The Multiphoton Imaging Facility is a sub-core of the Optical Core facility. It is housed in a dedicated single-use laboratory.

Multiphoton microscopy (MPM) is a nonlinear optical technique that utilizes femtosecond-pulsed near-infrared light as the illumination source. MPM can be used both for fluorescence imaging (tissue autofluorescence, cells expressing fluorescent proteins, as well as exogenous contrast agents), and for imaging utilizing a higher order scattering phenomenon termed Second Harmonic Generation (SHG, generated by oriented non-centrosymmetric tissue components such as collagen and oriented microtubules). Some advantages of MPM over conventional single-photon imaging include: (i) greater imaging depth (0.5 mm or more in tissue), (ii) imaging without the use of exogenous contrast agents (using tissue autofluorescence and SHG signals alone), and (iii) minimal photodamage to tissue, allowing high-resolution intravital imaging, including studies requiring repeated imaging.

Available Equipment

Human colonic mucosa showing crypts, separated by lamina propria. Cells are color coded green, and the collagen component of lamina propria is color-coded red. Small, bright green structures are lymphoid cells in the colon mucosa. No exogenous contrast used.

Custom-built Multiphoton Microscope, custom-assembled by Prof. Warren R. Zipfel, Associate Professor, Department of Biomedical Engineering, Cornell University, Ithaca, and Director, Developmental Resource for Biophysical Imaging Opto-electronics (DRBIO) program, also at Cornell University. Built around an upright Olympus BX61WI frame, with an adjustable height ASI stage. Excitation source is a femtosecond pulsed Ti-Sapphire laser (Mai Tai HP, Spectra-Physics, Mountain View, CA), tunable from 700 to 1020 nm. A BioRad 1024 scan system provides raster scanning of the sample. Images can be simultaneously acquired in three detection channels. In addition to 3 Hamamatsu Bialkali PMTs, we also have a GaAsP detector for specimens with relatively long wavelength emissions. The system also has an Olympus DP72 color CCD camera for capturing widefield images.

Olympus FluoView FV1000MPE Multiphoton Microscope, composed of a BX61W1 frame and an adjustable height Prior stage. The excitation source is a Mai Tai DeepSee, with automated dispersion compensation, and is tunable between 690 - 1040 nm. The system has four non-descanned detectors (NDD). Thus, non-overlapping signals from four different tissue sources can be simultaneously imaged. In addition to the Mai Tai DeepSee, the system also has a 405 nm UV laser, which can be used for uncaging and photoactivation, in conjunction with Multiphoton imaging. In addition to standard objectives, we have specialized IR-transmitting objectives for MPM, as well as two microprobe objectives from Olympus (6x and 20x; tip diameter: 1.3 mm).

Stereo Dissection Microscope, the Olympus SZX16, with zoom up to 16X. Both transmitted light and fluorescence illumination are available. Equipped with a DP20-5E digital color camera (shared with the histology microscope).

Human bladder biopsy showing low grade papillary carcinoma. Urothelial cells are color-coded green, and the collagen component of the fibrovascular core is color-coded red. No exogenous contrast used.

Animal anesthesia and surgery (for intravital imaging) with a dissection area that contains:

• Mouse/rat anesthesia equipment (inhalation anesthesia using isofluorane from VetEquip);
• Dissection microscope (see above) for microsurgery
• Carbon dioxide euthanasia setup for mice and rats
• Mouse pulse oxymeter
• Basic set of veterinary surgical tools and supplies.

Histology Microscope, an Olympus BX41 inverted microscope, equipped with a DP20-5E digital camera (shared with the dissection microscope).

Biosafety for imaging human tissue via a laminar flow biosafety chamber from NuAire (Plymouth, MN).

Image analysis workstation, equipped with MetaMorph image analysis software, Adobe Photoshop CS4 and Microsoft Office, IQBase image management database from Media Cybernetics.