Confocal imaging was performed on an A1R high speed laser scanning confocal system on a TiE inverted optical microscope platform (Nikon, Canada) with appropriate laser lines and filter sets. Transmitted light images were acquired on an inverted TiE microscope (Nikon, Canada) with phase contrast optics. Images were analyzed using ImageJ open access software (http://rsbweb.nih.gov/ij/ ). Brightness and contrast adjustments were the only manipulations performed to images.
Inverted tie microscope
Manufactured by Nikon
Sourced in Japan
The Inverted TiE microscope is a specialized laboratory instrument designed for optical microscopy. It features an inverted configuration, where the sample is placed above the objective lens, providing a unique perspective for observations. The core function of this microscope is to enable high-resolution imaging and analysis of specimens.
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7 protocols using inverted tie microscope
1
Confocal Microscopy for Imaging Analysis
2
Fluorescence Recovery After Photobleaching
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FRAP was performed as previously described (Maddox et al., 2000 (link); Molk et al., 2004 (link)). In brief, cells expressing either CDC10-GFP or GFP-2X(PHOsh2) were imaged at 25°C using an inverted TiE microscope (Nikon) equipped with a Xyla CMOS camera (Andor) and a 100× PlanApo 1.4 NA objective. Photobleaching was performed using a Coherent 488 nm Sapphire 50-mW laser through an LU4A laser module (Nikon) controlled by Nikon Elements software. Three prebleach images (600-ms exposure) were acquired to establish a baseline control for the initial fluorescence. A single focused 50-ms laser pulse was used to photobleach a Cdc10-GFP–containing horseshoe in sporulating cells (or, for GFP-2X(PHOsh2), a well-decorated portion of the PSM). The photobleaching treatment eliminated 75–95% of the original fluorescence. Immediately thereafter, single plane images (600-ms exposures) were acquired every 10 s for ∼2 min to follow any recovery. Such photobleaching experiments were performed on 15 different cells. Using ImageJ analysis software (National Institutes of Health), the observed fluorescence intensity values were corrected for background and photobleaching during image acquisition and displayed as the mean relative value for the bleached and unbleached regions for all 15 cells.
3
Multicolor Live-cell Imaging of Cellular Dynamics
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All images were captured by Volocity software (PerkinElmer) on an inverted TiE microscope (Nikon) attached to an UltraVIEW VOX system spinning-disc (PerkinElmer) with an EMCCD camera (Hamamatasu, C9100-13). For imaging, cells were grown on 35-mm glass-bottomed dishes. The microscope stage incubation chamber was maintained at 37 °C and 5% CO2. mVenus, mCerulean, RFP and DAPI were excited with 514-nm (25 mW), 440-nm (40 mW), 561 nm (50 mW) and 405 nm (50 mW) lasers, respectively. All channels were collected with a N.A. 1.25 TIRF 100×oil objective (Nikon CFI APO). An 8-μm z-stack of all channels with a step-size of 0.4 μm was acquired for all images. In the long-term time-lapse tracking of cell division, an 8-μm z-stack of both channels with a step-size of 0.4 μm was acquired every 15 min for ~12 h. To reduce the photo-damage effect on the cells, all 405, 561, and 514 nm lasers were set to 10% transmission.
4
Imaging Mitotic and Interphase eRPE1 Cells
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eRPE1 cells were grown on 35-mm glass bottom μ-dishes (ibidi, Cat#50-305-807). Cells were imaged in culturing media; 37°C and 5% CO2 was maintained using a cage incubator and a stage top chamber (OkoLab). Time-lapse z-stack images were captured on an inverted Ti-E microscope (Nikon) equipped with a CSU-X1 spinning disk confocal (Yokogawa), motorized XY stage with Z piezo (ASI), 4 line laser launch (Vortan), Lambda 10–3 emission filter wheel (Sutter), quad-band dichroic ZET 405/488/561/640x (Chroma), with Plan Apo VC 100x/1.4NA and Plan Apo VC 60x/1.3NA oil objectives, and a Photometrics Prime95B sCMOS camera (Teledyne). eRPE1 cells were imaged using a 488-nm laser and ET525/50m emission filter (Chroma). z-stacks were acquired every 20–30 sec for 1 hr for mitotic cells (Figs. 2 , 3 , 5 ) using the 100x/1.4NA objective, and every 15–30 min for up to 10 hours for interphase cells (Fig. 4 ) using the 60x/1.3NA objective. Image processing was conducted using Imaris software.
5
Cell Viability Assay Protocol
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To further investigate the viability of cells, a live/dead cell assay kit (Dojindo Laboratories, Kumamoto, Japan) was applied according to the manufacturer's instructions. RSC96 cells were cultured on various collagen membranes for 5 days before being examined. Membranes with cells seeded were washed with 1x PBS for 5 min and incubated with Calcein-AM 2 μM plus EthD-1 4 μM for 15 min at 37°C in the dark. After incubation, the resulting samples were then washed with 1x PBS for 5 min and then analyzed by an Inverted Ti–E microscope (Nikon, Japan).
6
High-Resolution Live-Cell Imaging Setup
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Images were acquired using a Nikon inverted TI‐E microscope via a coolsnap HQ2 camera. Commercially available software (Metamorph) controlled the stage, microscope, camera, and shutters. Fluorescent illumination was provided by a Sola Light Engine LED source (Lumencor). Temperature was kept at 37°C using an enclosed microscope chamber (Nikon) attached to a temperature sensitive heat exchanger. All experiments used a Phase 100x Plan Apo (NA 1.4) objective. Filter sets used were Chroma #41027 (mCh), Chroma #41028 (YFP), and Chroma #31044 v2 (CFP).
7
Immunofluorescence Staining of Oocytes
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Oocytes were permeabilized with 0.5% Triton X-100/PHEM (60 mM PIPES, 25 mM Hepes, pH 6.9, 10 mM EGTA, 8 mM MgSO 4 ) for five minutes, and then were fixed in 3.7% FPA in PHEM for 20 minutes at room temperature. After being washed with PBS/0.05% PVP (polyvinylpyrrolidone) three times at 10 minutes each, oocytes were blocked in blocking buffer (100 mM glycine and 1% BSA in PBS) for one hour at room temperature. Primary antibodies were then diluted in blocking buffer and oocytes were incubated in it overnight at 4.0 °C. Fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories) were used at 7.5 μg / ml. DNA was stained with 0.3 μg / ml Hoechst 33258. Next, buffers were were slowly and gentely removed from around the oocytes, which immobilized the oocytes on the slide; then a drop (about 5-10 μl) of anti-fade solution (0.25% n-propyl gallate and 90% glycerol in PBS)
was mounted onto the oocytes and oocytes were covered by a coverglass. To avoid the deformation of the oocytes, a double-stick tap was pre-placed between the slides and coverslips. Specimens were imaged with IQ2 on an Andor Revolution spinning-disk confocal system (Andor Technology PLC, Belfast, UK) mounted on an inverted TiE microscope (Nikon, Japan) with a 60 ×, 1.4 NA objective and captured with cold CCD camera (Andor). Most images are displayed as maximum intensity projections of the captured z stacks.
was mounted onto the oocytes and oocytes were covered by a coverglass. To avoid the deformation of the oocytes, a double-stick tap was pre-placed between the slides and coverslips. Specimens were imaged with IQ2 on an Andor Revolution spinning-disk confocal system (Andor Technology PLC, Belfast, UK) mounted on an inverted TiE microscope (Nikon, Japan) with a 60 ×, 1.4 NA objective and captured with cold CCD camera (Andor). Most images are displayed as maximum intensity projections of the captured z stacks.
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