SUPA, University of Glasgow, G12 8QQ, UK; b School of Engineering, University of Glasgow, G12 8LT, UK; c H. H. Wills Physics Laboratory, University of Bristol, BS8 1TL, UK; d Division for Biomedical Physics, Innsbruck Medical University, A-6020 Innsbrusck, Austria ABSTRACT We present a new type of stereo microscopy which can be used for tracking in 3D over an extended depth. The use of Spatial Light Modulators (SLMs) in the Fourier plane of a microscope sample is a common technique in Holographic Optical Tweezers (HOT). This set up is readily transferable from a tweezer system to an imaging system, where the tweezing laser is replaced with a camera. Just as a HOT system can diffract many traps of different types, in the imaging system many different imaging types can be diffracted with the SLM. The type of imaging we have developed is stereo imaging combined with lens correction. This approach has similarities with human vision where each eye has a lens, and it also extends the depth over which we can accurately track particles. Keywords: Spatial Light Modulator, Stereo Particle Tracking, Microscopy 1. INTRODUCTION Spatial Light Modulators (SLMs) are controllable, pixellated devices which provide phase or amplitude manip- ulation of light fields. In optical microscopy, this has been used to manipulate a microscope’s Point Spread Function (PSF). 1, 2 Here, an SLM is positioned in the back focal plane of the microscope’s objective lens. In this plane, also termed the Fourier plane, a sample’s spatial frequencies are present. For example, if the sample was a sine grating illuminated with a monochromatic plane wave, the intensity of light in the Fourier plane would be two bright spots offset from a central DC spot. The offset distance is related to the grating period, and the DC spot corresponds to undiffracted light. The SLM is capable of modulating the phase and amplitude of these spatial frequencies and, hence, provide aberration correction, defocus, darkfield and phase contrast imaging. It is also possible to create more elaborate modulations such as spiral phase contrast, 3 double-helix point spread functions, 4 Spatial Light Interference Microscopy (SLIM), 5 depth of field multiplexing 6, 7 and Gabor filters 8 amongst others. 9–11 A blazed grating on the SLM means that the image of the object is diffracted off axis. This can be used to separate the modulated image from the (unwanted) zero order image, which is also generated by the SLM due to imperfect diffraction efficiency. By using multiple gratings simultaneously, many images can be diffracted and detected on the same camera chip. 2, 6, 7 In this work, we describe the combination of these image enhancement modulations with stereo microscopy. 2 By illuminating a sample from two directions and Fourier filtering, the detected image is as if it was observed from two different directions. 12 Indeed, it is possible to achieve a similar effect with two objective lenses and a single light source. The advantage of the two distinct illumination directions is that the Fourier components of the two views are well defined in the SLM plane. This is also is compatible with high magnification objectives, although the images from the two light sources overlap in the image plane. To separate the two images, a Fourier filter has to be manufactured consisting of two prisms close to two apertures. The function of the prisms is to Further author information: M.P.L.: E-mail: m.lee.2@research.gla.ac.uk, Telephone: +44 141 330 2047 Optical Trapping and Optical Micromanipulation X, edited by Kishan Dholakia, Gabriel C. Spalding, Proc. of SPIE Vol. 8810, 881022 · © 2013 SPIE · CCC code: 0277-786X/13/$18 · doi: 10.1117/12.2027734 Proc. of SPIE Vol. 8810 881022-1 Downloaded From: http://spiedigitallibrary.org/ on 03/31/2014 Terms of Use: http://spiedl.org/terms