Particle tracking in confocal microscopy

The ability to image and analyze single molecules is a powerful tool in molecular biology. Most methods rely on the use of time-lapse imaging of wide-field fluoroescence images coupled with single molecule localization techniques such as fitting the measured intensity pattern of each sub-diffraction limit particle with a Gaussian. In general, such methods are limited to tracking motion in or near the focal plane. Moreover, due to the need to collect a relatively large number of photons to obtain a good signal-to-noise ratio (SNR), they are somewhat limited in temporal resolution with respect to the time scale of the motion of many biologically relevant single molecules.

To overcome these limitations, we are developing an alternative approach based on a single- or multi-photon microscope. Such microscopes rely on point detectors rather than CCD cameras. As a result, they have better SNR and temporal resolution but at the cost of a much smaller detection volume (on the order of femtoliters). To obtain trajectory information, we utilize the system to directly track the motion of single and multiple particles. Combing a novel position estimation algorithm which converts the measurements of fluorescence intensity obtained at different positions into a measurement of the location of the fluorescing particle with a standard Linear Quadratic Gaussian (LQG) control framework, we can track sub-diffraction limit particles with high spatial and temporal resolution.

The block diagram and physical apparatus in our lab are shown below. Fluorescence excitation is provided by a focused laser and actuation of the relative position of the particle and the focal volume of the microscope is (currently) achieved using a 3-D nanostage.


The CCD camera shown is not used for tracking but only to verify tracking and quantify performance. Under perfect tracking, the position of the fluorescent particle in the CCD image frame should remain fixed. In the movies below, we show experimental results from tracking a fixed particle (left) and a freely diffusing particle (right). Without tracking, the diffusing particle would have quickly moved out of the image frame. (Note that in the diffusing case, the changing rings indicate that the particle is diffusing along the optical axis.)

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Tracking a fixed particle

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Tracking a diffusing particle

Experiments to date have demonstrated tracking of single particles in 2-D with diffusion constants above 0.1 μm/s2 and simultaneous tracking of three particles. Ongoing efforts include implementing the controller algorithm for tracking in 3-D on a DSP to greatly increase the control loop rate and optimizing the scheme for tracking multiple particles.


  1. Z. Shen and S.B. Andersson, “Tracking multiple fluorescent molecules in two dimensions in a confocal microscope,” IEEE Conference on Decision and Control, pp. 6052-6057, 2009. download
  2. Z. Shen and S.B. Andersson, “LQG-based tracking of multiple fluorescent particles in two-dimensions in a confocal microscope,” American Control Conference, pp. 2266-2271, 2009.
  3. S.B. Andersson and T. Sun, “Linear optimal control for tracking a single fluorescent particle in a confocal microscope,” Applied Physics B: Lasers and Optics, vol. 94, no. 3, pp. 403-409, 2009. link to journal
  4. S.B. Andersson, “Localization of a fluorescent source without numerical fitting,” Optics Express vol. 16, no. 23, pp. 18714-18724, 2008. link to journal
  5. S.B. Andersson, “Tracking a single fluorescent molecule with a confocal microscope,” Applied Physics B: Laser and Optics, vol. 80, no. 7, pp. 809-816, 2005. link to journal