A home-built system was established to perform ASF wide-field microscopic imaging of cerebral vessels in mice. 915 nm CW laser beam as the excitation light source passing a 900 nm long-pass filter (FELH0900, Thorlabs) was incident on a 900 nm short-pass dichroic mirror (#69-222, Edmund Optics) and then reflected, irradiating onto the mouse brain through an infrared antireflection water immersion objective (XLPLN25XWMP2, 25×, NA = 1.05, Olympus). The excited ASF was collected by the same objective, and then passed through the same 900 nm short-pass dichroic mirror and a combination of a 900 nm short-pass filter (FESH0900, Thorlabs) and an 800 nm long-pass filter (FELH0800, Thorlabs). Finally, ASF signals were focused on a wide spectral responsive Si-based camera (GA1280, 1280 pixels × 1024 pixels, TEKWIN SYSTEM, China) through the built-in tube lens in the trinocular to visualize the cerebral vessels of the mouse. The system was equipped with an electric control module, which could control the objective (together with the whole microscope unit) to move along the Z-axis direction and collect the signals at different depths of the mouse brain for depth tomography. It could also control the loading platform to move in the X-Y direction and change the imaging field of view.
Xlpln25xwmp2
Manufactured by Olympus
Sourced in Japan
The XLPLN25XWMP2 is a high-numerical-aperture, water-immersion objective lens designed for Olympus microscopes. It has a magnification of 25x and a numerical aperture of 1.05. The lens is optimized for use with water-based samples and provides high-resolution imaging capabilities.
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Lab products found in correlation
Ba460 500, by Olympus (3 mentions)
Ba520 560, by Olympus (3 mentions)
Insight deepsee, by Spectra-Physics (3 mentions)
Dm505, by Olympus (3 mentions)
Dm570, by Olympus (3 mentions)
Maitai, by Spectra-Physics (3 mentions)
Lcv mpe, by Olympus (2 mentions)
Rdm690, by Olympus (2 mentions)
Fvmpe rs two photon microscope, by Olympus (2 mentions)
Insight x3 femtosecond pulsed infrared laser, by Spectra-Physics (2 mentions)
Mousestat, by Kent Scientific (2 mentions)
Filters f air cs, by Braintree Scientific (2 mentions)
Transfer pipette, by Thermo Fisher Scientific (2 mentions)
Petroleum jelly, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Evans blue, by Merck Group (2 mentions)
Fvmpe rs system, by Olympus (2 mentions)
Uplsapo30xs, by Olympus (2 mentions)
Lcv110 mpe incubator microscope, by Olympus (2 mentions)
Insight deepsee laser, by Spectra-Physics (2 mentions)
41 protocols using xlpln25xwmp2
1
Wide-Field Microscopy of Cerebral Vasculature
2
Label-free Imaging of Plant Cell Walls
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Label-free imaging of cellulose and hemicellulose was performed with a home-built stimulated Raman scattering microscope48 (link). A tunable picosecond two-color laser (picoEmerald-S, APE GmbH, Germany) served as the light source. It generated two synchronized 80 MHz picosecond laser beams. The 1032 nm Stokes beam was modulated at 20 MHz. The pump beam was tuned to 926.5 nm to detect cellulose and hemicellulose. Stokes and pump beams were coupled into a laser-scanning upright microscope (FVMPE, Olympus, Japan) equipped with a 25X water-immersion objective lens (XLPLN25XWMP2, Olympus, Japan). SRS signals were collected by a photodiode and lock-in amplifier module (customized from APE GmbH, Germany). Fresh maize leaf epidermal strips were placed on a glass slide with water and covered with a cover glass. SRS images (512 × 512 pixels) were acquired. The pixel dwell time was 2 to 4 µs. Each image was line-averaged 3 to 5 times to reduce noise. The signal intensity corresponds to the content of cellulose and hemicellulose.
3
High-resolution Perilimbal Imaging Protocol
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Samples were imaged and processed as described before [13 (link),14 (link)]. Briefly, full-thickness perilimbal scans were acquired with a confocal microscope designed for high-speed ribbon-scanning and large scale image stitching (RS-G4, Caliber I.D., Andover, MA, USA). The system was fitted with a scanning stage (SCANplus IM 120 × 80, #00-24-579-0000; Märzhäuser Wetzlar GmbH & Co. KG, Wetzlar, Germany) and an Olympus 25×, 1.05 NA water immersion objective (XLPLN25XWMP2; Olympus). Volumetric scans were acquired with a voxel size of 0.365 × 0.365 × 2.43 μm. A scan-zoom of 1.5 was used during acquisition to achieve the desired resolution. Images were acquired over a single channel with an excitation wavelength of 561 nm and an emission filter of 630/60. Laser percentage, high voltage (HV), and offset were held constant throughout the volume at 7, 85, and 15, respectively.
4
Two-Photon Microscopy Imaging Protocol
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ChroMS imaging was performed on a lab-built laser scanning two-photon microscope equipped with a vibrating-blade microtome23 (link) using a water-immersion objective (25× 1.05 NA, XLPLN25XWMP2, Olympus) and the wavelength mixing method described in ref. 53 (link). Imaging depth was set from 117 to 160 µm and slicing from 80 to 120 µm, with a XYZ sampling of 0.4–0.46 × 0.4–0.46 × 1.5 µm.
5
Long-term Cochlear Imaging Technique
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For long-term organ-scale imaging, we partially cut off the capsule adjacent to the apex tip of cochlear duct using tweezers carefully and the semicircular canals were removed. The isolated cochlea was put onto the dish as described above. For microscopy, we used an incubator-integrated multiphoton fluorescence microscope system (LCV-MPE, Olympus) with a × 25 water-immersion lens (NA = 1.05, WD = 2 mm, XLPLN25XWMP2, Olympus). The excitation wavelengths were set to 840 nm for CFP (InSight DeepSee, Spectra-Physics). Imaging conditions for the FRET biosensor were as follows: scan size: 800 × 800 pixels; scan speed: 10 μs/pixel; IR cut filter: RDM690 (Olympus); dichroic mirrors: DM505 and DM570 (Olympus); and emission filters: BA460-500 for CFP and BA520-560 for FRET detection (Olympus).
6
Multimodal Imaging and Patch Clamp Microscopy
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These experiments were performed using a home-built multimodal microscope with a patch clamp add on, the design of which has been described recently (39 ). Epifluorescence imaging was performed using two laser beams at 488 nm (OBIS 488 LX, Coherent) and 532 nm (MLL-III-532, CNI) focused onto the back aperture of a 25X objective (XLPLN25XWMP2, Olympus). Illumination power of 11.2 mW/mm2 and 37.23 mW/mm2 were used for brightness screening for 488 nm and 532 nm, respectively. For patch clamp characterization, the 532 nm intensity was 87.6 mW/mm2. The emission light was filtered using a multiband dichroic mirror (Di03-R405/488/532/635-t3-32x44, Semrock) and a 552 to 779.5 bandpass filter (FF01-731/137-25, Semrock). The images were acquired at a frame rate of 100 or 500 Hz using an sCMOS camera (ORCA Flash4.0 V3, Hamamatsu; 2048 x 2048 pixels, 6.5 μm pixel size). The voltage pulses, illumination, and camera recording were synchronized using the National Instruments DAQ (USB-6363). All software for controlling the hardware, image acquisition and analysis were custom written in Python (39 ).
7
Multimodal SLAM Microscopy Protocol
8
Two-Photon Microscopy for Live-Cell Imaging
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Images were acquired with a FVMPE RS two-photon microscope (Olympus) equiped with a 25 × , numerical aperture (NA) 1.05 water-immersion objective (XLPLN25XWMP2, Olympus) and an InSight X3 femtosecond-pulsed infrared laser (Spectra-Physics) for optimal fluorescence excitation and emission separation. Images were taken with a digital zoom of 7.2 at each image session using 0.75 μm step with a scanning dwell time of 2.55 μsec per pixel. Laser power was adjusted with the depth of imaging and kept below 50 mW. Time-lapse acquisition were done with galvanometric scanning mode and conventional raster scanning every 30 s for 5–10 min interspaced of 20 min during 1 h. Each image is a stack of scans over 20 μm depth. For Mito-dsred and GFP, a single track at 1040 nm excitation wavelength is used to obtain both GFP (em. 509 nm) and dsRed (em. 583 nm) images at the same time point. For ATeam, 850 nm excitation wavelength is used to obtain both CFP (em. 475 nm) and YFP (em. 527 nm) images at the same time point. For roGFP, the two images were acquired for each time point using alternating tracks 940 nm and 800 nm. Change of track was set after each stack. Each scan was obtained with constant laser intensity at 512 × 512 pixel resolution and microscope-imaging parameters were maintained between sessions.
9
Light Sheet Microscopy Protocol
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For light sheet microscopy, custom-made sample chambers were 3D-printed (Prusza) using PLA. A glass plate was bonded to the bottom of each chamber. Then, 10 mL of LB medium with 5 mM nickel oxide was prepared to OD600 = 0.05 and injected into the chamber. The chamber was sealed with the help of a plastic cover so that the medium would not spill during the shaking in the incubator. The samples were incubated for 8, 16, and 32 h at 37 °C and 120 rpm. After incubation, the chambers were washed 2× with 1% PBS and fixed with 4% PFA for 30 min to inactivate the bacteria. Afterwards, the chamber was washed 2 more times with 1% PBS and air-dried. Imaging was done using a custom-made inverted light sheet microscope. To create the light sheet, a Bessel beam was scanned and a special excitation objective (Thorlabs TL20X-MPL) was used to project the sheet into the sample. The collection of the fluorescence light was done with a 25× 1.05 NA objective (Olympus-XLPLN25XWMP2), a 250 mm tube lens to roughly achieve a 34× magnification, and a CMOS camera (IDS-µeye 3270). Sample movement was achieved with a closed-loop linear piezo stage (Piezosystem Jena, nanoSX 800). The image stacks were fed into a simple but custom-made algorithm to create a single image.
10
3PL Imaging of Nanoparticles and Tissues
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The 1550 nm fs laser was coupled to an upright confocal microscope (Olympus, BX61W1-FV1000). After passing through a scan lens and a tube lens, the laser beam was focused onto the sample by a water-immersed microscope objective (XLPLN25XWMP2, Olympus, 25 1.05 NA). The imaging sample could be a glass capillary tube filled with an aqueous dispersion of PS-PEG@TB NPs or the brain or ear of a live mouse. 3PL signals were epi-collected with the same objective and then passed through a customized 1035 nm short-pass dichroic mirror and a 590 nm long-pass filter (removing the excitation light and ambient noise). Then, the remaining fluorescence signals were collected using an external photomultiplier tube (HPM-100-50 Becker & Hickl GmbH) via non-descanned detection (NDD). Pictures were collected every 5 μm along the Z-axis, and 3D imaging was reconstructed by Z-scan stacks.
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