High-speed wide-field 3D images were acquired on a Nikon TiE microscope equipped with a 100× 1.49 NA TIRF objective and a Lumencor Spectra X (470-nm illumination) Photometrics 95B scientific CMOS (sCMOS) camera (Lumencor Inc., Beaverton, OR). Samples were mounted in a Tokai Hit environmental chamber (37°C with 5% CO2) and imaged as 3D stacks (20 frames/s, 0.8 s per stack,12-ms exposure, 15 z positions, 0.3-μm separation). An extended depth of focus projection was generated for each time point. No other processing was performed to images. During imaging of neurons, low magnification of the cells was used to distinguish neuronal axons from dendrites based on their established distinctive morphologies (e.g., identification of the axon initial segment that gives rise to the axon). Once identified, images of dendrites were subsequently acquired. Kymographs of the images were generated using NIS-Elements.
Ti e microscope
Manufactured by Lumencor
The Ti-E microscope is a high-performance laboratory microscope designed for advanced imaging applications. It features a motorized nosepiece, which allows for easy switching between multiple objectives, and a large, high-resolution display for detailed observation and analysis.
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Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Plan apo λ, by Nikon (4 mentions)
Sola se u nir light engine, by Lumencor (2 mentions)
Orca flash 4.0 v3 scmos camera, by Hamamatsu Photonics (2 mentions)
Perfect focus system, by Nikon (2 mentions)
Em ccd luca r camera, by Oxford Instruments (2 mentions)
Orca flash 4, by Hamamatsu Photonics (2 mentions)
Nis software, by Nikon (1 mentions)
6 protocols using ti e microscope
1
High-Speed 3D Imaging of Neurons
2
Fluorescence Microscopy for Cell Dynamics
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Fluorescence microscopy experiments were carried using a Nikon Ti-E microscope with CFI Plan Apochromat λ DM 100× objective, Lumencor Spectra X LED light source, Andor Zyla 4.2 CMOS camera, and a Lumencor SpectraX filter set. Images were collected with Nikon NIS software and analyzed using FIJI software (32 (link)), microbeTracker suite (33 (link)) and custom Matlab scripts and functions. Sample sizes are as follows: Figure 4C : - aTc t0 = 378 cells, t1 = 402, t2 = 732, t3 = 1100, t4 = 1993, + aTc t0 = 378, t1 = 567, t2 = 379, t3 = 398, t4 = 864. Figure 4D : - aTc t0 = 228, t1 = 140, t2 = 255, t3 = 375, t4 = 544, + aTc t0 = 228, t1 = 224, t2 = 158, t3 = 90, t4 = 170.
3
Multiplexed RNA and Protein Imaging
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We imaged RNA FISH samples on an inverted Nikon TI-E microscope equipped with a SOLA SE U-nIR light engine (Lumencor), an ORCA-Flash 4.0 V3 sCMOS camera (Hamamatsu), 20X Plan-Apo λ (Nikon MRD00205), 40X Plan-Fluor (MRH00401) and 60X Plan-Apo λ (MRD01605) objectives, and filter sets for DAPI, Cy3, Alexa Fluor 594, and Atto647N. For barcode ClampFISH and barcode HCR, we first acquired tiled images in a single Z-plane (scan) at 20X or 40X magnification, then, after identifying positions containing cells positive for resistant barcodes, we returned to those positions to acquire a Z-stack at 60X magnification. For subsequent rounds of single-molecule RNA FISH and ERK immunofluorescence, we acquired Z-stacks at 60X magnification. For scans, we used a Nikon Perfect Focus system to maintain focus across the imaging area.
4
Multiplexed RNA and Protein Imaging
5
Microscopy Techniques for Cell Imaging
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For all experiments except pH measurements, cells in the observation device were imaged using an inverted widefield microscope (Zeiss Axio Observer Z1). Fluorescence illumination was achieved using LED lights (precisExcite, CoolLed) and the light was collected using a ×63 (N.A. 1.4) objective and an EM-CCD Luca-R camera (Andor). Standard GFP and mCherry filters were used.
For experiments using the pHluorin cytosolic pH probe, a Nikon Ti-E microscope was used along with a LED light (Lumencor) fluorescence illumination system. The fluorescence was measured using two excitation wavelengths using a standard roGFP2 filter set (AHF, peak excitation wavelengths 390 /18 and 475/28 nm, beamsplitter 495 nm, and emission filter 525/50 nm). Emitted light was collected using a ×60 N.A. 1.4 objective and a CMOS camera (Hamamatsu Orca Flash 4.0).
We used motorized stages to follow up to 64 positions in parallel throughout the experiment. Single plane images were acquired every 15 , 30 , 60 , or 240 min depending on the phase of the culture (high sampling rate during FP versus lower acquisition frequency in SP) to limit photodamage.
For experiments using the pHluorin cytosolic pH probe, a Nikon Ti-E microscope was used along with a LED light (Lumencor) fluorescence illumination system. The fluorescence was measured using two excitation wavelengths using a standard roGFP2 filter set (AHF, peak excitation wavelengths 390 /18 and 475/28 nm, beamsplitter 495 nm, and emission filter 525/50 nm). Emitted light was collected using a ×60 N.A. 1.4 objective and a CMOS camera (Hamamatsu Orca Flash 4.0).
We used motorized stages to follow up to 64 positions in parallel throughout the experiment. Single plane images were acquired every 15 , 30 , 60 , or 240 min depending on the phase of the culture (high sampling rate during FP versus lower acquisition frequency in SP) to limit photodamage.
6
Multiparametric imaging of live cells
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For all experiments except pH measurements, cells in the observation device were imaged using an inverted widefield microscope (Zeiss Axio Observer Z1). Fluorescence illumination was achieved using LED lights (precisExcite, CoolLed) and the light was collected using a 63× (N.A. 1.4) objective and an EM-CCD Luca-R camera (Andor). Standard GFP and mCherry filters were used. For experiments using the pHluorin cytosolic pH probe, a Nikon Ti-E microscope was used along with a LED light (Lumencor) fluorescence illumination system. The fluorescence was measured using two excitation wavelengths using a standard roGFP2 filter set (AHF, peak excitation wavelengths 390nm/18nm and 475/28nm, beamsplitter 495nm, emission filter 525/50 nm). Emitted light was collected using a 60x N.A. 1.4 objective and a CMOS camera (Hamamatsu Orca Flash 4.0). We used motorized stages to follow up to 64 positions in parallel throughout the experiment. Single plane-images were acquired every 15 min, 30min, 60min or 240min depending on the phase of the culture (high sampling rate during FP versus lower acquisition frequency in SP) to limit photodamage.
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