All images were captured using an inverted fluorescence microscope (Eclipse Ti-E, Nikon) equipped with either an electron-multiplying charge-coupled device camera (ImagEM C9100-13, Hamamatsu; or iXon Ultra 897, Andor) or a scientific complementary metal-oxide-semiconductor camera (ORCA-flash 4.0 C11440-22C, Hamamatsu). We used a light-emitting diode light source (SPECTRA X Light Engine, Lumencor) to provide illumination with filter sets (from Semrock or Nikon): (i) excitation (Ex), 390/40 nm; dichroic, 405 nm; emission (Em), 452/45 nm (for DiFMU); (ii) Ex, 427/10 nm; dichroic, 458 nm; Em, 483/32 nm (for mseCFP); (iii) Ex, 480/40 nm; dichroic, 505 nm; Em, 535/50 nm (for mNeonGreen, and fluorescein); (iv) Ex, 504/12 nm; dichroic, 515 nm; Em, 542/28 nm (for mVenus); (v) Ex, 554/23 nm; dichroic, 573 nm; Em, 609/54 nm (for tdTomato, and mRuby2); and (vi) Ex, 630/38 nm; dichroic, 655 nm; Em, 694/44 nm. A 60× objective lens (Plan Apo VC; numerical aperture, 1.4; Nikon) was used for imaging experiments.
Imagem c9100 13
Manufactured by Hamamatsu Photonics
Sourced in Japan
The ImagEM C9100-13 is a high-performance, back-illuminated, electron-multiplying charge-coupled device (EM-CCD) camera. It is designed for low-light imaging applications, providing high quantum efficiency and excellent signal-to-noise ratio. The camera features a 1,024 x 1,024 pixel sensor with a pixel size of 13 μm.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti e inverted microscope, by Nikon (5 mentions)
Metamorph software, by Molecular Devices (4 mentions)
Uapon 150xotirf, by Olympus (4 mentions)
Pad cmv v5 dest gateway vector, by Thermo Fisher Scientific (3 mentions)
Ff01 427 10 excitation filter, by IDEX Corporation (3 mentions)
Hcimage software, by Hamamatsu Photonics (3 mentions)
Volocity software, by PerkinElmer (3 mentions)
Eclipse ti e, by Nikon (2 mentions)
Aquacosmos, by Hamamatsu Photonics (2 mentions)
Nis elements software, by Nikon (2 mentions)
Aquacosmos ratio system, by Hamamatsu Photonics (2 mentions)
Autoquant x3, by Media Cybernetics (2 mentions)
Delta vision elite microscope system, by GE Healthcare (2 mentions)
Plapon, by Olympus (2 mentions)
Ix3 zdc2, by Olympus (2 mentions)
Stage top incubator, by Tokai Hit (2 mentions)
H7422 40, by Hamamatsu Photonics (2 mentions)
P 721 pifoc, by Physik Instrumente (2 mentions)
H10425, by Hamamatsu Photonics (2 mentions)
Ff01 624 40 25 bandpass filter, by IDEX Corporation (2 mentions)
35 protocols using imagem c9100 13
1
Fluorescence Microscopy Imaging Protocols
2
Electron Microscopy Imaging with Patch Clamp
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Cell imaging was performed using an electron multiplying charge-coupled device camera (ImageM C9100-13, Hamamatsu Photonics) mounted on an inverted epifluorescence microscope equipped with a 60× oil immersion objective lens (Plan ApoN, Olympus), a stable xenon lamp (U-LH75XEAPO, Olympus), and a filter set that included a BP542 (excitation filter), BP620 (emission filter), and DM570 (dichroic mirror; Olympus) along with the patch clamp setup. Image acquisition was controlled by a computer using MetaMorph (Molecular Devices). We obtained phase contrast (exposure time, 100 ms) and fluorescence images (exposure time, 1,000 ms) just before the final approach of the glass pipette to a target cell. All phase contrast images included the target cell and pipette edge (data not shown).
3
Automated Multimodal Microscopy for Cell Analysis
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All measurements were performed with a completely automated inverted microscope (ECLIPSE Ti-E; Nikon, Tokyo, Japan) equipped with a high NA 60× objective lens (TIRF 60 × H; NA, 1.49; Nikon). The microscope was customized to introduce external laser beams (635 nm; Radius 635-25; Coherent, Santa Clara, CA, USA) at the indicated incident angle to actualize TIRF illumination. The following sets of excitation (Ex) and emission (Em) filters and a dichroic mirror (DM) were used with a high-pressure xenon lamp: for SYTOX, Ex = FF01-448/20-25, Em = FF01-482/25-25, and DM = Di02-R442-25x36; and for calcein, Ex = FF02-472/30-25, Em = FF01-520/35-25, and DM = FF495-Di03-25x36. The following Ex and Em filters and DM were used with the 632 nm laser for the CF660R dye: Ex = FF02-628/40-25, Em = FF01-692/40-25, and DM = FF660-Di02-25x36. These optical filters were purchased from Semrock (Rochester, NY, USA). Each image was projected on an EM-CCD camera (ImagEM C9100-13; Hamamatsu Photonics K.K., Sizuoka, Japan) through a 0.7× lens (C-0.7x DXM Relay Lens; Nikon). A stage-top incubator (ONICS; Tokai Hit Co., Shizuoka, Japan) was used to control temperature, humidity, and gas concentration.
4
FRET and Fluorescence Microscopy Imaging
5
Imaging Mitochondrial ATP Dynamics
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Mitochondrial ATP was imaged using a FRET-based ATP indicator based on the ε-subunit for the analytic measurement biosensor targeted at the mitochondrial matrix (mitAT1.03). cDNA of mitAT1.03 was ligated and cloned into the SalI/NotI sites of a pAd-CMV-V5-DEST Gateway vector (ThermoFisher Scientific), and the adenovirus vector containing mitAT1.03 cDNA (Ad-mitAT1.03) was constructed as previously described (20 (link)). Cultured HPAECs were infected with 50 plaque-forming units/cell of Ad-mitAT1.03.
Three to five days after Ad-mitAT1.03 infection, HPAECs (maintained at 37°C) were imaged on an ECLIPSE Ti-E inverted microscope (Nikon) with ×60 and ×100 Apo total internal reflection fluorescence oil objectives (1.49 numerical aperture) using a water-cooling electron multiplier charge-coupled device camera (ImagEM C9100-13, Hamamatsu) controlled by HCImage software (version 4.3, Hamamatsu). The dual-emission ratio imaging of mitAT1.03 used a FF01-427/10 excitation filter (Semrock), FF458-Di01 dichroic mirror, and Dual View Multichannel Imaging System (DV2, Photometrics) equipped with two emission filters [FF01-483/32 for cyan fluorescent protein (CFP) and FF01-542/27 for yellow fluorescent protein (YFP)]. Images were analyzed using the MetaMorph software program (version 7.7, Molecular Devices).
Three to five days after Ad-mitAT1.03 infection, HPAECs (maintained at 37°C) were imaged on an ECLIPSE Ti-E inverted microscope (Nikon) with ×60 and ×100 Apo total internal reflection fluorescence oil objectives (1.49 numerical aperture) using a water-cooling electron multiplier charge-coupled device camera (ImagEM C9100-13, Hamamatsu) controlled by HCImage software (version 4.3, Hamamatsu). The dual-emission ratio imaging of mitAT1.03 used a FF01-427/10 excitation filter (Semrock), FF458-Di01 dichroic mirror, and Dual View Multichannel Imaging System (DV2, Photometrics) equipped with two emission filters [FF01-483/32 for cyan fluorescent protein (CFP) and FF01-542/27 for yellow fluorescent protein (YFP)]. Images were analyzed using the MetaMorph software program (version 7.7, Molecular Devices).
6
Tagging SpAvt3p with GFP in S. pombe and S. cerevisiae
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To tag the SpAvt3p protein with green fluorescence protein (GFP) at its N-terminus, the open reading frame was amplified by PCR and subcloned into pTN54, a derivative of the thiamine-repressible expression vector pREP41 [29 (link)], thereby yielding pTN54-avt3+. S. pombe cells transformed with pTN54-avt3+ were grown in MM medium without leucine and thiamine at 30°C for 20 h, and then labeled with FM4-64, a lipophilic dye for vacuolar membrane staining, as described previously [30 (link)].
To construct the avt3+ expression plasmid for S. cerevisiae, the GFP-tagged avt3+ was subcloned into pRS416GPD [31 (link)], thereby obtaining pGPD-GFP-avt3+. A single point mutation, avt3E469A, was constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Staining with FM4-64 in S. cerevisiae cells was performed as described previously [32 (link)]. Cells were observed with an IX71 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a cooled charge-coupled device camera (ImagEMC9100-13; Hamamatsu, Japan). Images were acquired using Metamorph software (Universal imaging, West Chester, PA).
To construct the avt3+ expression plasmid for S. cerevisiae, the GFP-tagged avt3+ was subcloned into pRS416GPD [31 (link)], thereby obtaining pGPD-GFP-avt3+. A single point mutation, avt3E469A, was constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Staining with FM4-64 in S. cerevisiae cells was performed as described previously [32 (link)]. Cells were observed with an IX71 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a cooled charge-coupled device camera (ImagEMC9100-13; Hamamatsu, Japan). Images were acquired using Metamorph software (Universal imaging, West Chester, PA).
7
Epifluorescence Microscopy Imaging Protocol
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For epifluorescence microscopy an Olympus IX81-ZDC fluorescence microscope (Zilly et al., 2011 (link)) with a 60 × 1.49 NA apochromat objective or a 10 × 0.4 NA universal plan apochromat objective was used. For fluorescence excitation and detection we employed a MT20E Illumination system (Olympus) with a 150 W Xenon lamp and the filter sets F36-500 DAPI HC (for DAPI or TMA-DPH), F36-525 EGFP HC (for GFP or Alexa488), and F46-009 Cy5 ET (for Atto647N; all filters from AHF Analysentechnik, Tuebingen, Germany). For imaging, an EMCCD camera (ImagEM C9100-13, Hamamatsu Photonics, Hamamatsu, Japan) was used. Fixed samples were imaged in PBS containing TMA-DPH (ThermoFisherScientific, #T204) that visualizes membranes, or in PBS without TMA-DPH when DAPI (Sigma-Aldrich, #D9542) was used for staining of cell nuclei. Live cells in the GFP pH quenching experiments were imaged in phosphate salt solutions (different from PBS, see below). Images were analysed with ImageJ (for details see below) and are shown at arbitrary scaling, if not stated otherwise.
8
Calcium Imaging of Sperm Motility
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For Ca2+ imaging, Fluo-8H AM (Fig. 7 ) or Fluo-4 AM (Fig. 8 ) was used as a fluorescent probe. The dye-loaded sperm was prepared as described previously24 (link). Fluorescent images of the sperm were observed by a microscope (IX71, Olympus, Tokyo, Japan), and captured on a PC connected to a digital CCD camera (ImagEM, C9100-13; Hamamatsu Photonics, Hamamatsu, Japan) at 32.5 frames/sec using Aquacosmos (Hamamatsu Photonics), or a digital CCD camera (Retiga Exi; QImaging) at 50 frames/sec using an imaging application (TI workbench57 (link)), as described previously24 (link),59 (link). For fluorescence illumination, a stroboscopic lighting system with a power LED was used as described24 (link). Fluorescent signal intensity and sperm flagellar bending were also analyzed using the Bohboh software58 .
9
Spinning-Disk Confocal Imaging of Calcein-Stained Crystals
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In this experiment, we mainly used a spinning-disk confocal imaging system equipped with an Eclipse Ti-U inverted epifluorescence microscope (Nikon, Tokyo, Japan), hand-made reflection light, CSU-X1 laser-scanning unit (Yokogawa, Tokyo, Japan), and ImagEM C9100-13 electron-multiplying charge-couple device (EM-CCD) camera (Hamamatsu Photonics, Hamamatsu, Japan). The system was operated by a Hamamatsu Photonics AQUACOSMOS/RATIO system. During time-lapse confocal imaging, the exposure time was set to 200 msec, and calcein signals were recorded at 10-min intervals using a 488 nm excitation light and a 505–540 nm bandpass filter. We used another confocal system (A+confocal microscope system; Nikon) that was equipped with a high-resolution galvano scanner and operated by NIS Elements software (Nikon) to visualize crystallization at the cellular level. Calcein was excited at 480 nm and fluorescence was detected at 510–530 nm. Each individual specimen was placed on a glass-based dish and filled with 2 mL of FSW-calcein (100 µM) at room temperature (approximately 26 °C). To set the Z=0 µm on the surface of the glass substrate, we marked the crystals on the glass cover slip [8] (link).
10
Yeast Fluorescence Microscopy Protocols
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For fluorescence microscopy, yeast cells were grown in SD+CA medium containing appropriate supplements unless otherwise indicated. Cells were analyzed using two different fluorescence microscopy systems, as described previously (Mochida et al., 2020 (link)). The images in Figs. 1 D , S1 A , S5 B , and S6 were acquired using an inverted microscope (IX81; Olympus) equipped with an electron-multiplying charge-coupled device camera (ImagEM C9100-13; Hamamatsu Photonics), a 150× objective lens (UAPON 150XOTIRF, NA/1.45; Olympus), a Z drift compensator (IX3-ZDC2; Olympus), appropriate lasers and filters, and MetaMorph software (Molecular Devices). For time-lapse imaging, cells were grown in the glass-bottom dish and kept at 30°C using a stage-top incubator (TOKAI HIT). The images in Fig. S5 B were deconvoluted by AutoQuant X3 software (Media Cybernetics). All other fluorescence microscopy images were acquired using a Delta Vision Elite microscope system (GE Healthcare) equipped with a scientific complementary metal-oxide-semiconductor camera (pco.edge 5.5; PCO AG), a 60× objective lens (PLAPON, NA/1.42; Olympus), a 100× objective lens (UPlanSApo, NA/1.40; Olympus), and SoftWoRx software. Images acquired by a Delta Vision were deconvoluted using SoftWoRx software. All acquired images were analyzed using Fiji.
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