Maitai ehp

We performed in vivo two-photon imaging using a Prairie Ultima IV two-photon microscope (Bruker Nano, Inc., formerly Prairie Technologies) and a Ti: Sapphire laser (Mai Tai eHP, Spectra Physics). A 25× objective lens (1.1 N.A., Olympus) with zoom 4× scanning model was used for high-resolution dendritic imaging, which covered a field of view of 136 × 136 μm. Fast resonant scanning mode (512 by 512-pixel resolution, up to 30 frames per second and averaging every four frames) was used in all functional characterization experiments. An even faster scanning mode (64 by 64 pixel at 226 frames per second) while zoomed 8× achieved high temporal resolution imaging (without frame averaging, Fig. 5e). To obtain the orientation map, we imaged with a 16× objective lens (0.8-N.A., Nikon) under zoom 1× , covering a field of view of 850 × 850 μm. We collected 23 neurons in total from three monkeys, sampling neurons with both spiny and smooth dendrites. As we used the hSyn promoter, sampled neurons were either excitatory or inhibitory neurons. The only criteria we used in picking neurons was that the dendritic morphology should be distinct from the background fluorescence.

Label-free multi-photon imaging was used to study mouse skin and skull. To perform SHG image acquisition, we utilized an upright multi-photon excitation microscope (A1R-MP, Nikon, Inc.). The microscope was equipped with a water immersion objective lens (CFI75 Apo 25 × W MP, NA:1.1, Nikon, Inc.) and a Ti:Sapphire laser oscillator system (MaiTai eHP, Spectra-Physics, Inc.) with no additional optical modules for generating polarized light. The images were acquired as z-stack image sequences with a step size of 5 µm ranging from the deepest portions to the surface of the tissue. All SHG images were acquired at an excitation wavelength of 960 nm. To acquire the SHG signal and autofluorescence from tissue, we divided signals at 495 nm by employing a dichroic mirror^{54 (link),55 (link)}. The field of view of the acquired images was 0.5 mm × 0.5 mm, and the resolution was 1024 × 1024 pixels, i.e., the pixel size was 0.5 µm.

In order to perform SHG image acquisition, we utilized an upright multi-photon excitation microscope (A1R-MP, Nikon, Inc.). The microscope was equipped with a water immersion objective lens (CFI75 Apo 25 × W MP, NA:1.1, Nikon, Inc.) and a Ti:sapphire laser oscillator system (MaiTai eHP, Spectra-Physics, Inc.) with no additional optical modules for generating polarized light. To observe intact cartilage tissues, excised femurs were embedded in 1% agarose and the medial condyle was exposed under the objective lens of the microscopy system as previously described^{14 (link)}. The images were acquired as z-stack image sequences with a step size of 3 μm ranging from the deepest portions to the surface of the cartilaginous tissue. To observe tissue sections, sections mounted on glass slides were placed on the microscope stage before acquiring xy-images. All SHG images were acquired at an excitation wavelength of 880 nm. To detect the SHG signal, we employed a dichroic mirror at 458 nm and an emission filter at 440/40 nm (center wavelength/bandwidth). The field of view of the acquired images was 0.5 mm × 0.5 mm and the resolution was 2048 × 2048 pixels, i.e. the pixel size was 0.25 μm. The images originally recorded as 12-bit gray level images were converted to 8-bit gray level images for GLCM computation.

SHG imaging was performed by multi-photon microscopy (A1R-MP, Nikon, Inc.) wherein the microscope was equipped with a water immersion lens (CFI75 Apo 25 × W MP, NA:1.1, Nikon, Inc.) and a Ti:sapphire laser oscillator (MaiTai eHP, Spectra-Physics, Inc.) as described previously^{20 (link),48 (link)}. Excitation wavelengths of 950 nm with emission filter sets, the dichroic mirror 495 nm, and the shortpass filter 492 nm were used to detect the SHG signal. The images were acquired as z-stack image sequences with a step size of 2 μm. For observing whole tissue sections, 4 × 4 or 5 × 5 images (each 0.5 mm × 0.5 mm field of view, size 512 × 512 pixels) were recorded and stitched to create large images using NIS Elements software (Nikon, Inc.). The images were originally recorded as 12-bit gray-level images and subjected to the maximum intensity projection.

After a recovery period of 10 days after the second surgery, the animals were trained to maintain eye-fixation. Two-photon imaging was performed using a Prairie Ultima IV (In Vivo) 2P microscope (Bruker Nano, Inc., FMBU, formerly Prairie Technologies) powered by a Ti: Sapphire laser (Mai Tai eHP, Spectra Physics). The wavelength of the laser was set at 1000 nm. With a 16 × objective (0.8 N.A., Nikon), an area of 850 μm × 850 μm was imaged. A standard slow galvonometer scanner was used to obtain static images of cells with high resolution (1024 × 1024). The fast and resonant scan (up to 32 frames per second) was used to obtain images of neuron activity. The images were recorded at 8 frames per second by averaging each 4 frames. Infected cells of up to 700 μm in depth were imaged. We primarily focused on cells that were 160 μm to 180 μm deep, which included a high density of infected cells.

For TR-PL experiments samples were excited with the wavelength-tunable output of an OPO (Radiantis Inspire HF-100) (Radian, Barcelona, Spain), itself pumped by the fundamental of a Ti:sapphire fs-oscillator (Spectra Physics MaiTai eHP) at 820 nm. The repetition rate of the fs pulses was adjusted by a pulse picker (APE Pulse Select). Typical pulse energies were in the range of several nJ. The PL of the samples was collected by an optical telescope (consisting of two plano-convex lenses) and focused on the slit of a spectrograph (PI Spectra Pro SP2300) and detected with a Streak Camera (Hamamatsu C10910) (Hamamatsu Photonics, Hamamatsu, Japan) system with a temporal resolution of 1.4 ps. The data was acquired in photon counting mode using the Streak Camera software (HPDTA) measured at room temperature and exported to Origin Pro 2015 for further analysis.

Images were acquired as described [15 (link), 17 (link), 18 (link)], with slight modifications. Cells and matrices in the in vitro reconstruction system were observed by 2-photon microscopy every week (7 days ± 1 day) with a multiphoton confocal microscopy system (A1R + MP, Nikon, Tokyo) with a titanium-sapphire laser (wavelengths: 680–1050 nm, repetition rate: 80 MHz, pulse width: 70 fs; Mai Tai eHP, Spectra-Physics), a water-immersion objective lens (numerical aperture: 1.1; CFI75 Apo 25 × WMP, Nikon) and the following emission filters: 492-nm short-pass for second harmonic generation (SHG), 525/50-nm band-pass for EGFP, and 575/25-nm band-pass and 629/56-nm band-pass for tdTomato.
The SHG from collagen fibers, the emissions from EGFP expressed in osteoblasts and osteocytes, and the tdTomato emission from osteoclasts were all observed using excitation light at a wavelength of 930–950 nm. Three-dimensional images were taken at Z-steps of 1 µm. Some zoom-up images were captured at Z-steps of 0.5 µm. The observation began at co-culture week 0 and ended at co-culture week 5. The same position was observed weekly. Images were taken from six regions in each dish. After each observation, fresh medium was added after the cells had been rinsed twice with PBS to prevent contamination. Three series of experiments were conducted.