We used an inverted laser-scanning microscope (FV1200 MPE, Olympus) optimized for near-infrared (near-IR) throughput and a 25× water objective (XLPlan N, 1.05 N.A., MP, Olympus) with high near-IR transmission for SRS microscopic imaging. A picoEMERALD system (Applied Physics & Electronics) supplied synchronized pulse pump beam (with tunable 720–990 nm wavelength, 5–6 ps pulse width, and 80-MHz repetition rate) and Stokes (with fixed wavelength at 1064 nm, 6 ps pulse width, and 80 MHz repetition rate). Stokes was modulated at 8 MHz by an electronic optic modulator. Transmission of the forward-going pump and Stokes beams after passing through the samples was collected by a high N.A. oil condenser (N.A. = 1.4). A high O.D. bandpass filter (890/220, Chroma) was used to block the Stokes beam completely and to transmit the pump beam only onto a large area Si photodiode for the detection of the stimulated Raman loss signal. The output current from the photodiode was terminated, filtered, and demodulated by a lock-in amplifier at 8 MHz to ensure shot-noise-limited detection sensitivity. The demodulated signal was fed into analog channel of the FV1200 software FluoView 4.1a (Olympus) to form image during laser scanning.
Xlplan n
Manufactured by Olympus
Sourced in Japan, United States
The XLPlan N is a high-performance optical microscope objective lens designed for a wide range of laboratory applications. It features a numerical aperture of 0.95 and a working distance of 0.2 mm, providing excellent optical performance and resolution. The lens is constructed with premium optical components to ensure durability and consistent image quality.
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Lab products found in correlation
Fv1200mpe, by Olympus (3 mentions)
Mai tai ti sapphire multi photon laser, by Spectra-Physics (3 mentions)
Fm1 43, by Thermo Fisher Scientific (2 mentions)
Fv1000mp microscope, by Olympus (2 mentions)
Ix61wi, by Olympus (2 mentions)
Uplansapo, by Olympus (2 mentions)
Fds1010, by Thorlabs (2 mentions)
Hf2li, by Zurich Instruments (2 mentions)
Fv1000mpe, by Olympus (2 mentions)
Bx51wi, by Olympus (2 mentions)
Tm4000, by Hitachi (1 mentions)
Fvmpe rs, by Olympus (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Fluo4 am calcium dye, by Thermo Fisher Scientific (1 mentions)
Pluronic acid f 127, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fast green dye, by Merck Group (1 mentions)
Fv1000, by Olympus (1 mentions)
Ultima 4, by Bruker (1 mentions)
Fv1000 laser scanning confocal, by Olympus (1 mentions)
20 protocols using xlplan n
1
Stimulated Raman Scattering Microscopy Setup
2
Visualizing Cell-Seeded Scaffolds with Multiphoton Microscopy and SEM
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Cell seeded scaffolds were visualized using a custom-built multiphoton microscope previously reported [39 (link)]. Coherent anti-stokes Raman scattering (CARS) imaging at 2911 cm-1 was used to image the PCL scaffold fibres, whilst simultaneously exciting two photon fluorescence (TPEF) from Phalloidin and DAPI stained cells. All images were acquired using a 25x/1.05 N. A water immersion lens (XLPlanN, Olympus), providing a maximum field of view of 509 μm on the sample. To measure infiltration of cells into the scaffold Z-stacks were acquired with 1 μm steps.
Cell seeded scaffolds were also visualized using SEM using a previously described osmium based method [40 (link)]. These osmium stained scaffolds were visualized using a Hitachi TM4000 tabletop SEM (Hitachi) with a 15 kV accelerating voltage and a 10mm working distance. These scaffolds were sputter coated with an Elmscope FLM-007 using gold.
Cell seeded scaffolds were also visualized using SEM using a previously described osmium based method [40 (link)]. These osmium stained scaffolds were visualized using a Hitachi TM4000 tabletop SEM (Hitachi) with a 15 kV accelerating voltage and a 10mm working distance. These scaffolds were sputter coated with an Elmscope FLM-007 using gold.
3
Intravital Imaging of Murine Liver
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Mice were anaesthetized via i.p. injection of Avertin (240 mg/kg). The antibodies and fluorescent dyes were injected via tail vein. Surgical preparation for liver intravital imaging was performed as previously described32 (link).The liver was observed by two-photon microscopy (FVMPE-RS, OLYMPUS) equipped with two infrared lasers (MAITAI HPDS-OL: 690 nm–1040 nm; INSIGHT X3-OL: 690 nm−1300 nm). The MAITAI laser was tuned to 940 nm for excitation of FITC or Dendra2. INSIGHT laser excitation was tuned to 1200 nm for simultaneous excitation of PE, eF 660 or DiD. Emitted light was detected using a 25 × 1.05 NA water lens (XLPlan N, OLYMPUS) coupled to a 4-color detector array. The operating room temperature was normally kept between 20 °C and 24 °C.
4
Multiphoton Imaging of Biological Samples
5
Fluorescence Lifetime Imaging and Confocal Microscopy
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FLIM was done as described previously (McCullock et al., 2020 (link)). Cells were transfected in either 35 or 60 mm culture dishes and imaged using a water immersion 25× objective (XL Plan N, 1.05 NA) mounted on an Olympus IX61WI upright microscope. A Mai Tai Ti:Sapphire multi-photon laser (Spectra Physics) was used for excitation with an 860 nm wavelength, a repetition rate of 80 MHz and pulse width of approximately 100 fs. Donor emission was filtered by a 480–20 filter and measured by a H72422P Hamamatsu hybrid avalanche photodiode. Time-correlated single photon counting was done using a Becker and Hickl card with a resolution of 25 ps. Using VistaVision software (ISS), donor fluorescence from the plasma membrane from individual cells was binned and fit with a single exponential function, consistent with the lifetime of CFP variant mTurquoise2 (Goedhart et al., 2012 (link)).
For confocal imaging, cells were transfected with Lck-CFP or CFP-tagged cASIC1 in 35 mm dishes. After 2 days, cells were stained with 2 mL of 7.5 µM FM1–43 (Invitrogen) immediately prior to imaging with an Olympus FV1000MP microscope using a 60× water immersion objective (U Plan SApo, 1.20 NA). CFP and FM1–43 were simultaneously excited with a 440 nm laser and emissions between 465 and 495 nm collected as CFP and 575 and 675 nm collected as FM1–43.
For confocal imaging, cells were transfected with Lck-CFP or CFP-tagged cASIC1 in 35 mm dishes. After 2 days, cells were stained with 2 mL of 7.5 µM FM1–43 (Invitrogen) immediately prior to imaging with an Olympus FV1000MP microscope using a 60× water immersion objective (U Plan SApo, 1.20 NA). CFP and FM1–43 were simultaneously excited with a 440 nm laser and emissions between 465 and 495 nm collected as CFP and 575 and 675 nm collected as FM1–43.
6
Widefield and Two-photon Calcium Imaging
7
Intraventricular Calcium Imaging of Embryos
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Prior to imaging, embryos were injected intraventricularly with Fluo-4AM calcium dye (17 μg of calcium dye Fluo-4AM, Molecular Probes) dissolved in 3 μl of 20% F-127 pluronic® acid (Sigma) in dimethyl sulfoxide (DMSO, Sigma) that was then diluted in artificial cerebrospinal fluid (ACSF, in mM: 125 NaCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 5 KCl, 20 D-glucose, 10 HEPES) to reach a final concentration 0.4 mM of Fluo with 13 mM FastGreen dye (Sigma) added for injection guidance.
Two-photon imaging was performed with Olympus Fluo View 1000MP system at the rate 1.2 fps at a resolution 512 × 512 via 25X objective (XLPlan N, Olympus). Excitation light was produced by Mai-Tai DeepSee laser (Spectra Physics).
Two-photon imaging was performed with Olympus Fluo View 1000MP system at the rate 1.2 fps at a resolution 512 × 512 via 25X objective (XLPlan N, Olympus). Excitation light was produced by Mai-Tai DeepSee laser (Spectra Physics).
8
Coherent Anti-Stokes Raman Scattering Microscopy
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Synchronized pump (tunable from 720–990 nm) and Stokes (fixed at 1064.2 nm) beams with both 6-ps pulse width and 80-MHz repetition rate are provided by a picoEmerald system from APE (Applied Physics & Electronic, Inc.). The intensity of the 1,064 nm Stokes beam was modulated sinusoidally by a built-in electro-optic modulator (EOM) at 8 MHz with a modulation depth of more than 95%. Spatially and temporally-overlapped pump and Stokes beams were coupled into an inverted laser-scanning microscope (FV1200MPE, Olympus). A 25× water objective (XLPlan N, 1.05 NA, MP, Olympus) was used. The forward-going pump and Stokes beams after passing through the samples were collected in transmission with a condenser (oil immersion, 1.4 NA, Olympus). A large-area (10 mm ×10 mm) Si photodiode (FDS1010, Thorlabs) was used for pump intensity detection after filtering the Stokes beam completely with two high-optical-density bandpass filter (890/220 CARS, Chroma Technology). The output current of the photodiode was then sent to a fast lock-in amplifier (HF2LI, Zurich Instruments) for signal demodulation.
9
Photostimulation-Driven Circuit Mapping
10
Ultrafast Laser Ablation Microscopy
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