Two- or Three-photon microscopy uses two (or three) photons in the infrared spectral range instead of one photon in the visible spectral range to excite fluorophores. Due to less scattering of infrared light this microscopy technique can image much deeper into the tissue than conventional “single”-photon techniques. In addition, two (or three) - photon microscopes excite fluorophores only in the plane of focus and thus provide z-resolution comparable to confocal microscopy. Multi-photon-microscopes are therefore ideally suited for all kinds of photo-manipulation experiments. The LMF has most of the accessory equipment like heating pads, isofluran vaporizer, puls oximeter, and dental drill for surgery and imaging of narcotized as well as awake animals.
For details please see section Confocal Scanning Microscopes
Zeiss LSM7MP (two setups)
Our Zeiss LSM7MP are upright two-photon setup with an infrared lasers and up to four ultra sensitive non-descanned detectors (NDDs) based on gallium arsenide phosphide (GaAsP) technology. A pair of them is directly mounted to the objective holder for the most efficient collection of scattered fluorescence. The LSM7MP-2 uses a Chameleon UltraII laser (Coherent) where wavelengths from 700 nm to 1050 nm can be chosen and the LSM7MP is equipped with an InSight X3 laser (SpectraPhysics) where wavelengths from 700 to 1300 nm are available. The setups are ideally suited for in vivo imaging of narcotized or awake animals. In addition, a broad selection of specialized clearing objectives (BABB, CUBIC, Scale etc.) are available and match almost every clearing strategy. Thus, high resolution imaging of cleared specimen is possible on these setups as well.
Thorlabs resonant setup
This custom made microscope is mounted hanging below an anti-vibration table. The unique design allows to image behaving animals in huge and complex arenas underneath the table. The infrared laser (700-1050nm) is attenuated by an EOM which provides blanking and thus reduces phototoxicity. The system is equipped with two ultra-sensitive gallium arsenide phosphide (GaAsP) detectors in a non descanned arrangement. By combining the resonant scanner (8kHz – 30Hz@512lines) with a piezo driven z-stepper, 3D imaging with high temporal resolution is possible.
Thorlabs 3P Setup
Our 3Photon setup is a custom-made microscope system based on Thorlabs components with a 2P laser (680 and 1040nm) and a 3P laser (NOPA/Spirit from Spectra Physics, 4Mhz >1.2mJ pulse energy). Wavelengths around 1300 nm and 1700 nm can be used for imaging green or red fluorescent dyes in the three-photon mode. The two-photon mode can be used as well for comparison reasons, for example. Compared to two-photon microscopy three-photon microscopy can penetrate much deeper into scattering tissue and provides significantly increased signal-to-noise ratios. Thus, the setup is optimized for deep tissue imaging of awake animals.
LaVision TRIM ScopeII with OPO
Our LaVision Trim ScopeII with OPO is a two-photon setup with a novel design which generates a large space around the objective (invivo bridge). Therefor, it is ideally suited for imaging behaving animals in various arenas. The microscope incorporates three excitation paths. Two Ti:Sapphire lasers deliver near infrared light (680-1040nm with a split at 820nm) light which can be used for imaging or stimulation. An optical parametric oscillator (OPO) delivers far infrared (>1040nm) radiation for the excitation of red fluorescent dyes. Each laser line can be guided to a galvo scanner or a resonant scanner, which are independent. The alignment of the focal spots allows simultaneous imaging and stimulation. The resonant scanner (8kHz) is perfect for the acquisition of time-lapse at high frame rates (30Hz @ 512 lines). A combination with a piezo driven z-stepper allows fast 3D imaging. The fluorescence is detected by two high sensitive gallium arsenide phosphide detectors. Four non-descanned detectors (NDD) can be utilized to collect the emission generated by galvo scanner light path. Every detector is ultraAsensitive in a specific spectral range. Of course the setup is ideally suited for in vivo imaging of narcotized animals (mouse and rat), too. The LMF can provide the accessory equipment like heating pads, isofluran vaporizor, puls oximeter, and dental drill for surgery and imaging of narcotized animals.
For details see the Super Resolution section