Fluorescent flatmount images were acquired with Nikon Ti inverted widefield microscope with a Prior ProScanIII motorized stage. The objective used was Plan Apo Lambda 10 x/0.45 Air DIC N1 objective, and the camera used was Hamamatsu ORCA-Flash 4.0 V3 Digital CMOS camera. Fluorescent retina section images were acquired with W1 Yokogawa Spinning disk confocal microscope with 50 µm pinhole disk and 488, 561, and 640 laser lines. The objectives used were either Plan Apo 20 x/0.75 air or Plan Apo 60 x/1.4 oil objectives, and the camera used was Andor Zyla 4.2 Plus sCMOS monochrome camera. Nikon Elements Acquisition Software (AR 5.02) was used for image acquisition and Fiji or Adobe Photoshop CS6 was used for image analysis.
Ti inverted widefield microscope
Manufactured by Nikon
The Nikon Ti Inverted Widefield microscope is a laboratory equipment designed for microscopy applications. It features a widefield optical system and an inverted configuration, allowing for versatile sample observation and handling. The core function of this microscope is to provide high-quality, wide-field imaging capabilities for various scientific and research applications.
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Lab products found in correlation
Gel loading pipette tips, by Thermo Fisher Scientific (2 mentions)
1 j1 camera, by Nikon (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Collagen 1, by Corning (2 mentions)
Nis elements imaging software, by Nikon (2 mentions)
Proscan 3, by Nikon (1 mentions)
Orca flash4.0 v3 digital cmos camera, by Hamamatsu Photonics (1 mentions)
4.2 plus scmos monochrome camera, by Oxford Instruments (1 mentions)
Lsm 710 confocal scanning microscope, by Zeiss (1 mentions)
Hoechst stain, by Thermo Fisher Scientific (1 mentions)
Elements software, by Nikon (1 mentions)
5 protocols using ti inverted widefield microscope
1
Fluorescent Retina Imaging Protocols
2
Retinal Imaging and Cone Analysis
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Retinal flat mounts were imaged using a Nikon Ti inverted wide-field microscope (×10 or ×20 air objective). Retinal cross-sections were imaged using a Zeiss LSM710 scanning confocal microscope (×20 air objective or ×40 oil objective). All image analysis was performed using ImageJ (NIH). To quantify GFP-positive or cone arrestin–positive cones, custom ImageJ modules were used as previously described (9 (link), 10 ). For each flat mount, the locations of the optic nerve head and 4 retinal leaflets were first manually defined. The number of GFP-positive or cone arrestin–positive objects within the region corresponding to the central retina was then automatically counted and used to represent the number of cones in that sample.
3
Embedding Organoids in Collagen Droplets
4
Live-Imaging of Biofilm Formation
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Live-imaging experiments were performed based on methods previously described for P. aeruginosa biotic biofilms (17 (link)). Briefly, CF AECs were seeded onto glass coverslips and cultured for 7 days. For virus coinfection studies, CF AECs were infected with RSV for 24 h prior to addition of bacteria. For imaging, control or RSV-infected CF AECs on coverslips were transferred to FCS2 live-cell imaging chambers (Bioptechs), stained with Hoechst stain (Molecular Probes), and then infected with S. aureus USA100 expressing GFP. Bacteria were allowed to attach for 1 h without flow and then grown for up to 6 h in MEM under flow conditions (rate of 50 ml/h). Images from 5 fields per chamber were acquired at desired time points on a Nikon Ti inverted wide-field microscope. Bacterial biomass was quantified with Nikon Elements software.
5
Embedding Organoids in Collagen Droplets
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To embed organoids in collagen droplets, a sheet of Parafilm was soaked in 70 % ethanol, air dried, and pressed against the holes of a box of gel-loading pipette tips (1-200 μl, Fisher Scientific 02-707-138) to create a dimpled mold31 (link). One organoid was placed in each dimple and 30 μL of 7 mg/mL collagen I (Corning) was added. The droplets were incubated 25 minutes at 37°C and carefully resuspended in 3 ml of RB media in an untreated 6-well plate. The media was changed weekly and the droplets were imaged after 2 weeks of incubation using a Nikon Ti Inverted Widefield microscope and a Nikon 1 J1 Camera. Droplet diameters were measured using NIS Elements imaging software (Nikon) and normalized to the diameter of empty droplets from the same set, for a total of three sets. Droplets that failed to undergo compaction (∼ 20 % in control and ∼ 50 % in PKD samples) were excluded. Droplets were fixed with 4% paraformaldehyde for 20 minutes at room temperature, incubated 16 hours in 30 % sucrose (Sigma) in water, mounted in Tissue-Tek (Sakura), flash frozen, and cryosectioned onto SuperfrostPlus slides (Fisher). Sections were stained in Picro-sirius red solution (Sky-Tek laboratories) for one hour, rinsed in two changes of 0.5% acetic acid solution, and dehydrated in two changes of absolute ethanol before mounting. Immunofluorescence was performed as described below.
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