Perfect focus system

Manufactured by Oxford Instruments

The Perfect Focus system is a laboratory equipment designed to maintain the focus of microscope images during long-term observations. It continuously monitors and adjusts the focus to compensate for drift, maintaining a stable and consistent image throughout the experiment.

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Lab products found in correlation

Ultra 897 emccd camera, by Oxford Instruments (2 mentions) Eclipse ti inverted microscope, by Oxford Instruments (2 mentions) Diskovery tirf borealis widefield illuminator, by Oxford Instruments (2 mentions) Diskovery platform, by Oxford Instruments (2 mentions) Ti spinning disk confocal microscope, by Nikon (1 mentions) Emccd camera, by Oxford Instruments (1 mentions) Csu x1, by Yokogawa (1 mentions) Apo tirf objective, by Nikon (1 mentions) Ti e microscope, by Nikon (1 mentions) Neo scmos camera, by Oxford Instruments (1 mentions) Ixon ultra 888 emccd camera, by Oxford Instruments (1 mentions) Ti e inverted wide field fluorescence microscope, by Excelitas (1 mentions) Csu x1 spinning disk, by Nikon (1 mentions)

Close spelling variants of this product

Perfect Focus 3 system (3 citations) Ti-E perfect focus system (2 citations)

8 protocols using perfect focus system

Quantifying Single Molecule Dynamics

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Cells were grown in 96-well glass bottom dishes and were imaged on an inverted Nikon TI spinning disk confocal microscope with the Nikon Perfect Focus system, 100× 1.45 NA objective, an Andor EM-CCD camera, and Micro-Manager software (Edelstein et al., 2010 ). Images in Figures 4A,C were acquired using widefield illumination, a 20x air objective, and a Hamamatsu CMOS Flash 4.0 camera.
To determine the number of antibodies bound to a single peptide array, a sfGFP-linker-mCherry fusion protein was imaged using image acquisition parameters for which GFP and mCherry fluorescence intensities were equal. Using the same acquisition parameters, fluorescence intensity ratio of the mito-mCherry-peptide array and GFP-tagged antibody was determined.
To measure fluorescence intensities, a circular region of interest (ROI) with a diameter of 0.5 µm was applied and an average background (same ROI measured in five different areas of the cell that did not contain a fluorescent foci) was subtracted. Kinesin run lengths and speeds were determined by kymographs analysis (Sirajuddin et al., 2014 ). Single molecule tracking was performed in 2D using Fiji Trackmate. MSD plots were generated in Matlab using the X,Y tracking coordinates generated by Trackmate. Diffusion coefficients were calculated based on MSD plots.

Tanenbaum M.E., Gilbert L.A., Qi L.S., Weissman J.S, & Vale R.D. (2014). A protein tagging system for signal amplification in gene expression and fluorescence imaging. Cell, 159(3), 635-646.

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Spinning Disk Confocal Imaging of mRNA Foci

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Cells were grown in 96-well glass bottom dishes (Matriplate, Brooks). Images were acquired using a Yokogawa CSU-X1 spinning disk confocal attached to an inverted Nikon TI microscope with Nikon Perfect Focus system, 100× NA 1.49 objective, an Andor iXon Ultra 897 EM-CCD camera, and Micro-Manager software (Edelstein et al., 2010 ). Single z-plane images were acquired every 30 s unless noted otherwise. During image acquisition, cells were maintained at a constant temperature of 36°C–37°C. Camera exposure times were generally set to 500 ms, unless noted otherwise. We note that stable expression of PP7-mCherry, either with or without the CAAX domain, also resulted in an accumulation of mCherry signal in lysosomes, but lysosomes could be readily distinguished from mRNA foci based on signal intensity and mobility.

Yan X., Hoek T.A., Vale R.D, & Tanenbaum M.E. (2016). Dynamics of Translation of Single mRNA Molecules In Vivo. Cell, 165(4), 976-989.

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High-Resolution TIRF Microscopy Imaging

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Total internal reflection fluorescence microscopy (TIR‐FM) was performed as previously described.⁶³ Briefly, cells were mounted in PBS and imaged using a 60×, 1.49 NA APO TIRF objective (Nikon) mounted on a fully motorized Nikon Ti‐Eclipse inverted microscope with Perfect Focus System and coupled to an Andor “Diskovery TIRF/Borealis widefield illuminator” equipped with an additional 1.8× tube lens (yielding a final magnification of ×108). TIR‐FM illumination was achieved using a Diskovery Platform (Andor Technology). For live cell experiments, cells were maintained at 37°C during imaging. Imaging sequences were acquired using a sCMOS camera with 6.5 μm pixel size (pco.edge).

Lakoduk A.M., Kadlecova Z, & Schmid S.L. (2020). A functionally neutral single chain antibody to measure beta‐1 integrin uptake and recycling. Traffic (Copenhagen, Denmark), 21(9), 590-602.

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Live-cell Imaging with Nikon Ti-E Microscope

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An inverted Nikon Ti-E microscope was controlled using Micromanager (Edelstein et al., 2014 (link)). The microscope was connected to a pre-equilibrated heating chamber set to 37°C and equipped with a Perfect Focus System, a Heliophor light engine (89 North) and an Andor sCMOS Neo camera. We utilized two objective lenses: a CFI Plan Apo Lambda x40 0.95 air objective lens; and a CFI Plan Apo Lambda x60 1.40 air objective lens. Acquisitions were performed on GFP and Cy5 channels as appropriate.

Kan W., Enos M.D., Korkmazhan E., Muennich S., Chen D.H., Gammons M.V., Vasishtha M., Bienz M., Dunn A.R., Skiniotis G, & Weis W.I. (2020). Limited dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction. eLife, 9, e55015.

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Live Cell Imaging with TIR-FM

Cited 1 time

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Total internal reflection fluorescence microscopy (TIR-FM) was performed as previously described.^{63 (link)} Briefly, cells were mounted in PBS and imaged using a 60×, 1.49 NA APO TIRF objective (Nikon) mounted on a fully motorized Nikon Ti-Eclipse inverted microscope with Perfect Focus System and coupled to an Andor “Diskovery TIRF/ Borealis widefield illuminator” equipped with an additional 1.8× tube lens (yielding a final magnification of ×10⁸). TIR-FM illumination was achieved using a Diskovery Platform (Andor Technology). For live cell experiments, cells were maintained at 37°C during imaging. Imaging sequences were acquired using a sCMOS camera with 6.5 μm pixel size (pco.edge).

Lakoduk A.M., Kadlecova Z, & Schmid S.L. (2020). A functionally neutral single chain antibody to measure beta‐1 integrin uptake and recycling. Traffic (Copenhagen, Denmark), 21(9), 590-602.

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Quantitative CRISPR Imaging Protocols

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CRISPR imaging data were acquired on a Nikon Ti-E inverted wide-field fluorescence microscope equipped with a ×100 NA 1.40 PlanApo oil immersion objective, an LED light source (Excelitas X-Cite XLED1), an sCMOS camera (Hamamatsu Flash 4.0), and a motorized stage (ASI) with stage incubator (Tokai Hit). Acquisitions were controlled by MicroManager. All images were taken as z stacks at 0.4 μm steps and with a total of 15 steps and were projected in maximum intensity. Long-term live cell imaging was performed on Andor Dragonfly (high-speed confocal microscopy) based on Nikon TI microscope with Nikon Perfect Focus system, ×60 NA 1.40 objective, an Andor iXon Ultra 888 EM-CCD camera. Images were taken as z stacks at 0.5 μm steps (7 steps) and at a frame rate of 5 Hz. Interval time was set as 10 min. Cells were imaged for 4–6 h. During image acquisition, cells ware maintained at constant temperature of 37 °C and 5% CO₂ within an incubation box. All the fluorescence imaging data were analyzed by Image J. Signal-to-noise ratio was defined as the ratio of the intensity of a fluorescent signal and the power of background noise as following formula:

SNR = \frac{P_{signal}}{P_{noise}} = \frac{Max intensity of GFP spot - Mean intensity of background GFP}{Std. dev. of background signal}

Chen B., Zou W., Xu H., Liang Y, & Huang B. (2018). Efficient labeling and imaging of protein-coding genes in living cells using CRISPR-Tag. Nature Communications, 9, 5065.

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Confocal Imaging of DNA Damage Response

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All imaging datasets were acquired using a confocal spinning disk microscope (Andor Dragonfly) coupled to a Nikon Ti-2 inverted epifluorescence microscope with automated stage control, Nikon Perfect Focus System, and a Zyla PLUS 4.2Megapixel USB3 camera. Illumination was done with 100mW 405nm, 50mW 488nm, 50 mW 561nm, 140mW 640nm and 100mW 785nm solid state lasers. All hardware was controlled using Andor Fusion software. All images were taken with a NA 0.80 CFI60 Plan Apochromat Lambda D 20x objective (Nikon MRD70270), except for the foci features taken in the DDR364 base-editing screens. Foci features (RAD51, BRCA1, RPA2, γH2AX, 53BP1 and RAD18) and EPCAM in the DDR364-irradaition screen and foci features (RAD51, γH2AX, and RPA2) and EPCAM in the DDR364-chemo screen were taken with a NA 1.4 CFI60 Plan Apochromat Lambda 60x oil immersion objective (Nikon MRD01605). Lasers, laser powers, exposure times, objectives and experiment-specific acquisition parameters are summarized in Supplementary Table 4 & 5. Images were acquired with 4 z-slices at 1.5 μm intervals for the cultured cells and with 6 z-slices at 1.5 μm intervals for the tissue sections unless otherwise specified.

Gu J., Iyer A., Wesley B., Taglialatela A., Leuzzi G., Hangai S., Decker A., Gu R., Klickstein N., Shuai Y., Jankovic K., Parker-Burns L., Jin Y., Zhang J.Y., Hong J., Niu S., Chou J., Landau D.A., Azizi E., Chan E.M., Ciccia A, & Gaublomme J.T. (2023). CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue. bioRxiv.

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Quantifying mRNA Dynamics in Live Cells

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Cells were grown in 96-well glass bottom dishes (Matriplate, Brooks). Images were acquired using a Yokogawa CSU-X1 spinning disk confocal attached to an inverted Nikon TI microscope with Nikon Perfect Focus system, 100x NA 1.49 objective, an Andor iXon Ultra 897 EM-CCD camera, and Micro-Manager software (Edelstein et al., 2010 ). Single z-plane images were acquired every 30 s unless noted otherwise. During image acquisition, cells were maintained at a constant temperature of 36°C–37°C. Camera exposure times were generally set to 500 ms, unless noted otherwise. We note that stable expression of PP7-mCherry, either with or without the CAAX domain, also resulted in an accumulation of mCherry signal in lysosomes, but lysosomes could be readily distinguished from mRNA foci based on signal intensity and mobility.

Yan X., Hoek T.A., Vale R.D, & Tanenbaum M.E. (2016). Dynamics of Translation of Single mRNA Molecules In Vivo. Cell, 165(4), 976-989.

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