A left thoracotomy was performed as above and a 3D-printed titanium stabilization probe with imaging window (Allan-Rahill et al., 2020 (link); Jones et al., 2018 (link)) attached to the left ventricle free-wall using tissue adhesive (Vetbond). Texas Red-conjugated 70 kDa dextran (3% in saline; Thermo Fisher Scientific #D1830) was injected retro-orbitally to identify the vasculature. A Ti:Sapphire laser (Chameleon, Coherent) with wavelength centered at 950 nm was used to excite indicator molecules and images collected using a custom multiphoton microscope equipped with four detection channels and high-speed resonant scanners running ScanImage (Pologruto et al., 2003 (link)). Emission fluorescence was detected using long-pass dichroic mirrors and bandpass filters for GCaMP8 (517/65 nm) and Texas Red (629/56). Water was placed within a rubber O-ring of the stabilization probe to allow immersion of the microscope objective (Olympus XLPlan N 25 × 1.05 NA). ECG and respiratory signals were collected while imaging z-stacks (50–100 frames; 2 µm per z-step; 30 frames/s) in 4–5 different regions in each mouse. Image reconstruction was performed as previously described (Jones et al., 2018 (link)) by indexing image lines based on their acquired position within the cardiac or respiratory cycles.
Xlplan n 25
Manufactured by Olympus
Sourced in Japan
The XLPlan N 25 is a high-performance microscope objective lens designed for scientific and industrial applications. It features a numerical aperture of 0.25 and a working distance of 10.6 mm, providing excellent optical performance and resolution.
Automatically generated - may contain errors
Lab products found in correlation
Fvmpe rs, by Olympus (2 mentions)
B scope, by Thorlabs (1 mentions)
Ff03 525 50, by IDEX Corporation (1 mentions)
Ff562 di03, by IDEX Corporation (1 mentions)
Ff01 607 70, by IDEX Corporation (1 mentions)
Texas red dextran, by Thermo Fisher Scientific (1 mentions)
Fvmpe, by Olympus (1 mentions)
Methoxy x04, by Bio-Techne (1 mentions)
Insight ds, by Spectra-Physics (1 mentions)
Bx61wi, by Olympus (1 mentions)
Fv1000, by Olympus (1 mentions)
Maitai, by Spectra-Physics (1 mentions)
Fv31s sw, by Olympus (1 mentions)
Vetbond, by 3M (1 mentions)
Fluoview, by Olympus (1 mentions)
6 protocols using xlplan n 25
1
Cardiac Calcium Imaging with 3D-Printed Probe
2
Two-Photon Imaging in Anaesthetized Mice
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Two-photon imaging was performed with urethane-anaesthetized adult mice (as above), using a resonant scanner-based B-Scope (Thorlabs) with a Chameleon Vision 2 laser (Coherent, wavelength 920 nm) and an Olympus objective lens (XLPlan N × 25). The B-Scope is equipped with a reverse dichroic mirror (ZT405/488/561/680-1100rpc, Chroma) and the emission light was separated by using a dichroic mirror (FF562-Di03, Semrock), with band-pass filters FF03-525/50 and FF01-607/70 (both from Semrock) for the green and red channels, respectively. Images were acquired using the ThorImage software with a frame rate of 30 Hz.
3
Imaging Pial Vessel CAA in Mice
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We imaged pial vessel CAA using 2-photon microscopy. Optical access to the brain was achieved through a polished and reinforced thinned skull preparation sealed with cyanoacrylate glue and a cover glass [26 (link), 33 (link)]. Mice were allowed at least two weeks to recover from window implantation. To label Aβ deposits, methoxy-X04 (Tocris, dissolved in DMSO at 100 mM) was intraperitoneally injected one day before imaging at a dose of 1 mg/100 g [44 (link)]. To fluorescently label the blood vessel, Texas Red dextran (40 μl, 2.5%, molecular weight (MW) = 70,000 kDa, Thermo Fisher Scientific) in saline was injected retro-orbitally immediately before imaging. Imaging was performed on a commercial 2-photon microscope (FVMPE; Olympus) with XLPlan N 25 × 1.05 NA objective. Excitation pulses came from a solid-state laser (InSight DS + ; Spectraphysics) set at 830 nm wavelength. Image stacks were acquired through Fluoview software. During imaging, anesthesia was maintained with ~ 1.5% isoflurane in an oxygen/nitrogen mix (21% oxygen), with slight adjustments made to the isoflurane to maintain the respiratory rate at ~ 1 Hz. Animals were kept at 37 °C with a feedback-controlled heating pad. Two photon images are average projection of three-dimensional stacks using ImageJ (NIH).
4
In Vivo Two-Photon Imaging of Marmoset Neurons
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In vivo two-photon imaging of neurites was performed using an upright microscope (BX61WI; Olympus) equipped with a laser scanning microscope system (FV1000; Olympus) and a water-immersion objective lens (XLPlanN, 25×, NA 1.05; Olympus). The system included mode-locked, femtosecond-pulse Ti:sapphire lasers (MaiTai; Spectra Physics) set at a wavelength of 980 nm.
Imaging sessions were conducted every 3 days. A marmoset was pretreated with atropine and anesthetized with sevoflurane inhalation (3–5%). The marmoset was laid in the prone position and its head was fixed using the imaging chamber and brass posts (Fig.1a ). In each case, the imaging location was identified based on blood vessel morphology. Tuft dendritic branches (<100 µm depth) of layer 2/3 pyramidal neurons were used for two-photon imaging experiments. An objective lens correction collar was manually set just before imaging so as to minimize spherical aberrations (i.e., to acquire the brightest image possible). The reciprocal scan mode was used to scan each xy-image (256 by 256 pixels; 65 msec/frame). Three-dimensional fluorescent images with 51 xy-images, each separated by 0.5 µm, were obtained at each imaging site.
Imaging sessions were conducted every 3 days. A marmoset was pretreated with atropine and anesthetized with sevoflurane inhalation (3–5%). The marmoset was laid in the prone position and its head was fixed using the imaging chamber and brass posts (Fig.
5
Longitudinal Calcium Imaging in Mouse S1 Cortex
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All surgical operations were conducted under combined anesthesia with Zoletil (30 mg/kg) and xylazine (10 mg/kg). A cranial window, measuring 2 × 2 mm, was crafted above the left S1 cortical hind paw region (positioned laterally by 1.5 mm and posteriorly by 0.5 mm from Bregma) to facilitate longitudinal calcium observations. We utilized a surgical blade (#11) for this. The S1 was injected with the Adeno-associated virus showcasing GCaMP6s (from the University of Pennsylvania Gene Therapy Program Vector Core). After the viral injection, a thin cover glass (sourced from Matsunami, Japan) was placed over the cranial window, sealed securely with Vetbond (3M) and dental cement.
Over a period of 2 weeks, the mice underwent acclimatization on a treadmill while their heads were stabilized, spending 40 min daily. The imaging process used a two-photon microscope (FVMPE-RS, Olympus, Tokyo, Japan) with a water immersion objective lens (XLPlan N 25, NA = 1.05, Olympus, Tokyo, Japan). The GCaMP6s indicator was excited by900 nm light provided by a Ti: sapphire laser system (Chameleon, Coherent, USA). Capturing of the imaging frames was done via the FLUOVIEW (FV31S-SW, Olympus, Tokyo, Japan) at a rate of approximately 5 Hz.
Over a period of 2 weeks, the mice underwent acclimatization on a treadmill while their heads were stabilized, spending 40 min daily. The imaging process used a two-photon microscope (FVMPE-RS, Olympus, Tokyo, Japan) with a water immersion objective lens (XLPlan N 25, NA = 1.05, Olympus, Tokyo, Japan). The GCaMP6s indicator was excited by900 nm light provided by a Ti: sapphire laser system (Chameleon, Coherent, USA). Capturing of the imaging frames was done via the FLUOVIEW (FV31S-SW, Olympus, Tokyo, Japan) at a rate of approximately 5 Hz.
6
Two-Photon Calcium Imaging of Neuronal Activity
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A two-photon microscope (FVMPE-RS, Olympus) equipped with a water immersion objective lens (XLPlan N 25, NA=1.05, Olympus, Tokyo, Japan) was used for calcium imaging. To excite GcaMP6s, a Ti: sapphire laser (Chameleon, Coherent, USA) was tuned to 900 nm. All time-lapse images (512×512 pixels, 0.99 µm/pixel) were acquired at 5.11 Hz using FLUOVIEW (Olympus, Tokyo, Japan). The brain surface was imaged after the first session. By matching the blood vessel patterns on the brain surface, relocation to the previously imaged region was possible across different days.
To identify the region corresponding to the right side of the neck on S1, tactile stimulation was applied using a brush during in vivo calcium imaging (Fig. 1E~G ). During imaging, mice were lightly anesthetized with isoflurane. Mechanical stimulation was applied to the hind paws and neck.
To record spontaneous activities in awake condition, the mice were acclimated under head-fixed conditions on a treadmill for 30 min per day for 1 week (Fig. 2A ). Imaging for each treatment was performed on different days for four consecutive days. The order of treatment was randomly selected. Data were acquired 6~7 min after PBS or 5-HT injection and 1~2 min after 5% EtOH or CAP injection. Before imaging, we manually corrected the imaged region to minimize location differences caused by mouse movement.
To identify the region corresponding to the right side of the neck on S1, tactile stimulation was applied using a brush during in vivo calcium imaging (
To record spontaneous activities in awake condition, the mice were acclimated under head-fixed conditions on a treadmill for 30 min per day for 1 week (
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