Tracking single particles: a user-friendly quantitative evaluation

Abstract

As our knowledge of biological processes advances, we are increasingly aware that cells actively position sub-cellular organelles and other constituents to control a wide range of biological processes. Many studies quantify the position and motion of, for example, fluorescently labeled proteins, protein aggregates, mRNA particles or virus particles. Both differential interference contrast (DIC) and fluorescence microscopy can visualize vesicles, nuclei or other small organelles moving inside cells. While such studies are increasingly important, there has been no complete analysis of the different tracking methods in use, especially from the practical point of view. Here we investigate these methods and clarify how well different algorithms work and also which factors play a role in assessing how accurately the position of an object can be determined. Specifically, we consider how ultimate performance is affected by magnification, by camera type (analog versus digital), by recording medium (VHS and SVHS tape versus direct tracking from camera), by image compression, by type of imaging used (fluorescence versus DIC images) and by a variety of sources of noise. We show that most methods are capable of nanometer scale accuracy under realistic conditions; tracking accuracy decreases with increasing noise. Surprisingly, accuracy is found to be insensitive to the numerical aperture, but, as expected, it scales with magnification, with higher magnification yielding improved accuracy (within limits of signal-to-noise). When noise is present at reasonable levels, the effect of image compression is in most cases small. Finally, we provide a free, robust implementation of a tracking algorithm that is easily downloaded and installed.

Control of cell membrane tension by myosin-I

“…Membrane tethers were then formed by translating the piezoelectric stage to move the sample away from the trapped bead. Forces exerted by membrane tethers on the bead were derived from microsphere position data (15), acquired at video rate using a CCD camera; position data were converted to force using the stiffness of the optical trap (k Trap ), which for our studies ranged from 0.1-0.4 pN/nm (Fig. S1).…”

“…Images of trapped particles were captured with a Sony Exwave HAD color CCD using a National Instruments PCI-1410 image acquisition card with custom software written in Lab-VIEW 8.5. Time-lapse images acquired at video rate were used to obtain the position of trapped particles with software developed by Carter et al (15). Trap stiffness calibration was performed by measuring the excursion of a trapped bead in response to different viscous drag forces applied by moving the flow chamber with the piezoelectric stage.…”

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

“…Differential interference contrast (DIC) microscopy is based on variations in the index of refraction. All these techniques, if used in an optimized way, can achieve an accuracy of the order of 5 nanometers [64]. Other techniques that do not require labels are based on harmonic generation [385] and coherent Stokes Raman scattering (CARS) microscopy [180].…”

On the use of in vivo cargo velocity as a biophysical marker

“…The data presented here is from analysis of 13 wild-type and 7 Δ(halo) embryos [1,23]. Video-enhanced differential interference contrast (DIC) microscopy recordings of moving droplets were acquired onto videotape with a 100x 1.4 NA plan-apo objective and a 2.5x magnifying lens in front of the video camera [1,16,25]. Sequences from the recording (usually of approximately 1.5 minute duration) were then analyzed.…”

“…Sequences from the recording (usually of approximately 1.5 minute duration) were then analyzed. Location of individual droplets as a function of time was determined with few nanometer-level resolution [25] using centroid analysis for droplets that traveled a minimum distance of 0.5 μm perpendicular to the apical edge of the embryo. See Supplement for further details.…”