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Tetraspeck bead

Manufactured by Thermo Fisher Scientific
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TetraSpeck beads are fluorescent microspheres used for calibration and quality control in microscopy and flow cytometry applications. They are available in multiple fluorescent colors and sizes to meet various experimental requirements.

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Spelling variants of this product

TetraSpeck (34 citations) TetraSpeck fluorescent beads (13 citations) Tetraspeck 100 nm beads (4 citations) TetraSpeck microsphere beads (3 citations) Multicolour tetraspeck beads (2 citations) 0.2 µm TetraSpeck™ beads (2 citations)

86 protocols using tetraspeck bead

1

Fluorescent Bead Imaging Protocol

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TetraSpeck beads (0.1 μm) (Thermo Fisher Scientific, Waltham, MA) were diluted in water and immobilized on imaging slides by addition of PBS buffer followed by quick washing with clean buffer. For measurement, an isolated bead was selected and a z-stack of 200 XY images was acquired with z-steps of 50 nm. Each XY image (256 × 256 pixels) was taken with a pixel resolution of 26.6 nm.
Angert I., Karuka S.R., Mansky L.M, & Mueller J.D. (2022). Partitioning of ribonucleoprotein complexes from the cellular actin cortex. Science Advances, 8(33), eabj3236.
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2

Single-particle Tracking via TIRFM

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Qdots, Cy3-labelled anti-IgM Fabs, and TetraSpeck beads (100 nm diameter; Thermo Fisher, catalogue #T7279) were diluted in MilliQ water, plated onto clean coverslips, and dried to obtain a dispersed layer of individually stuck particles. The coverslips were gently washed to remove any free-floating particles and the stuck particles were then imaged by TIRFM for 10 s at 33 Hz. Tracks generated from >300 adhered particles were analyzed to determine a distribution of their diameter, i.e. the maximum distance between any two points of the track. The 95th percentile value of this distribution was set as the ‘immobility threshold’, which was used to remove effectively immobile particles from each set of trajectories prior to conducting all analysis.
Abraham L., Lu H.Y., Falcão R.C., Scurll J., Jou T., Irwin B., Tafteh R., Gold M.R, & Coombs D. (2017). Limitations of Qdot labelling compared to directly-conjugated probes for single particle tracking of B cell receptor mobility. Scientific Reports, 7, 11379.
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3

Tetra-Speck Bead Characterization Protocol

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Tetra-Speck beads (0.75 μl from stock, catalog no. T7279, Thermo Fisher) were diluted in 360 μl H2O, mixed with 40 μl 1 M MgCl2 and put on a coverslip in a custom manufactured sample holder. After 10 min, the mix was replaced with 400 μl H2O. For experiments where the Si-Oil objective was used, the mix was replaced by 400 μl refractive index matched buffer consisting of 55% Glycerol (85%) and 45% H2O. Using Micro-Manager, about 20 positions on the coverslip were defined and the beads were imaged acquiring z-stacks (-1 μm to 1 μm, 10 nm step size) using the same filters as used in the intended experiment. In SMAP, an average PSF model was calculated from tens of beads30 (link).
Diekmann R., Kahnwald M., Schoenit A., Deschamps J., Matti U, & Ries J. (2020). Optimizing imaging speed and excitation intensity for single molecule localization microscopy. Nature methods, 17(9), 909-912.
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4

Immobilization of TetraSpeck Beads

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We used TetraSpeck beads (T7279; Thermo Fisher Scientific) with a diameter of ∼100 nm for image registration. To prepare the beads for imaging we added 10 µL of 1 mg/mL poly-D-lysine (P6407; Sigma) in Milli-Q water to the flow cell and incubated for 3 min, washed with 20 µL of BRB80 [80 mM Pipes (pH 6.8), 1 mM EGTA, and 1 mM MgCl2], and then added 10 µL of 1:1,000 diluted TetraSpeck beads in BRB80 and incubated for 5 min. Finally, we washed the flow cell with 40 µL of BRB80.
Niekamp S., Sung J., Huynh W., Bhabha G., Vale R.D, & Stuurman N. (2019). Nanometer-accuracy distance measurements between fluorophores at the single-molecule level. Proceedings of the National Academy of Sciences of the United States of America, 116(10), 4275-4284.
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5

Mapping Single-Molecule Fluorescence Dynamics

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SHREC experiments were performed as previously described (20 (link)). To generate a mapping, images of TetraSpeck beads (Thermo Fisher) were collected as previously described (21 (link)). A mapping was generated using collected bead images using the previously reported program codes (22 (link)). The mapping was used to map the location of fluorescent molecules in the Alexa 488 channel onto the mApple channel to confirm the correct mapping of bead images (Fig. S3B) and to calculate the distances of two motor domains of Myo10 labeled with mApple and Alexa 488. The point spread functions of single molecules were fitted into a two dimensional Gaussian to localize their centroids (23 (link), 24 ). Steps were determined based on a MATLAB program (developed in Ahmet Yildiz’s lab at University of California, Berkeley). After the steps were detected, step sizes were calculated as the displacements between neighbored averaged positions. The distances were calculated as Euclidean distances between two positions of two motor domains of Myo10. The multipeak Gaussian fitting to histograms of step sizes or intermotor distances were performed using the normalMixEM procedure of the mixtool package, a multipeak fitting tool programmed by R language (12 , 25 ).
Qin X., Yoo H., Man Cheng H.C., Nguyen Q.Q., Li J., Liu X., Prunetti L., Chen X., Liu T., Sweeney H.L, & Park H. (2020). Simultaneous tracking of two motor domains reveals near simultaneous steps and stutter steps of myosin 10 on actin filament bundles. Biochemical and biophysical research communications.
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6

Imaging Lipid Nanotubes with His-tagged Fluorescent Proteins

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Lipid nanotubes comprised of Galactose-ceramide:Ni-NTA DGS lipids (99:1 mol%) were formed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl buffer as previously reported54 (link) and labelled with His-tagged FPs by incubating the FP and lipid nanotubes at 4 °C for 1 hour.
Labelled lipid nanotubes were applied to the carbon support of freshly glow-discharged C-Flat grids (2/1-2C, Electron Microscopy Science, USA). Grids were then washed three times, to wash away unbound FPs, by blotting from the backside of the grid and quickly rehydrating with fresh buffer. In the last washing step 100 nm tetraspeck beads (ThermoFisher Scientific) were added. The grids were then illuminated with blue light to deactivate the FPs on the grids as described prior to blotting from the reverse side of the grid and plunge-freezing. Grids were stored in liquid nitrogen.
Tuijtel M.W., Koster A.J., Jakobs S., Faas F.G, & Sharp T.H. (2019). Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins. Scientific Reports, 9, 1369.
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7

Super-Resolution Imaging of Cellular Structures

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SIM images were taken with an N-SIM system (Nikon) attached to a Ti2-E inverted microscope (Nikon) with a CMOS camera (ORCA-Flash 4.0 V3; Hamamatsu Photonics) using a Plan Apochromat 100×/1.35 NA silicon-immersion objective lens at a step size of 0.12 μm. The chromatic aberration of the system was calibrated and corrected by 0.1 μm TetraSpeck beads (T7279; Thermo Fisher Scientific) in the mounting medium. Images were reconstructed and analyzed using NIS elements AR (Nikon) according to the manufacturer's protocol.
Tocan V., Hayase J., Kamakura S., Kohda A., Ohga S., Kohjima M, & Sumimoto H. (2021). Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl. The Journal of Biological Chemistry, 297(6), 101354.
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8

Fluorescent Bead-Based Image Registration

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To register the three channels, we used TetraSpeck beads (T7279; Thermo Fisher Scientific) based on a previously described protocol for two-color image registration (36 (link)). To this end, we prepared one of the three flow chambers of our flow cells with dynein (or with the DNA-origami nanoruler; SI Appendix, Fig. S1) and another flow chamber on the same flow cell with TetraSpeck. The beads were immobilized by adding 10 µL of 1 mg/mL Poly-d-lysine (P6407; Sigma) in Milli-Q water to the flow cell, followed by a 3-min incubation and a wash with 20 µL of BRB80 (80 mM Pipes [pH 6.8], 1 mM EGTA, and 1 mM MgCl2). Afterward, we added 10 µL of 1:300 diluted TetraSpeck beads in BRB80 and incubated for 5 min. Finally, the flow cell was washed with 40 µL of BRB80.
Niekamp S., Stuurman N., Zhang N, & Vale R.D. (2021). Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility. Proceedings of the National Academy of Sciences of the United States of America, 118(31), e2101391118.
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9

Multichannel Imaging and Spatial Bead Registration

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Each round of imaging included imaging with the 405-nm channel which included the DAPI stain of the cell along with imaging in the 647-nm, 561-nm and 488-nm channels of TetraSpeck beads’ (T7279, Thermo Fischer) and seqFISH+ probes. In addition, a pre-hybridization image was used to find all beads before the readouts were hybridized. Bead locations were fit to a 2D Gaussian. An initial estimate of the transformation matrix between the DAPI image for each serial hybridization round and the only beads image was found using imregcorr (Matlab). Using this estimate transformation, the bead coordinates were transformed to each serial hybridization image, where the location of the bead was again fit to a 2D gaussian. A final transformation matrix between each hybridization image and the only beads image was then found by applying fitgeotrans (Matlab) to the sets of Gaussian fit bead locations. For the tissue samples no beads were used and registration was based on DAPI alone.
Eng C.H., Lawson M., Zhu Q., Dries R., Koulena N., Takei Y., Yun J., Cronin C., Karp C., Yuan G.C, & Cai L. (2019). Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+. Nature, 568(7751), 235-239.
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10

Polarity Site Tracking and Analysis

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Polarity site tracking was performed as in [21 (link)] using Volocity software (Quorum Technologies Inc., Puslinch, ON, Canada). The 3D centroid of each polarity site was located by thresholding the Spa2-mCherry signal. Centroids were tracked until the Spa2-mCherrry signal overlapped with the Spa2-mCherry signal of the partner’s polarity site. Fluorescent 0.2 µm TetraSpeck beads (Thermo Fisher Scientific, Waltham, MA, USA) were added to the slab to account for stage drift; the centroid of the bead was used as an origin to track the centroid of the polarity site at each timepoint. Analysis excluded timepoints where no polarity site was detected or where multiple polarity sites were detected.
Jacobs K.C, & Lew D.J. (2022). Pheromone Guidance of Polarity Site Movement in Yeast. Biomolecules, 12(4), 502.
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