Improved nanometer-scale particle tracking in optical microscopy using microfabricated fiduciary posts.

Image-based particle tracking methods have numerous applications in biological studies at the cellular and molecular level. For example, tracking thermal motions of micron-sized spherical particles embedded in the cell cytoplasm reveal microheterogeneity of cellular rheology (1). Fluorescent microspheres conjugated with protein analogs as well as colloidal gold beads have been used to study mobility of lipids and proteins in the cell membranes (2) and to track motions of kinesin molecules on microtubules (3). Improved instrumentation and multiple particle tracking algorithms now permit high-throughput, nanometer-scale positional resolution using time-lapse differential interference contrast (DIC) (4,5) and fluorescence imaging (4). However, there remain impediments to accurate tracking at the nanometer scale. Despite advances in motorized stage design, stages still exhibit lowfrequency positional drift due to thermal cooling of stepper motors and high-frequency vibrations arising from relative motion of sample and imaging instrumentation [e.g., charged-coupled device (CCD) cameras]. Although immobilized beads adhered to glass coverslips have been used as stationary markers (6), it is difficult to control their precise location and stability during cell culture. Furthermore, any fiduciary markers confined to the coverslips will not be in focus when imaging just a few microns away and are thus unsuitable for correcting tracking errors at the tops of cells. Similarly, immobilizing large beads is not feasible since they have small attachment area to the glass surface and can easily become dislodged. Other avenues to mitigate mechanical instability are expensive piezo stages, highperformance vibration isolation tables, and remote cooling of camera CCD chips. Even with these improvements, there will always remain some uncertainty as to whether particle trajectories represent true motion or coverglass motion artifacts. To overcome uncertainty of particle position at both high and low frequencies (e.g., vibration and stage drift, respectively) and to enable tracking of coverslip motion relative to cell organelle motion, even at microns away from the coverslip, we microfabricated 10-μm-high and 3-μm-diameter cylindrical SU-8 posts on glass coverslips to serve as fiduciary markers during tracking. Microposts were fabricated using SU-8 2010, a negative epoxy-type photoresist (MicroChem, Newton, MA, USA), on 40-mm circular no.1 glass coverslips (Bioptechs, Butler, PA, USA) using standard photolithography techniques (7). Glass coverslips were initially cleaned using, sequentially, acetone for 10 min, isopropanol for 10 min, and deionized (DI) water for 10 min. An adhesion layer (OmniCoatTM; MicroChem) was spin-coated on the glass surface using a spin coater (PWM32; Headway Research, Garland, TX, USA) followed by a baking process on a hot plate at 200°C for 1 min. SU-8 2010 was then spin-coated on top of the adhesion layer at 5000 rpm for 30 s to obtain a 10-μm-thick uniform photoresist layer. The resulting layer was baked at 65°C for 10 min and at 95°C for 20 min. A chromium mask was designed in AutoCAD® (Autodesk, San Rafael, CA, USA) and fabricated commercially (MEMS and Nanotechnology Improved nanometer-scale particle tracking in optical microscopy using microfabricated fiduciary posts