Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 May 21;6(6):2181-90.
doi: 10.1364/BOE.6.002181. eCollection 2015 Jun 1.

Electrically tunable lens speeds up 3D orbital tracking

Paolo Annibale  1 , Alexander Dvornikov  1 , Enrico Gratton  2
Affiliations

Electrically tunable lens speeds up 3D orbital tracking

Paolo Annibale et al. Biomed Opt Express. .

Abstract

3D orbital particle tracking is a versatile and effective microscopy technique that allows following fast moving fluorescent objects within living cells and reconstructing complex 3D shapes using laser scanning microscopes. We demonstrated notable improvements in the range, speed and accuracy of 3D orbital particle tracking by replacing commonly used piezoelectric stages with Electrically Tunable Lens (ETL) that eliminates mechanical movement of objective lenses. This allowed tracking and reconstructing shape of structures extending 500 microns in the axial direction. Using the ETL, we tracked at high speed fluorescently labeled genomic loci within the nucleus of living cells with unprecedented temporal resolution of 8ms using a 1.42NA oil-immersion objective. The presented technology is cost effective and allows easy upgrade of scanning microscopes for fast 3D orbital tracking.

Keywords: (110.0110) Imaging systems; (180.0180) Microscopy.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
a) Picture and schematics of the casing of the electrical lens, that allows it to be installed at the rear of any RMS objective. b) The focal length changes with the Current, with a slope depending on the magnifying power of the objective. A 20x objective yields an axial field of view of over one mm, which reduces to 200 μm if a 60x objective is used.
Fig. 2
Fig. 2
Comparative performances of an objective piezo-stage and of the electrical lens. a) Schematics of the orbital tracking configuration. A full 3D tracking period is achieved by performing 4 radial periods: 2 above and 2 below the particle. b) Piezo and lens performance compared for an orbit period of 8.192 ms. The up-down period for orbital tracking 32.77 ms. Comparing the displacement of the focal plane achieved by movement of the objective (Piezo, blue line, 2) and by the use of the ETL (Lens, red line, 1) c) Comparing piezo and lens performance for an orbital period of 2.048 ms. d) Demodulation of the piezo response for decreasing orbit period. The ideal square wave response turns to a triangular wave response, as the device is not able to perform the entire axial excursion in the required time e) Lens focal offset of ETL response for increasing axial frequency (i.e. decreasing orbit period, as indicated in the top axis). A resonance at about 500Hz is observed.
Fig. 3
Fig. 3
a) Reconstructed trajectory of a 1 μm fluorescent bead moved in a periodic 3D spiral pattern by the stepper-motor stage over a FOV of 15 μm (x-y) and 30 μm (z). Axial tracking is performed using the objective piezo-stage. b) Reconstructed trajectory of a 1 μm fluorescent bead moved in a periodic 3D spiral pattern by the stepper-motor stage over a FOV of 15 μm (x-y) and 30 μm (z). Axial tracking is performed using the ETL. For both experiments reported in a and b a 40x 0.8 NA water objective was used. c) Frequency distribution (log-scale) of the measured distance between scanners position and localized particle position for the test trajectories generated in a and b. d) Sedimentation profile of a 15 μm fluorescent bead over an axial range of 350 μm followed using the electrical lens and a 20x 0.4NA objective.
Fig. 4
Fig. 4
Bottom-up Picture of Arabidopsis Thaliana plants rooting in an agar plate with a glass-bottom. b) 3D reconstruction of a portion of the root from a 2-photon excitation z-stack performed by using 750 nm excitation. c) Trajectory of the root axis reconstructed from 3D orbital particle tracking, performed by ramping the position of the collimated laser beam along the axial direction using the ETL over a range exceeding 400 μm. d) Z-Section of a root highlighting a root hair protrusion. e) Pattern of the rosette orbit performed in the XZ plane to reconstruct the shape of the root hair. f) nSpiro reconstruction of the root hair. All experiments were performed with a 20x 0.4NA objective.
Fig. 5
Fig. 5
a) Trajectory of two fluorescently labeled DNA loci tracked in 3D using a 60x 1.42 NA oil immersion objective in combination with the ETL. b) Mean Squared Displacement (MSD) of the distance difference between the two loci.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dupont, Lamb D. C., “Nanoscale three-dimensional single particle tracking,” Nanoscale 3(11), 4532–4541 (2011).10.1039/c1nr10989h - DOI - PubMed
    1. Chenouard N., Smal I., de Chaumont F., Maška M., Sbalzarini I. F., Gong Y., Cardinale J., Carthel C., Coraluppi S., Winter M., Cohen A. R., Godinez W. J., Rohr K., Kalaidzidis Y., Liang L., Duncan J., Shen H., Xu Y., Magnusson K. E., Jaldén J., Blau H. M., Paul-Gilloteaux P., Roudot P., Kervrann C., Waharte F., Tinevez J. Y., Shorte S. L., Willemse J., Celler K., van Wezel G. P., Dan H. W., Tsai Y. S., Ortiz de Solórzano C., Olivo-Marin J. C., Meijering E., “Objective comparison of particle tracking methods,” Nat. Methods 11(3), 281–289 (2014).10.1038/nmeth.2808 - DOI - PMC - PubMed
    1. Ragan T., Huang H., So P., Gratton E., “3D particle tracking on a two-photon microscope,” J. Fluoresc. 16(3), 325–336 (2006).10.1007/s10895-005-0040-1 - DOI - PubMed
    1. Enderlein J., “Tracking of fluorescent molecules diffusing within membranes,” Appl. Phys. B 71(5), 773–777 (2000).10.1007/s003400000409 - DOI
    1. Kis-Petikova K., Gratton E., “Distance measurement by circular scanning of the excitation beam in the two-photon microscope,” Microsc. Res. Tech. 63(1), 34–49 (2004).10.1002/jemt.10417 - DOI - PubMed

LinkOut - more resources