Cultured primary mouse neurons were imaged by automated fluorescent microscopy (26 (link)–28 (link), 30 (link)). The system consists of an inverted microscope (Nikon Eclipse Ti) equipped with the Perfect Focus System (Nikon), a high-numerical aperture 20× objective lens, a digital camera, a Lambda XL lamp (Sutter) and an ASI MS2000 stage to automatically control the platform. Neurons were imaged inside a thermo chamber, in which temperature and CO2 concentration was maintained at 37°C and 5% respectively.
Lambda xl lamp
Manufactured by Sutter Instruments
The Lambda XL lamp is a versatile light source designed for laboratory applications. It provides a stable and consistent output across a wide range of wavelengths, making it suitable for various experimental setups. The lamp's core function is to generate light that can be used for illumination, spectroscopy, or other light-based analyses.
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Lab products found in correlation
Em ccd camera, by Hamamatsu Photonics (3 mentions)
Nis elements software, by Nikon (2 mentions)
Cooled scmos camera, by Hamamatsu Photonics (2 mentions)
Eclipse ti, by Nikon (1 mentions)
Perfect focus system, by Nikon (1 mentions)
Bx 2 microscope, by Olympus (1 mentions)
Eos 4000d, by Canon (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Zyla scmos camera, by Oxford Instruments (1 mentions)
Observer z1 inverted microscope, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Ti s inverted microscope, by Nikon (1 mentions)
9 protocols using lambda xl lamp
1
Automated Fluorescent Imaging of Primary Mouse Neurons
2
In Vivo Calcium Imaging of Neuronal Activity
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Wide-field imaging was used to monitor the expression levels of the Ca2+ sensors and quality of the cranial windows. The mice were head-fixed on a custom 3D-printed running wheel using optical posts that were mounted to the optical table, holding clamps (Standa) and modified ball-joints (Thorlabs GmbH) allowing for adjustments in AP elevation. Single images were acquired by a Canon EOS 4000D camera through a ×5 Mitutoyo long working distance objective (0.14 NA) in an Olympus BX-2 microscope. The light source was a xenon arc Lambda XL lamp (Sutter Instruments) with 480/545 nm and 560/635 nm filters (#39002 and #39010, Chroma). All animals were imaged using two sets of parameters at each time-point, with exposure times of 600 and 2000 ms, and ISO of 100 and 400, respectively. The mice ran freely in darkness during imaging. In addition, wide-field videos were captured at 25 Hz during both spontaneous activity (in darkness) and with visual stimulation.
3
Imaging Neuronal Calcium Dynamics in Cultured Neurons
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All experiments were conducted at 37°C within a whole-microscope incubator (In Vivo Scientific) at DIV16–19 as we described previously (Maschi and Klyachko, 2017 (link)). Neurons were perfused with bath solution (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 15 mM Glucose, 50 mM DL-AP5, 10 mM CNQX, pH adjusted to pH 7.4). Fluorescence was excited with a Lambda XL lamp (Sutter Instrument) through a 100 × 1.45 NA oil-immersion objective and captured with a cooled EMCCD camera (Hamamatsu). With this configuration the effective pixel size was 80 nm. Focal plane was continuously monitored, and focal drift was automatically adjusted with 10 nm accuracy by an automated feedback focus control system (Ludl Electronics). Field stimulation was performed by using a pair of platinum electrodes and controlled by the software via Master-9 stimulus generator (A.M.P.I.). Images were acquired using two frames with an acquisition time of 40 ms, one 45 ms before stimulation and one coincidently (0 ms delay) with stimulation. In some experiments (Figure 2A-C ) imaging was performed using a cooled sCMOS camera (Hamamatsu). With this configuration, the effective pixel size was 60 nm and images were acquired at a frame rate of 200 ms.
4
Calcium Imaging of DRG Neurons
5
Fura-2 and F-tractin Imaging of Ca2+ Dynamics
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Transfected cells were loaded with 2.5 µM fura-2/AM at 22–25oC for 30 min in RPMI 1640 without phenol red or sodium bicarbonate. After washing, cells remained for 30 min in RPMI before they were resuspended in Ringer’s solution immediately prior to loading onto coverslips. Cells were stimulated on anti-CD3 in 0.5 mM Ca2+o, followed by either Ca2+o removal or application of 100 µM 2-APB in 0.5 mM Ca2+o, then perfusion with 2, 5, or 10 mM Ca2+. F-tractin-P-tdTom and fura-2 were imaged on a Zeiss Observer Z1 inverted microscope at 37°C using an αPlan-Apochromat 100X, 1.46 N.A. oil immersion DIC objective (Carl Zeiss). Fura-2 imaging was performed using a Lambda XL lamp (Sutter), 380/15 and 357/10 excitation filters, 400-nm dichroic and 480-nm long pass emission filter (Omega Optical, Brattleboro, VT). Data are displayed as the ratio of emissions in response to excitation at 357 and 380 nm (357/380 ratio). F-tractin-P-tdTom images were acquired using through-the-objective TIRF with 561-nm laser excitation and a Zeiss 74HE filter set and an ImagEM-1K EMCCD camera (Hamamatsu, Hamamatsu City, Japan), and all equipment was controlled using Zeiss Axiovision software.
6
Microscale Neuronal Activity Imaging
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All experiments were conducted at 37°C within a whole-microscope incubator (In vivo Scientific) at DIV16–19 as described previously (Maschi et al., 2018 ). Neurons were perfused with bath solution (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 15 mM Glucose, 50 mM DL-AP5, 10 mM CNQX, pH adjusted to pH 7.4). Fluorescence was excited with a Lambda XL lamp (Sutter Instrument) through a 100x 1.45 NA oil-immersion objective and captured with an EMCCD camera (Hamamatsu) or cooled sCMOS camera (Hamamatsu). Focal plane was continuously monitored, and focal drift was automatically adjusted with 10 nm accuracy by an automated feedback focus control system (Ludl Electronics). Field stimulation was performed by using a pair of platinum electrodes and controlled by the software via Master-9 stimulus generator (A.M.P.I.). Images were acquired using two frames with an acquisition time of 40ms, one 45ms before stimulation and one coincidently (0ms delay) with stimulation.
7
Intracellular Calcium Imaging of Islets
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The microfluidic device was secured onto the stage of a Ti-S inverted microscope (Nikon Instruments, Inc., Melville, NY). A Lambda XL lamp fitted with a shutter and filter wheel (Sutter Instruments, Novato, CA) containing appropriate emission filters was used to excite intracellular Fura PE3-AM at 340 and 380 nm. Fluorescence images were collected with a CCD camera (Cascade, Photometrics, Tucson, AZ) controlled by Nikon NIS Elements software (Nikon) every 20 s after a 150 ms exposure (S1 Fig ). The ratio of fluorescence emission at 520 nm after the 340 nm excitation to that after 380 nm, F340/F380, was used to calculate [Ca2+]i for each islet using calibration constants [50 (link),51 (link)] that were calculated previously using a Ca2+ calibration kit [52 (link)].
8
Imaging Calcium Dynamics in Neurons
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All experiments were conducted at 37°C within a whole-microscope incubator (In Vivo Scientific) at DIV16–19. Neurons were perfused with bath solution (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 15 mM glucose, 50 μM DL-AP5, 10 μM CNQX adjusted to pH 7.4). Asynchronous release events were recorded using the same solutions, except that 3 mM Sr2+ and 0 mM CaCl2 were used in the bath. Fluorescence was excited with a Lambda XL lamp (Sutter Instrument) through a 100 × 1.45 NA oil-immersion objective and captured with a cooled CMOS camera (Hamamatsu). With this configuration, the effective pixel size was 65 nm. The focal plane was continuously monitored, and focal drift was automatically adjusted with ~10 nm accuracy by an automated feedback focus control system (Ludl Electronics). Field stimulation was performed by using a pair of platinum electrodes and controlled by the software via Master-9 stimulus generator (AMPI). Images were acquired using an acquisition time of 40 ms, one 45 ms before stimulation and one coincidently with stimulation (0 ms delay).
9
Quantifying Neurotransmitter Release Dynamics
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Neurotransmitter Release Measurements: All experiments were conducted at 37°C within a whole-microscope incubator (In Vivo Scientific) at DIV16-19 as described previously (Maschi et al., 2018) . Neurons were perfused with bath solution (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 15 mM Glucose, 50 mM DL-AP5, 10 mM CNQX, pH adjusted to pH 7.4). Fluorescence was excited with a Lambda XL lamp (Sutter Instrument) through a 100x 1.45 NA oil-immersion objective and captured with an EMCCD camera (Hamamatsu) or cooled sCMOS camera (Hamamatsu). Focal plane was continuously monitored, and focal drift was automatically adjusted with 10 nm accuracy by an automated feedback focus control system (Ludl Electronics). Field stimulation was performed by using a pair of platinum electrodes and controlled by the software via Master-9 stimulus generator (A.M.P.I.). Images were acquired using two frames with an acquisition time of 40ms, one 45ms before stimulation and one coincidently (0ms delay) with stimulation.
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