Dye-loaded lobules were placed in a perfusion chamber on cover slips which were coated with cell tak (from BD Bioscience, Bedford, MA) in solution containing: 130 NaCl, 5.4 KCl, 1CaCl2, 10 HEPES, 1 MgCl2, 10 glucose, pH 7.4. The clusters were imaged using a Leica SP2 confocal mounted on a DM IRE2 inverted microscope with 20 × (0.4 NA) dry objective and images were acquired every 1`s. Both Calcein and Fluo2 AM were excited with the 488-nm line of an argon ion laser with emission at 510 nm. In experiments employing multiphoton microscopy, Fluo2 loaded lobules were excited at 810 nm using a Spectra Physics tunable fS pulsed Ti-Sapphire laser controlled by Fluoview software on a Olympus FV1000MP microscope using a 25× water immersion objective (1.03 NA). The clusters were stimulated with 1 μm carbachol (CCh) (Sigma, St. Louis, MO, U.S.A.) and maintained at 37 °C. Changes in Ca2+ in the medium are indicated in the text as is time of CCh addition. Images were acquired at a resolution of 512 × 512 pixels. Regions of interest were selected and fluorescence intensity in that region was determined as a function of time and expressed relative to the initial fluorescence.
Fv1000mp microscope
Manufactured by Olympus
Sourced in Japan
The FV1000MP microscope is a high-performance multiphoton imaging system designed for advanced biological research. It features a powerful laser source, high-resolution optics, and sophisticated software to enable detailed analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fm1 43, by Thermo Fisher Scientific (2 mentions)
Ix61wi, by Olympus (2 mentions)
Xlplan n, by Olympus (2 mentions)
Uplansapo, by Olympus (2 mentions)
Cell tak, by BD (1 mentions)
Carbachol cch, by Merck Group (1 mentions)
Mai tai ti sapphire multi photon laser, by Spectra-Physics (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Olympus (1 mentions)
6 protocols using fv1000mp microscope
1
Intracellular Calcium Dynamics in Lobules
2
Fluorescence Lifetime Imaging and Confocal Microscopy
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FLIM was done as described previously (McCullock et al., 2020 (link)). Cells were transfected in either 35 or 60 mm culture dishes and imaged using a water immersion 25× objective (XL Plan N, 1.05 NA) mounted on an Olympus IX61WI upright microscope. A Mai Tai Ti:Sapphire multi-photon laser (Spectra Physics) was used for excitation with an 860 nm wavelength, a repetition rate of 80 MHz and pulse width of approximately 100 fs. Donor emission was filtered by a 480–20 filter and measured by a H72422P Hamamatsu hybrid avalanche photodiode. Time-correlated single photon counting was done using a Becker and Hickl card with a resolution of 25 ps. Using VistaVision software (ISS), donor fluorescence from the plasma membrane from individual cells was binned and fit with a single exponential function, consistent with the lifetime of CFP variant mTurquoise2 (Goedhart et al., 2012 (link)).
For confocal imaging, cells were transfected with Lck-CFP or CFP-tagged cASIC1 in 35 mm dishes. After 2 days, cells were stained with 2 mL of 7.5 µM FM1–43 (Invitrogen) immediately prior to imaging with an Olympus FV1000MP microscope using a 60× water immersion objective (U Plan SApo, 1.20 NA). CFP and FM1–43 were simultaneously excited with a 440 nm laser and emissions between 465 and 495 nm collected as CFP and 575 and 675 nm collected as FM1–43.
For confocal imaging, cells were transfected with Lck-CFP or CFP-tagged cASIC1 in 35 mm dishes. After 2 days, cells were stained with 2 mL of 7.5 µM FM1–43 (Invitrogen) immediately prior to imaging with an Olympus FV1000MP microscope using a 60× water immersion objective (U Plan SApo, 1.20 NA). CFP and FM1–43 were simultaneously excited with a 440 nm laser and emissions between 465 and 495 nm collected as CFP and 575 and 675 nm collected as FM1–43.
3
Monitoring FRET in Transduced NRVMs
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NRVMs were transduced with an nDKAR-expressing adenovirus. After 48 h, FRET was analyzed by confocal microscopy on an Olympus FV1000MP microscope. Transduced cells were identified by exciting CFP at 440 nm and emission monitored at 480 nm. FRET was determined by measuring the ratio of YFP emission at 535 nm to CFP emission at 480 nm after CFP excitation at 440 nm. Because the YFP and CFP are fused in this construct, it is not necessary to correct for expression or spectral bleedthrough of the various constructs. Data were normalized to the initial YFP/CFP ratio in each set at an arbitrary value of 1.
4
Fluorescent Protein Imaging in Myocytes
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Myocytes transfected with plasmids or transduced adenoviruses expressing enhanced GFP (EGFP)–, YFP-, or mCherry-tagged proteins were analyzed in an Olympus FV1000MP microscope in confocal mode with a LUMPLFL 40×/0.8 numerical aperture W (Olympus, Tokyo, Japan) lens. EGFP was excited at 488 nm and emission monitored at 510 nm, YFP was excited at 515 nm and emission monitored at 527 nm, mCherry and monomeric red fluorescent protein were excited at 559 nm and emission monitored at 618 nm, CFP was excited at 440 and emission monitored at 476 nm, and 4′,6-diamidino-2-phenylindole was excited at 405 nm and emission monitored at 461 nm. During imaging, cells were in culture medium containing serum.
5
Cardiac Myocyte ROS Quantification
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Neonatal rat cardiac myocytes were incubated with ET1 in the presence or absence of N-acetyl cysteine (NAC, 3.5 mM) for 24 h prior to imaging. Production of ROS in cultured cardiac myocytes was quantified using the cell-permeant fluorescence probes CM-H2DCFDA (DCF; Cat.#C6827, Thermo Fisher) and MitoSOX Red (Cat.#M36008), using an Olympus FV1000 MP microscope and 20X water immersion lens as described previously (23 (link)). Beating cardiac myocytes were incubated with DCF (5 μM) and MitoSOX Red (5 μM) for 30 min at 37°C, washed with PBS, prior to imaging at 37°C. We used an excitation wavelength of 800 nm. Emitted light was collected by three photomultiplier tubes fitted with bandpass filters. 512 × 512 pixel images were collected and image analysis was performed using Image J (NIH, http://rsb.info.nih.gov/ij/ ). Signal quantification was performed by drawing a region of interest (ROI) and calculating mean fluorescence intensity which was normalized to fluorescent area. DCF and Mitosox fluorescence was calculated from 10 randomly selected regions from three biological replicates (~300 cardiac myocytes).
6
Fluorescent Lifetime Imaging and Confocal Microscopy
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Fluorescent lifetime imaging (FLIM) was done as described previously 39 (link) . Cells were transfected in either 35mm or 60 mm culture dishes and imaged using a water immersion 25x objective (XL Plan N, 1.05 NA) mounted on an Olympus IX61WI upright microscope. A Mai Tai Sapphire multi-photon laser (Spectra Physics) was used for excitation with an 860 nm wavelength, a repetition rate of 80 MHz and pulse width of ~ 100 fs. Donor emission was filtered by a 480-20 filter and measured by a H72422P Hamamatsu hybrid avalanche photodiode (APD). Time-correlated single photon counting (TCSPC) was done using a Becker and Hickl card with a resolution of 25 ps. Using VistaVision software (ISS), donor fluorescence from the plasma membrane from individual cells was binned and fit with a single exponential function, consistent with the lifetime of CFP variant mTurquoise2 40 (link) .
For confocal imaging, cells were transfected with Lck-CFP or CFP-tagged cASIC1 in 35 mm dishes. After two days, cells were stained with 2 mL of 7.5 µM FM1-43 (Invitrogen) immediately prior to imaging with an Olympus FV1000MP microscope using a 60x water immersion objective (U Plan SApo, 1.20 NA). CFP and FM1-43 were simultaneously excited with a 440 nm laser and emissions between 465-495 nm collected as CFP and 575-675 nm collected as FM1-43.
For confocal imaging, cells were transfected with Lck-CFP or CFP-tagged cASIC1 in 35 mm dishes. After two days, cells were stained with 2 mL of 7.5 µM FM1-43 (Invitrogen) immediately prior to imaging with an Olympus FV1000MP microscope using a 60x water immersion objective (U Plan SApo, 1.20 NA). CFP and FM1-43 were simultaneously excited with a 440 nm laser and emissions between 465-495 nm collected as CFP and 575-675 nm collected as FM1-43.
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