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W1 spinning disk unit

Manufactured by Oxford Instruments
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The W1 Spinning Disk unit is a piece of lab equipment designed for fluorescence microscopy applications. It features a spinning disk that enables rapid image acquisition. The core function of the W1 Spinning Disk unit is to provide a platform for high-speed, low-phototoxicity fluorescence imaging.

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Lab products found in correlation

Ti e microscope, by Nikon (2 mentions) Ti e microscope, by Oxford Instruments (2 mentions) Catalase, by Merck Group (1 mentions) Glycerol, by Thermo Fisher Scientific (1 mentions) Glucose oxidase, by Merck Group (1 mentions) Trolox, by Merck Group (1 mentions) Cell tak, by Thermo Fisher Scientific (1 mentions)

4 protocols using w1 spinning disk unit

1

Quantifying Cellular GFP Expression

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Isoclonal populations were washed with 10 mL PBS solution and then immobilized on Cell-Tak coated 8-well chambered image dish. For the GFP standard curve, soluble eGFP (Cell Biolabs, San Diego, CA) standards (in PBS) of known concentration were imaged under the same conditions as cellular GFP. Both GFP standards, and cellular GFP were imaged on a Nikon Ti-E microscope equipped with a W1 Spinning Disk unit, an Andor iXon Ultra DU888 1k x 1k EMCCD camera and a Plan Apo VC 100x/1.4 oil objective in the UCSF Nikon Imaging Center, exposure time was 200 ms with 20% laser power. Approximately 10 xy locations were randomly selected for each isoclonal population. After background and auto-fluorescence subtraction from cellular GFP images, the cellular GFP concentration was calculated from the GFP standard curve (Figure S7A). Using the measured cellular volume and cellular GFP concentration, the absolute number of GFP molecules per cell was calculated. For a review on molecular counting see (Coffman and Wu, 2014 (link)).
Hansen M.M., Desai R.V., Simpson M.L, & Weinberger L.S. (2018). Cytoplasmic amplification of transcriptional noise generates substantial cell-to-cell variability. Cell systems, 7(4), 384-397.e6.
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2

Imaging Single mRNA and Transcriptional Centers

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To minimize photo bleaching, cells were imaged in a photo-protective buffer containing 50% glycerol (Thermo Fisher Scientific, Waltham, MA), 75 μg/mL glucose oxidase (Sigma Aldrich, Darmstadt, Germany), 520 μg/mL catalase (Sigma Aldrich, Darmstadt, Germany), and 0.5 mg/mL Trolox (Sigma Aldrich, Darmstadt, Germany). Images were taken on a Nikon Ti-E microscope equipped with a W1 Spinning Disk unit, an Andor iXon Ultra DU888 1k x 1k EMCCD camera and a Plan Apo VC 100x/1.4 oil objective in the UCSF Nikon Imaging Center. Approximately 10 xy locations were randomly selected for each isoclonal population. For each xy location, Nyquist sampling was performed by taking ~30, 0.4 um steps along the z-plane. The exposure times for TAMRA (100% laser power), and DAPI (50 % laser power) channels were 500 ms, and 50 ms for single mRNA analysis and 50 ms, and for transcriptional center (TC) analysis. For each z-plane in a 3-D stack images for both single mRNA analysis and TC analysis were taken.
Hansen M.M., Desai R.V., Simpson M.L, & Weinberger L.S. (2018). Cytoplasmic amplification of transcriptional noise generates substantial cell-to-cell variability. Cell systems, 7(4), 384-397.e6.
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3

Quantitative GFP Imaging of Isoclonal Cells

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Isoclonal populations were incubated with shield for 20 hours (if applicable). Approximately 6 x 105 cells were washed with 2 mL of PBS solution and then immobilized on a Cell-Tak (Fisher) coated 8-well chambered imaging dish, using the manufacturer’s protocol. Both soluble GFP standards and cellular GFP were imaged on a Nikon Ti-E microscope equipped with a W1 Spinning Disk unit, an Andor iXon Ultra DU888 1k x 1k EMCCD camera, and a Plan Apo VC 100x/1.4 oil objective in the UCSF Nikon Imaging Center; the exposure time was 500 ms with 50% laser power. Approximately 15 xy locations were randomly selected for each isoclonal population. After background and autofluorescence subtraction from the cellular GFP images, the cellular GFP concentration was determined from the GFP standard curve. The cellular volume was approximated from the measured cellular dimensions, assuming a spherically shaped cell.
Razooky B.S., Cao Y., Hansen M.M., Perelson A.S., Simpson M.L, & Weinberger L.S. (2017). Nonlatching positive feedback enables robust bimodality by decoupling expression noise from the mean. PLoS Biology, 15(10), e2000841.
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4

Imaging Single mRNAs and Transcriptional Centers

Cited 1 time
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Cells were imaged in a previously described buffer to minimize photo-bleaching (50% glycerol, 75 μg/mL glucose oxidase, 520 μg/mL catalase, and 0.5 mg/mL Trolox) (Waks et al., 2011 (link)). Images were taken on a Nikon Ti-E microscope equipped with a W1 Spinning Disk unit, an Andor iXon Ultra DU888 1k x 1k EMCCD camera and a Plan Apo VC 100x/1.4 oil objective in the UCSF Nikon Imaging Center. Approximately 10 xy locations were randomly selected for each condition. For each xy location, Nyquist sampling was performed by taking ~30, 0.4 μm steps along the z-plane. The exposure times for Quasar 670 (100% laser power), TAMRA (50% laser power), and DAPI (50% laser power) channels were 500 ms, 500 ms, and 50 ms for single mRNA analysis and 50 ms, 50 ms, and 50 ms for transcriptional center (TC) analysis. For each z-plane in a 3-D stack images for both single mRNA analysis and TC analysis were taken. Cells were segmented manually. DAPI image stacks were used for nuclear segmentation, spot/TC identification and counting was performed using in-house MATLAB programs (available upon request).
Hansen M.M., Wen W.Y., Ingerman E., Razooky B.S., Thompson C.E., Dar R.D., Chin C., Simpson M.L, & Weinberger L.S. (2018). A Post-Transcriptional Feedback Mechanism for Noise Suppression and Fate Stabilization. Cell, 173(7), 1609-1621.e15.
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