The genes encoding NIR-GECO1 and GCaMP6s were expressed under the control of synapsin promoter in cultured rat hippocampal neurons. Neurons were stimulated using a custom-built field stimulator using a stimulus isolator (A385, World Precision Instruments) with platinum wires. Field stimuli (50 V, 83 Hz, 1 ms) were delivered in trains of 1, 2, 3, 5, 10, 20, 40, 80, 120 and 160 field stimuli to the cultured neurons. Neurons were imaged using a Nikon Eclipse Ti2 inverted microscope equipped with a 40× objective (Nikon, 1.4 NA). A quad bandpass filter (set number: 89000, Chroma) was used along with a 480 nm LED (Spectra X light engine, Lumencor) or a 640 nm LED (Spectra X light engine, Lumencor) to image GCaMP6s and NIR-GECO1 respectively. Fluorescence was collected using a sCMOS camera (Orca-Flash4.0, Hamamatsu) at 34 Hz. For GCaMP6s, the response amplitude (ΔF/Fmin) was quantified as the change in fluorescence divided by baseline fluorescence over 0.5 s period preceding the stimulus. For NIR-GECO1, the response amplitude was quantified as the change in fluorescence divided by peak fluorescence during the stimulus (-ΔF/Fmin). Signal-to-noise ratio (SNR) was quantified as peak change in fluorescence over the standard deviation of the signal over the 0.5 s period preceding stimulation.
Spectra x light engine
Manufactured by Lumencor
Sourced in United States, Germany
The SPECTRA X light engine is a compact, high-intensity, and versatile solid-state light source designed for a wide range of laboratory applications. It provides stable and uniform illumination across a broad spectrum, from ultraviolet to near-infrared wavelengths, making it suitable for various imaging, microscopy, and spectroscopy techniques.
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Lab products found in correlation
Axio observer z1, by Zeiss (12 mentions)
Eclipse ti, by Nikon (7 mentions)
Metamorph software, by Molecular Devices (6 mentions)
Orca flash 4, by Hamamatsu Photonics (5 mentions)
Nis elements software, by Nikon (4 mentions)
Pclamp 10, by Molecular Devices (4 mentions)
Eclipse ti2, by Nikon (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Ix83 microscope, by Olympus (3 mentions)
Ds qi2 camera, by Nikon (3 mentions)
Eclipse ti e, by Nikon (3 mentions)
Zyla 5.5 camera, by Oxford Instruments (3 mentions)
Digidata 1440a, by Molecular Devices (3 mentions)
Visiview software, by Visitron (3 mentions)
Dmi8 s microscope, by Hamamatsu Photonics (3 mentions)
C9100 em ccd camera, by Lumencor (3 mentions)
Zyla 4.2 scmos camera, by Oxford Instruments (3 mentions)
Ix73 epi fluorescent microscope, by Oxford Instruments (3 mentions)
X light spinning disk system, by Nikon (3 mentions)
Ti e base, by Nikon (3 mentions)
114 protocols using spectra x light engine
1
Calcium and Voltage Imaging of Neuronal Activity
2
Calcium and Voltage Imaging of Neuronal Activity
3
Dual Imaging of Fluorescent Worms
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Dual imaging was performed using an upright microscope (BX51WI; Olympus) and a 10 x objective (UplanSApo, NA 0.4; Olympus). For bright-field imaging, light emanating from a near-infrared (780 nm) LED (M780LP1 and driver LEDD1B; Thorlabs) was filtered using a (785/62 BrightLine HC; Semrock) and projected onto the sample via the bright-field illumination condenser. To excite fluorescence, the Teal line from an LED lamp (Spectra X light engine; Lumencor) was filtered (513/17 BrightLine HC; Semrock) and projected onto the sample using a 520 nm long-pass dichroic (FF520-Di02; Semrock). Transmitted and emitted light were filtered using a 532 long-pass filter (BLP01-532R; Semrock). To simultaneously record images in bright-field and fluorescence, a dual-camera device was used (DC2; Photometrics). Light was split into two channels using a 695 long-pass dichroic mirror (695DCXRUV; Photometrics) and images were projected into two cameras (acA3088-57um; BASLER). Fluorescent light was band-pass filtered (550/49 Brightline HC; Semrock) before reaching the camera sensor. The exposure time (6ms) of one camera served to synchronize the acquisition of the second camera and the Lumencor light engine. Individual worms were manually tracked using a 3-axis motorized stage (X-LSM150A; Zaber).
4
Screening Kohinoor Mutants for Bright and Fast Photoswitching
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We spread Escherichia coli JM109 (DE3) cells transformed with the DNAs of mutant Kohinoor library on 95-mm LB agar plates and incubated them at 37°C for 16 h. We screened mutants with a high brightness and fast photoswitching by using an in-house-built illumination system [7 (link)]. We irradiated the Kohinoor mutants expressed in the E. coli cells with an excitation light at 475 nm from an LED light source (SPECTRA X Light Engine; Lumencor, Beaverton, OR, USA) and took the fluorescence through a bandpass filter (center wavelength, 525 nm; FF01-525/45; Semrock, Rochester, NY, USA) with a charge-coupled device (CCD) camera (01-QIClick-F-M12; QImaging, Surrey, BC, Canada). We measured the photoswitching with irradiation at 475 and 386 nm for on-switching and off-switching, respectively. We analyzed the data by ImageJ-Fiji software [20 (link)].
5
Multicolor Fluorescence Imaging Protocol
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Imaging was performed using a standard epifluorescence microscope (Zeiss Axio Imager.Z2) connected to external LED source (Lumencor® SPECTRA X light engine). Light engine was setup with filter paddles (395/25, 438/29, 470/24, 555/28, 635/22, 730/40). Images were obtained with a sCMOS camera (2048 × 2048, 16-bit, ORCA-Flash4.0 LT Plus, Hamamatsu), automatic multi-slide stage (PILine, M-686K011), and Zeiss Plan-Apochromat objectives 20x (0.8 NA, air, 420650-9901), 40× (1.4 NA, oil, 420762–9900). Filter cubes for wavelength separation included quad band Chroma 89402 (DAPI, Cy3, Cy5), quad band Chroma 89403 (Atto425, TexasRed, AlexaFluor750), and single band Zeiss 38HE (AlexaFluor488). In SBL-based ISS, one of the base libraries was either conjugated to TexasRed or Atto425 fluorophores that requires substitution of the 555/28 filter paddle with a compatible 575 one to image TexasRed. Regions were outlined and saved in order to perform repetitive cycle imaging.
6
Quantifying Intracellular Calcium Dynamics
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WT and iRhom1/2 DKO MEFs cells were grown on fibronectin coated glass-bottomed dishes. Cells were washed twice with HBS and incubated with Calbryte 520 AM (2 μM, 20 °C) for 1 hr in HBS containing 0.02% Pluronic F-127 and probenecid (2.5 mM). Cells were then loaded with ci-IP3/PM (1 μM, 20 °C, 1 h) (SiChem, #cag-iso-2-145), washed twice and incubated in HBS for a further 30 min before imaging in HBS at 20 °C using an inverted Olympus IX83 microscope equipped with a ×60 objective. Calbryte 520 fluorescence was recorded in widefield (488 nm excitation, 525/50 nm emission), with illumination for 25 ms every sec. A flash of ultraviolet (UV) light (150 ms, 395/20 nm, SPECTRA X-light engine, Lumencor) was delivered after 10 s to photolyse ci-IP3. Cells were imaged, background-corrected and analysed using MetaMorph (Molecular Devices).
7
Dual-CCD Fluorescence Imaging System
8
Photocurrent Kinetics of Light-Activated Ion Channels
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To investigate the photocurrent kinetics, irradiation was carried out using a SpectraX light engine (Lumencor Inc., Beaverton, OR) controlled by computer software (pCLAMP 10.3, Molecular Devices) at wavelengths (nm, >90% of the maximum): 438±24 (blue), 475±28 (cyan) and 513±17 (teal). The power of the light was directly measured under a microscope by using a visible light-sensing thermopile (MIR-101Q, SSC Co., Ltd., Kuwana City, Japan). Every photocurrent was measured with a holding potential of −60 mV and at pH 7.4 outside. Two test pulses of light (20 ms and 100 ms) were applied before and after irradiation of 1-s strong light (blue: 438±24 nm, 5.1 mWmm−2 or cyan: 475±28 nm, 5.1 mWmm−2). Each irradiation protocol was applied every 60 s to enable full recovery from the desensitization.
9
Widefield and Confocal Microscopy of eL22-HA
10
Nikon Microscopy for Infection Kinetics
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Cells were imaged via a Nikon Eclipse Ti inverted microscope equipped with a QICAM Fast 1394 digital CCD camera (Q Imaging, Surrey, BC, Canada). The cells were maintained at 37°C, 5% CO2, and 85% relative humidity via a stage top incubator (In Vivo Scientific) and a custom outer chamber maintained at 37°C by an In Vivo Scientific temperature controller. Illumination was provided by a Spectra X light engine (Lumencor), and the experiment was controlled with NIS Elements (Nikon).
Imaging for individual cell tracking was performed with a Nikon Plan Apo 10x, 0.45 NA objective lens beginning 1.5 hpi and continued every 10 minutes for 25 hours. The imaging for the spreading infections was performed with a Nikon Plan Apo 4x, 0.20 NA objective lens beginning 3 hpi and continued every 2 hours for 27 hours.
Imaging for individual cell tracking was performed with a Nikon Plan Apo 10x, 0.45 NA objective lens beginning 1.5 hpi and continued every 10 minutes for 25 hours. The imaging for the spreading infections was performed with a Nikon Plan Apo 4x, 0.20 NA objective lens beginning 3 hpi and continued every 2 hours for 27 hours.
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