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2019 Tech Talk
The More (Photons) the Merrier: Intravital & Deep Tissue Imaging Using Non-Linear, Multiphoton Excitation
Basic Equipment Description for Grant Applications***
Multi Photon Intravital and Tissue Imaging (MITI) Core Facility
The MITI Core Facility, part of the Preclinical Imaging Center housed within the Fred & Pamela Buffett Cancer Center, is equipped with an Olympus FVMPE-RS Multiphoton Laser Scanning Microscope. This system is specially configured for deep tissue imaging in both living and fixed samples. Dual beam, multi photon excitation is accomplished using a Spectra Physics Dual line InSight X3 near infrared laser. The main output from the InSight laser is a tunable line ranging from 680 to 1300 nm with a second multiphoton excitation line at 1045 nm. This extended excitation range supports both traditional fluorophores (blue to long range red) and fluorescent proteins, as well as, second and third harmonic imaging. Potential signal crosstalk during simultaneous imaging is reduced by transitioning to sequential scanning (line or frame). Likewise, this system utilizes a Quadralign 4 axis alignment module to autocorrect to potential laser beam misalignment and pixel shift during multi-color excitation.
Equipped with 5, 10 and 25x objectives, Z-axis and xy stage motors, this system can image both intravital and fixed samples. Deep Tissue imaging is facilitated using a highly-specialized light path containing silver coated, high reflectance scanner mirrors equipped to increase detection of scattered and low intensity fluorescence. This system uses both a traditional Galvanometer scanner and a high-speed Resonant scanner capable of imaging 512 x 512 pixels at 30 frames per section or up to 438 frames per section when imaging 512 x 32 pixels. Two multi-alkaline and two gallium arsenide phosphide (GaAsP) are available for multichannel detection, the latter delivering optimum quantum efficiency and improved signal to noise ratios.
The Olympus FVMP-RS system is additionally equipped with 458 and 588 nm stimulation laser lines exclusively positioned for photobleaching, light stimulation and/or photoactivation of various fluorescent or caged constructs. In addition to automating a number of imaging procedures, the Olympus software is available for collecting and interacting with various types of data including optical Z-sectioning, 3D imaging, multi-location imaging at individual and multiple time points and fluorescence quantification.
Data Reproducibility and Rigor Statement for Grant Applications
To ensure consistent equipment performance, the MITI Research Core performs weekly maintenance on all instruments and maintains comprehensive preventative maintenance and service contracts with Olympus and Newport/Spectra Physics to maintain the research core’s microscope and laser systems in optimal condition. To further ensure consistent reproducibility and rigor during ongoing/long term imaging experiments (individual studies which may vary greatly in system configuration), both IR laser output (power at a given wavelength) and detector responses (intensity under identical collection parameters) are measured prior to each imaging session and are not allowed to vary by more than 10% throughout the complete imaging process. In addition to maintaining equivalent system functioning, all imaging configurations for a given experimental series are kept identical throughout the imaging series.
*** Please notify Dr. Jensen Smith to discuss research proposal development, inquiries regarding logistics of desired studies, and/or updates in proposed or active funding utilizing the imaging core in a timely fashion.
Tracking of grants, publications, presentations, and activities supported by and/or proposing use of the MITI Research Core is extremely important for maintaining and expanding the research core’s equipment and services.