Two-photon imaging was performed with a commercial microscope (B-scope, Thorlabs) with 925 nm excitation from a Ti-Sa laser (Spectra-physics). Images were acquired at a framerate of ∼28 Hz. Each imaging frame consisted of 512 × 512 pixels, and spanned 1132 × 982 μm. Images were acquired for ∼2.4-min-long segments, with intersegment intervals of 7 s. Trials with missing data (trials which overlapped with the intersegment intervals) were not analyzed. A custom (MATLAB, RRID:SCR_001622 ) program performed motion correction (Full-frame cross-correlation correction) on imaging frames.
Ti sa laser
Manufactured by Spectra-Physics
Sourced in United States
The Ti-Sa laser is a solid-state laser that utilizes a titanium-doped sapphire (Ti:sapphire) crystal as the active medium. The Ti-Sa laser can generate tunable wavelengths of light in the near-infrared region of the electromagnetic spectrum, typically between 650 and 1,100 nanometers.
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Lab products found in correlation
6 protocols using ti sa laser
1
Two-Photon Imaging Microscopy Protocol
2
Non-invasive Imaging of Parchment Artifacts
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A custom-built upright microscope based on a femtosecond Ti:Sa laser (Mai Tai, SpectraPhysics) tuned to 860 nm was used as previously described15 (link). A high numerical aperture air objective (20×, NA 0.75, Olympus) was used for noncontact imaging and 0.7 µm µm lateral by 4 µm axial resolutions near the sample surface were achieved with 860 nm excitation. Simultaneous 2PEF (GG400 high-pass filter, Schott) and SHG (427/10 interferential filter, Semrock) signals were epi-detected by using photon-counting photomultiplier tubes (P25PC, Electron Tubes). 2PEF and SHG signals are represented in false colors, respectively red and green. The acquisition pixel rate was 200 kHz that corresponds to around one frame per second for 640 × 640 pixel wide images (480 × 480 μm2 with 0.8 μm pixel size). Laser power at the objective focus was 8 to 20 mW, without any observable damage in the studied samples.
Several images were acquired in different regions of every sample: 5 images in untanned skin, 13 and 20 images in respectively preserved and altered areas of “1639” parchment. A large image of size 1.3 × 1.1 mm2 was acquired in the maritime map by stitching 4 × 3 images at sequential positions.
Several images were acquired in different regions of every sample: 5 images in untanned skin, 13 and 20 images in respectively preserved and altered areas of “1639” parchment. A large image of size 1.3 × 1.1 mm2 was acquired in the maritime map by stitching 4 × 3 images at sequential positions.
3
Fluorescence Lifetime Imaging of GFP
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Determination of fluorescence lifetime was done as previously described (Hofman et al., 2008 (link)). Cells were transfected with the indicated constructs and subsequently lifetime of GFP was determined using a Nikon PCM 2000 CSLM equipped with a fluorescence lifetime module (LiMo, Nikon), which captures four images in four consecutive time gates of approximately 2 ns each. The excitation light was provided by a frequency doubled picoseconds pulsed Ti:Sa laser (Tsunami, Spectra Physics). For imaging a NA=1.20/40x water emission objective and the medium-sized pinhole were used. The four-gate intensity decays recorded for each pixel were fitted with a monoexponential decay using the LiMO software, meaning that also for probes with multiexponential decays, one single average lifetime is observed. Lifetimes described in this study should therefore be considered as average lifetimes. Intensity thresholding was performed to remove background-(glass) and auto-fluorescence. To rule out effects of cell-to-cell variation in FRET efficiency, lifetimes of at least four cells were determined and a student T-test was performed to determine statistical significances. Fluorescent lifetimes were normalized against the lifetime of GFP alone.
4
Two-photon Imaging with High-Speed Acquisition
5
Multimodal Fluorescence Imaging of 3D Cell Cultures
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Fluorescence intensity and lifetime images of the 3D cell cultures were acquired using an LSM 880 (Carl Zeiss, Jena, Germany) laser scanning microscope equipped with a FLIM module Simple Tau 152 TCSPC (Becker & Hickl GmbH, Berlin, Germany). A water immersion objective C-Apochromat 40×/1.2 NA W Korr was used for image acquisition. During image acquisition, the cells were maintained at 37 °C and 5% CO2.
Two-photon fluorescence of NAD(P)H was excited with a femtosecond Ti:Sa laser (MaiTai, Spectra-Physics, Milpitas, CA, USA, repetition rate 80 MHz, pulse duration 140 fs) at 750 nm wavelength and registered in the range of 455–500 nm. The average power applied to the samples was ~6 mW, and the approximate rate of photon counting was 1–2 × 105 photons/s. Image collection time was 60 s. Two-photon excitation of DOX fluorescence was performed at 780 nm and emission was registered using 690/50 filter. The photons were collected for 90 s. The average power applied to the samples was ~6 mW, and the approximate rate of photon counting was 1–2 × 105 photons/s. In one-photon mode, DOX fluorescence was excited at 488 nm and registered in the range of 540–650 nm.
FLIM images of NAD(P)H and DOX were acquired sequentially from the same 5–7 randomly selected fields of view in each culture dish.
Two-photon fluorescence of NAD(P)H was excited with a femtosecond Ti:Sa laser (MaiTai, Spectra-Physics, Milpitas, CA, USA, repetition rate 80 MHz, pulse duration 140 fs) at 750 nm wavelength and registered in the range of 455–500 nm. The average power applied to the samples was ~6 mW, and the approximate rate of photon counting was 1–2 × 105 photons/s. Image collection time was 60 s. Two-photon excitation of DOX fluorescence was performed at 780 nm and emission was registered using 690/50 filter. The photons were collected for 90 s. The average power applied to the samples was ~6 mW, and the approximate rate of photon counting was 1–2 × 105 photons/s. In one-photon mode, DOX fluorescence was excited at 488 nm and registered in the range of 540–650 nm.
FLIM images of NAD(P)H and DOX were acquired sequentially from the same 5–7 randomly selected fields of view in each culture dish.
6
Longitudinal Imaging of Cortical Activity in Mice
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Mice (CaMK2-tTA::tetO-GCaMP6s) used for imaging experiments were implanted with a head plate and a cranial window over the forelimb region of M1 on their right hemisphere, and then underwent the recovery and water restriction procedures described above. After 2 to 7 days of task familiarization as described in the section “Behavioral training of the joystick task,” we started imaging cortical activity with excitation at 925 nm from a Ti-Sa laser (Spectra-Physics) at ~28 frames/s using a two-photon microscope (B-SCOPE, Thorlabs). For each mouse, a single field of view in the forelimb region of M1 (covering 472 μm by 508 μm at a depth of approximately 250 μm beneath the dura in layer 2/3) was longitudinally imaged over the course of 60-day training. Although a single field of view was imaged throughout the experiment, data from each day were processed independently without limiting our analyses to neurons present in all days. Only the imaging days with satisfying image clarity and no other technical issues were analyzed (54 ± 3 days, mean ± SD across five mice).
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