hiPSC-CMs were imaged in an onstage microscope incubator at 37 °C and 5% CO2 to maintain standard physiological conditions on a Nikon Ti-E inverted microscope with Andor Zyla 4.2+ digital CMOS camera. Videos of the beating hiPSC-CMs were recorded at 100 fps over 20 seconds in bright-field, and exported as a series of single-frame image files. The image series were then analyzed using in-house and open source motion tracking software19 (link) that calculates motion vectors based on block matching of macroblocks of pixels from one frame to the next. The software then generates motion waveforms that represent the contractile physiology of hiPSC-CMs.
Ti e inverted microscope
Manufactured by Oxford Instruments
The Ti-E inverted microscope is a high-performance optical microscope designed for advanced imaging applications. It features a robust and stable construction, allowing for precise and reliable observations. The Ti-E provides users with a core function of magnifying and illuminating samples for detailed analysis and investigation.
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Lab products found in correlation
Ultra 888 emccd camera, by Oxford Instruments (3 mentions)
Scmos digital camera, by Oxford Instruments (3 mentions)
Csu x1 spinning disk confocal system, by Nikon (2 mentions)
Vectashield medium, by Vector Laboratories (2 mentions)
Glass bottom plates, by USA Scientific (2 mentions)
Celldiscoverer 7 microscope, by Nikon (2 mentions)
Neo scmos camera, by Oxford Instruments (2 mentions)
Matrigel, by Corning (2 mentions)
Csu w1 confocal scanning unit, by Nikon (2 mentions)
Ultra 897 emccd camera, by Oxford Instruments (2 mentions)
Rmo270, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (2 mentions)
Alexa 568 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Air objective, by Leica Microsystems (1 mentions)
Andor neo scmos camera, by Oxford Instruments (1 mentions)
Metamorph, by Universal Imaging (1 mentions)
Csu wd, by Oxford Instruments (1 mentions)
Plan apo, by Oxford Instruments (1 mentions)
29 protocols using ti e inverted microscope
1
Imaging and Analysis of hiPSC-CMs
2
Epifluorescence and TIRF Microscopy Protocol
3
Immunofluorescence Staining Protocol
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After growth and treatment as indicated on glass‐bottom 96‐well plates, cells were fixed for 30 min at room temperature with a freshly prepared solution of 12% paraformaldehyde in PBS and permeabilized with 1% Triton X‐100. Samples were then stained with primary and secondary antibodies in PBS + 0.1% Triton X‐100 + 2% bovine serum albumin, and images were captured on a Nikon Ti‐E inverted microscope with a 20×/0.75 NA objective with an Andor Zyla 5.5 scMOS camera.
4
Live-cell Imaging of T. cruzi Amastigotes and Epimastigotes
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Live-cell imaging of SMP1-1–GFP-expressing T. cruzi was performed as follows.
(i) Amastigotes. Approximately 2 × 106 freshly isolated intracellular T. cruzi amastigotes in 150 μL of prewarmed imaging medium were placed directly on the glass coverslip of a 35-mm glass-bottom dish and allowed to settle at 37°C in a 5% CO2 incubator for 10 min. Seventy-five microliters of medium was removed prior to placing the dish on the microscope for imaging.
(ii) Epimastigotes. Approximately 2 × 105 epimastigotes in 150 μL of LIT growth medium were placed directly on the glass coverslip of a 35-mm glass-bottom dish and allowed to settle prior to imaging. Parasites were imaged using a Yokogawa CSU-X1 spinning disk confocal system paired with a Nikon Ti-E inverted microscope and an iXon Ultra 888 EMCCD camera (100× objective). Image processing, analysis, and display were performed using ImageJ Fiji software (55 (link)).
5
Mapping Actin and Keratin Dynamics
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Cells were transfected with Lifeact-GFP and keratin 18-mCherry and seeded on PRIMO micropatterned PDMS, as described previously. Images were taken every 4 s using a Nikon TiE inverted microscope with a spinning-disc confocal unit (CSU-WD, Yokogawa) and a Zyla scientific complementary metal–oxide–semiconductor camera (Andor) controlled by µManager71 (link),72 (link) using a ×60 objective (Plan Apo; NA, 1.2; water-immersion type). The local velocity fields of the actin and keratin fluorescence signals were measured by comparing each frame and its previous time point with a custom-made PIV software in MATLAB. A mask of each cell was drawn with respect to the F-actin signal in ImageJ73 (link). A radial coordinate, centred in the mask centroid, was assigned to each PIV data point, and normalized by the local radius of the cell-mask contour. Likewise, the local velocity fields were decomposed into their radial and tangential components. The distributions of the total and radial velocities inside each cell were then binned into equal-sized intervals of the normalized radial coordinate. The average total and radial velocity for each radial bin was then calculated.
6
Fluorescence Imaging of LAF-1 Condensates
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Fluorescence imaging was performed on an Nikon Ti-E inverted microscope equipped with an Andor iXon 897E EM-CCD camera, a Yokugawa CSU-X1 Spinning Disk Confocal Scan Head, and an Andor FRAPPA photomanipulation system. A 100x Nikon objective with a numerical aperature of 1.4 was used for all fluorescence imaging. LAF-1 condensates were formed in buffer containing 20 mM Tris pH 7.4 and 200 mM NaCl, along with 0.075 mg/mL PolyU RNA of various lengths and variable nucleotide concentration. Droplets were incubated for 8 minutes at room temperature and then transferred into a PDMS chamber adhered to a slide and pre-passivated with 2% w/v Pluoronic F-127. For FRAP experiments, a spot size of 3 pixels, corresponding to roughly 0.42 μm, was used for photobleaching.
Analysis of FRAP timecourses was performed using custom packages written in MATLAB and incorporated feature finding scripts from Maria Kilfoil’s MATLAB Point Tracking Software (Supplementary Information ).
Analysis of FRAP timecourses was performed using custom packages written in MATLAB and incorporated feature finding scripts from Maria Kilfoil’s MATLAB Point Tracking Software (
7
Immunofluorescence Labeling of Parasites
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Parasites were fixed in 1% PFA-PBS for 10 min, spotted onto poly-L-lysine coated slides and allowed to adhere for 30 min. Adhered parasites were washed with PBS and permeabilized with 0.1% Triton X-100-PBS for 10 min, rewashed again with PBS, and blocked with 3% BSA (Sigma-Aldrich) in PBS for 1 h. Then, parasites were incubated with anti-BIP antibodies in 1% BSA in PBS for 1 h (a generous gift from Dr J. D. Bangs, U. Buffalo) and washed three times with PBS before incubation for 1 h with goat anti-rabbit IgG secondary antibody Alexa fluor 594 (Thermo Fisher Scientific), and 100 ng/ml DAPI (Sigma-Aldrich). Finally, parasites were washed with PBS and mounted with ProLong Antifade (Thermo Fisher Scientific). EPIs were analyzed using a Yokogawa CSU-X1 spinning disk confocal system paired with a Nikon Ti-E inverted microscope equipped with an iXon Ultra 888 EMCCD camera. All images were acquired with the 100× objective and image processing was completed using ImageJ Fiji (https://fiji.sc ) (57 (link)).
8
Fluorescence Microscopy Imaging Protocol
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For microscopy, cells were grown to log phase in SD supplemented with appropriate amino acids mixes, and then briefly pelleted and resuspended to concentrate prior to imaging. Images for Fig 2 were captured using a DeltaVisionTM Elite system (GE Healthcare Life Sciences), equipped with an Olympus IX-71 inverted microscope, a sCMOS CAMERA, A 100x/1.4 Oil Super-Plan APO objective, and a DeltaVision Elite Standard Filter Set with the FITC filter (excitation:475/28, emission:525/48) for GFP and the TRITC filter (excitation: 542/27, emission: 594/45) for mCh. Image acquisition and deconvolution were performed with the program softWoRx. All other fluorescence imaging, was performed on a Nikon Ti-E inverted microscope with a 1.4 numerical aperture, 100× oil-immersion objective and an Andor iXon3 EMCCD camera. A Lumencor LED light engine was used for fluorophore excitation. Image acquisition was performed the MetaMorph acquisition software. For each field, 10-slice Z-stacks (0.25 μm apart) were captured. For image cropping and other figure preparation steps, the ImageJ version 1.50i (National Institutes of Health) software was used.
9
Cardiac Organoid Imaging and Analysis
10
Immunofluorescence Imaging of Trypanosoma cruzi
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Trypanosoma cruzi epimastigotes expressing CYP51::GFP or ∆CYP51 epimastigotes were fixed in 1% paraformaldehyde and permeabilized with TritonX-100. Parasites were placed on poly-L-lysine coated slides and stained with a rabbit α-BiP primary antibody (gift from Jay Bangs, 1:1,000 dilution; Bangs et al., 1993 (link)), and a goat α-rabbit secondary antibody conjugated to Alexa Fluor 647 (Invitrogen, Waltham, MA, United States of America 1:1,000 dilution). DAPI (Thermo Scientific, Waltham, MA, United States of America (1:5,000 dilution, 1 mg/ml stock) was used to identify parasite DNA. Parasites were mounted in ProLong Diamond (Thermo Fisher, Waltham, MA, United States of America) and cured for 24 h. Parasites were imaged on a Yokogawa CSU-X1 spinning disk confocal system paired with a Nikon Ti-E inverted microscope and an iXon Ultra 888 EMCCD camera. The 100x lens was used for imaging, and image processing, analysis, and display were completed in FIJI (Schindelin et al., 2012 (link)).
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