To evaluate changes in cytosolic Ca2+ concentrations, Fura2 (DOJINDO, Kumamoto, Japan) fluorescence signals of whole-cell voltage-clamped pyramidal cells were measured by adding 15 µmol/L of Fura2 to the internal pipette solution. Using a fast-switching multi-wavelength illumination system (Lambda DG-4; Sutter Instruments, CA, USA), Fura2 fluorescence signals were excited at 340 nm and 380 nm every 10 s and emission was filtered at 510 nm with a 500-nm dichroic mirror. To acquire these images, a × 40 water immersion objective lens (LUMPlanFI/IR; Olympus, Japan) and a CCD camera (ORCA-flash 4.0 v3; Hamamatsu Photonics, Shizuoka, Japan) were used. All images were saved in MetaMorph software (Molecular Devices, CA, USA) and for analyses, we measured a region of interest (ROI) defined as a circular area 5 µm in diameter with maximum fluorescence intensity near the center of the recording cell. Mean fluorescence intensity ratio for 340-nm and 380-nm excitations in the ROI was calculated.
Lumplanfi ir
Manufactured by Olympus
Sourced in Japan
The LUMPlanFI/IR is a long working distance objective lens designed for Olympus microscopes. It features a numerical aperture of 0.80 and a working distance of 3.3 mm, making it suitable for a variety of applications that require high-resolution imaging and long-distance observation.
Automatically generated - may contain errors
Lab products found in correlation
Lambda dg 4, by Sutter Instruments (2 mentions)
Fura 2, by Dojindo Laboratories (2 mentions)
Fura 2, by Sutter Instruments (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Bx51wi upright microscope, by Olympus (2 mentions)
Ff670 sdi01, by IDEX Corporation (2 mentions)
Optosource illuminator, by Cairn Research (2 mentions)
X10468 02, by Olympus (2 mentions)
Ac508 750 b, by Thorlabs (2 mentions)
Ac508 500 b, by Thorlabs (2 mentions)
Deepsee, by Spectra-Physics (2 mentions)
Optoscan monochromator, by Cairn Research (2 mentions)
Orca flash4.0 v3, by Hamamatsu Photonics (1 mentions)
4 protocols using lumplanfi ir
1
Measuring Cytosolic Calcium Dynamics in Pyramidal Cells
2
Measuring Cytoplasmic Calcium Changes
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To assess Ca2+ changes in cytoplasm, 15 µmol/L of Fura-2 (DOJINDO, Kumamoto, Japan) was added to the pipette solution and the Fura-2 fluorescence signal of whole-cell voltage-clamped pyramidal neurons was measured. Fura-2 was excited every 10 s at 340 nm and 380 nm using a fast-switching multi-wavelength illumination system (Lambda DG-4; Sutter Instruments, Novato, CA, USA). Fluorescence emission was long-pass filtered at 510 nm, and a 500 nm dichroic mirror was used. A × 40 water immersion objective lens (LUMPlanFI/IR, Olympus, Tokyo, Japan) and a CCD camera (CoolSNAP EZ; Photometrics, Tucson, AZ, USA) were used to acquire images. Illumination and image acquisition were regulated by MetaMorph software (Molecular Devices, San Jose, CA, USA). The region of interest (ROI) was defined as a circular area (5 µm in diameter) with maximum fluorescence intensity located near the center of the somatic cell. The ratio of mean fluorescence intensity (340 nm excitation/380 nm excitation) in the ROI was computed.
3
Holographic Photostimulation and Widefield Imaging
4
Holographic Photostimulation and Widefield Imaging
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An analogous holographic photostimulation path was coupled with widefield epifluorescence imaging on a second system, here denoted as setup 2 (see Supplementary Fig. S6 ).
This system was built around an Olympus BX51WI upright microscope, capable of widefield epifluorescence imaging using illumination with an arc lamp, (OptoSource Illuminator, Cairn Research, coupled with a monochromator, Optoscan Monochromator, Cairn Research), and an Orca Flash 4.0 Hamamatsu CCD camera for epifluorescence imaging. The native infrared differential-interference contrast (DIC) path of the Olympus microscope allowed DIC imaging on the CCD.
The holographic photoactivation laser source consisted of a conventional pulsed Ti:Sapphire laser, used at 920 nm (pulse width: 100 fs, repetition rate: 80 MHz, Mai-Tai, Deep-See, Spectra Physics).
The holographic path was analogous to the one described for setup 1: a beam expander enlarged the beam in front of the spatial light modulator (LCOS-SLM X10468-02), whose plane was projected at the back focal plane of a 40×-NA 0.8 objective (LUM PLAN FI/IR, Olympus) by an afocal telescope (f=750mm, Thorlabs #AC508-750-B and f=500mm Thorlabs #AC508-500-B). The holographic beam was coupled to the optical axis of the microscope by a dichroic mirror (FF670, SDi01, 25×36 mm, Semrock). Photostimulation light pulses were generated by a Pockels cell (350-80, Conoptics).
This system was built around an Olympus BX51WI upright microscope, capable of widefield epifluorescence imaging using illumination with an arc lamp, (OptoSource Illuminator, Cairn Research, coupled with a monochromator, Optoscan Monochromator, Cairn Research), and an Orca Flash 4.0 Hamamatsu CCD camera for epifluorescence imaging. The native infrared differential-interference contrast (DIC) path of the Olympus microscope allowed DIC imaging on the CCD.
The holographic photoactivation laser source consisted of a conventional pulsed Ti:Sapphire laser, used at 920 nm (pulse width: 100 fs, repetition rate: 80 MHz, Mai-Tai, Deep-See, Spectra Physics).
The holographic path was analogous to the one described for setup 1: a beam expander enlarged the beam in front of the spatial light modulator (LCOS-SLM X10468-02), whose plane was projected at the back focal plane of a 40×-NA 0.8 objective (LUM PLAN FI/IR, Olympus) by an afocal telescope (f=750mm, Thorlabs #AC508-750-B and f=500mm Thorlabs #AC508-500-B). The holographic beam was coupled to the optical axis of the microscope by a dichroic mirror (FF670, SDi01, 25×36 mm, Semrock). Photostimulation light pulses were generated by a Pockels cell (350-80, Conoptics).
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