vBack Focal Plane interferometry (BFPI) is a widely used technique in particle tracking, providing high-speed, highresolution measurements. We present Structured Back Focal Plane Interferometry (SBFPI), a modification to BFPI utilizing structured beams and structured detectors . Utilising Conical Wavefront Gaussian Beams and Annular Quadrant Photodetectors, SBFPI significantly increases tracking range while maintaining sensitivity and robustness to aberrations. We studied the variation in detection range and sensitivity over our parameters. A maximum increase in axial and lateral tracking range of 4.25-fold and 1.83-fold was found respectively, concomitant with sensitivity decreases of 53% and 61%. We also found parameter combinations with smaller range improvements but which retain sensitivity, in particular a 2.31 fold axial range increase with a 22.4% reduction in sensitivity, and these can be tuned to each experiment. We also studied the effect of aberrations on signal integrity.
Living cells adjust their cytoskeletal organization and mechanically change their overall shape by reacting to the changes of the microenvironment. The ability to quantify these dynamic events in micro and nanoscale in real-time at the same time contributes to our understanding of the functional response of living cells. The combination to achieve both microscale and nanoscale imaging simultaneous at volumetric speeds is challenging. Traditional TIRF microscopy has excelled in measuring surface interaction but yet limited in imaging depth and requires fluorescent labelling. Likewise, the ability to quantify the total volume and shape change of biological cells as they interact requires either confocal microscopy or lightsheet microscopy. In this paper, we propose an in toto label free approach through coherent optical interference to measure volumetric information and surface interaction at the same time to provide a full view of the cell during dynamic activities.
In optical micromanipulation, back focal plane interferometry (BFPI) has remained the most widely used method for tracking dielectric and metallic microparticles with nanometer resolution precision at speeds of up to few a MHz. Basic BFPI employs Gaussian beams which severely limits the detection range of the technique unless the focal parameters are tuned by increase the width of the beam, but this is usually concomitant with significant loss in detection sensitivity. A challenge in BFPI is to extend its linear range while maintaining this detection sensitivity along each axis. We constructed a system which utilizes a combination of structured beam shaping and structured detection (Annular Quadrant Detection), which we called Structured Back Focal Plane Interferometry (S-BFPI). A Gaussian beam is shaped by a spatial light modulator by imparting a conical wavefront, which increases the depth of focus while simultaneously maintaining the Gouy phase shift and hence the sensitivity of detection. In addition, an annular QPD is used for detection. Using S-BFPI, we were able to achieve a 200% axial range extension with only a 4.6% reduction in insensitivity, and a 167% lateral range extension with a 45% reduction in sensitivity. SBFPI can tailor its detection range and sensitivity over an intermediate range of displacement and sensitivity improvements at hand. We finally demonstrated its robustness against aberrations common to optical systems. S-BFPI presents itself as a flexible, tunable option for use as an optical measurement tool.
Here we propose a region-recognition approach with iterative thresholding, which is adaptively tailored to extract the appropriate region or shape of spatial frequency. In order to justify the method, we tested it with different samples and imaging conditions (different objectives). We demonstrate that our method provides a useful method for rapid imaging of cellular dynamics in microfluidic and cell cultures.