Schematic diagram of the optical tweezer configuration. The laser beam was expanded by a Galelian telescope, lenses L1 and L2. L3 focused the image of the trapping plane crated by the infinity corrected objective on to the camera. BS: nonpolarizing 50:50 beam splitter, F1,2: 650 nm short-pass filters to protect the camera from backscattered IR laser light. The sample stage was mounted on a piezoelectric translation stage whose axis of motion was aligned in the y direction.
Images of a 2.85 μm bead taken as the microscope (NA = 1.25) was defocused, in order to simulate the effect of looking at particles at varying depths in the sample for a fixed focus. The numbers refer to the variation in depth (where 0 indicates the in-focus position).
Particles as imaged by the tracking camera and software. The bead on the right is held by the optical tweezers, while the bead on the left is stuck to the cover glass and appears out of focus. (a) Configuration as seen by the camera. (b) The same image after applying local inversion around the out-of-focus bead. (c) The same image overlayed with the tracking region masking. Red areas are excluded from the centroid calculation, inner unshaded areas contribute to the calculation. White scale bars in all images are 5 μm.
Dataflow through the various microchip devices forming our smart camera. The thickness of the arrows relates to the quantity of data, with the data rate being shown in MB/s (KB/frame inside the brackets). Images travel from the CMOS sensor to the FPGA where fixed pattern noise is removed. The datastream is then split with a “centroider” module measuring the positions of objects within all the images. These centroids are buffered in an external SRAM memory along with a down-selected subset of images, both of which are then transmitted to a PC over USB2.
Simultaneously recorded displacements of a settled reference bead (dashed line) and of an optically trapped probe bead (solid line) in the 0.8 mole fraction water–glycol mixture. The sample stage was driven sinusoidally at 2 Hz and the motion recorded at 250 Hz.
Achievable frame rates for tracking with our smart camera.
Results of active phase-shift viscosity measurements of water–ethanediol mixtures using our techniques compared with bulk values measured using a falling-cylinder viscometer (Ref. 20).
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