![]() ![]() Therefore a major limiting factor for further development of fast microscopy is the efficiency of the illumination system. ![]() At these extreme acquisition speeds an important parameter is the quantity of light which arrives from the sample at the sensor. The development of fast CCD and CMOS cameras allows today microsecond exposure time and, consequently, tens to hundred kHz sampling rate (i.e. In general, when fast dynamical processes (in particular out of equilibrium) have to be detected by a camera, the exposure time of the sensor must be minimized to avoid blurring, while the acquisition rate should be maximized to sample correctly the dynamics. These measurements can be used to obtain information about the particle local environment, the mechanical behavior of single molecules tethered to the particles, and the action of enzymes. Several implementations of these techniques employ the spatial distribution of the light scattered from microscopic particles to track their motion with nm-resolution or to reconstruct their shape –. Examples include single-molecule biophysics techniques, such as magnetic tweezers, , (holographic) optical tweezers – and tethered particle motion –, together with micro-rheology, , holography –, and micro-fluidic devices. In particular, an increasing number of applications based on video microscopy require to detect and track, with nanometer and microsecond resolution, the motion of scattering, non-fluorescent, micro- and nano-particles. In optical microscopy, it is today of key interest to combine the high spatial resolving power of microscopes with the ability to detect temporally fast dynamics. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. There are no patents, products in development or marketed products to declare. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: Xavier Hachair is employed by BBright. All relevant data are within the paper.įunding: FP acknowledges funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: The authors confirm that all data underlying the findings are fully available without restriction. Received: ApAccepted: AugPublished: September 24, 2014Ĭopyright: © 2014 Dulin et al. PLoS ONE 9(9):Įditor: Tom Waigh, University of Manchester, United Kingdom This comparison of sources can guide in choosing the most efficient illumination system with respect to the specific application.Ĭitation: Dulin D, Barland S, Hachair X, Pedaci F (2014) Efficient Illumination for Microsecond Tracking Microscopy. In particular, we show that the simple and inexpensive laser illumination enables less than s camera exposure time at high magnification on a large field of view without coherence image artifacts, together with a good hologram quality that allows nm-tracking of microscopic beads to be performed. Here we systematically compare the main illumination systems employed in holographic tracking microscopy, and we show that a superluminescent diode and a modulated laser diode perform the best in terms of image quality and acquisition speed, respectively. ![]() A main limiting factor for further development of fast video microscopy remains the illumination of the sample, which must deliver sufficient light to the camera to allow microsecond exposure times. The possibility to observe microsecond dynamics at the sub-micron scale, opened by recent technological advances in fast camera sensors, will affect many biophysical studies based on particle tracking in optical microscopy. ![]()
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