The IL is located in state-of-the-art, environmentally controlled laboratories in the Forchheimer Medical Science Building, the Golding Building and the Price Center for Genetic and Translational Medicine/Block Research Pavilion. The IL is staffed with engineers and physicists who develop novel software and instrumentation to address the needs of individual investigators’ research. Recent development projects in the IL include a two-laser, multiphoton microscope, a multiphoton FCS microscope, a high-speed single molecule detection microscope and a fast switching, multichannel TIRF microscope.
Intravital Multiphoton Station
The Intravital Multiphoton Station is a commercially available multi-photon/confocal microscope (Olympus FV1000-MPE) multi-photon station equipped with a Mai-Tai automated Ti:Sapph laser with a Deep-See GVD compensator, six confocal laser lines (405, 458, 488, 515, 559 and 635nm) and four simultaneously acquiring channels. The microscope has been fitted with custom detection filter sets, environmental controls and anesthesia equipment that make this system a near turn-key solution for intravital imaging. The standard 25x 1.05NA objective is optimized for multi-photon imaging and allows both low and high magnification imaging with a single lens and an environmental chamber allows long time-lapse sessions.
Two-Laser Multiphoton Station
The Two-Laser Multiphoton Station is a custom-built, point-scanning multiphoton microscope. Based around an Olympus IX71 stand, it is equipped with two laser systems (a Ti:Sapph laser and a separate Ti:Sapph pumped OPO), and can excite fluorophores in the range of 690-1040nm as well as 1100-1600nm. This system thus extends the capabilities of multiphoton microscopy to enable the use of far-red fluorophores (those fluors with excitation peaks >550nm). With 4 simultaneously acquiring detectors, it is able to capture cell-cell interactions deep within living tissues. Its use of a high NA, low magnification and long working distance objective, combined with its mosaicing capabilities, allows the rapid switching between large field of view histological type imaging and high-magnification single-cell imaging. These capabilities make it an ideal experimental platform for intravital imaging.
Fluorescence Fluctuation Spectroscopy Station
The two-photon dual-color fluorescence fluctuation spectroscopy (FFS) microscope is built around an Olympus IX-71 inverted body and Coherent Chameleon mode-locked Ti-Sapphire laser. FFS exploits the fluctuating fluorescence signal from a very small observation volume created by the two-photon excitation to characterize the behavior of fluorescently labeled molecules. FFS is a highly sensitive, specific, non-invasive and quantitative technique that is ideal to study dynamics and interactions in living cells. It has been used to measure translation and rotation diffusion, transport and chemical interaction.
The FRET station (also known as Digital Station 5 or DS-5) is a multi channel FRET station capable of imaging in 22 different modes of operation and allowing CFP, YFP, GFP, Cy3, Cy5, CFP/YFP FRET and GFP/Cy3 FRET imaging. The station is optimized for live cell imaging and has an environmental chamber and a continuous reflective interference feedback focus (CRIFF) adjuster for long time-lapse applications. A Dual View emission splitting system allows simultaneous acquisition of two channels for higher speed acquisitions.
The high-speed station uses wide field based laser excitation while emitted light is detected on two deep cooled EMCCDs. While the principal setup is rather simple, this simplicity optimizes the overall detection efficiency by reducing the number of surfaces between objective and camera. This combination of widefield excitation, dual CCD collection and the sensitive optical configuration allow for rapid time lapse acquisition of very low signals in two channels. For example, in the past this microscope has been used to follow single mRNA molecules move through nuclear pores with 20ms temporal resolution.
The photomanipulation station consists of a wide field microscope featuring custom excitation using 2 laser lines (491nm and 561nm) and a sensitive EM-CCD. It also features a 405nm laser line that can be used for photoactivation. The size of the photoactivated area can vary from a diffraction-limited spot to a full field excitation using an adjustable lens system. A highly sensitive stage is used to move the region of interest to the photoactivating spot. Sinultaneous 2-color imaging on a single camera chip can be achieved using a dual view system (Photometrics).
The multiline TIRF station is an objective based total internal reflection fluorescence microscope built by modifying the Olympus single-arm TIRM system. The original laser sources have been replaced by five separate laser lines that can be rapidly and independently controlled (shuttered and intensity varied) via an acousto-optic tunable filter (AOTF). Custom mechanics and a motorized fiber positioned allow independent setting of TIRF angles for each laser line as well as rapid switching between epi and the separate TIRF modes. The station also features a motorized XY stage, environmental control chamber and a micro-injection apparatus.
The primary design consideration for the uncaging microscope is the ability to quantitatively photolyze UV-sensitive caging groups and image simultaneously. The uncaging source is a Q-switched tripled Nd:YLF laser, producing 5 ns pulses at 349 nm with ~ 100 µJ energy and a repetition rate varying from single shot to 1 kHz. The pulsed laser enables careful control of the uncaging dose delivered to the sample. The laser can be expanded to produce a collimated beam which overfills the back aperture, producing a diffraction-limited uncaging spot, or the beam can be defocused and apertured to produce a roughly circular uncaging spot of arbitrary size. The dichroics are designed for high reflectance of the UV laser, and the excitation filters are positioned directly in front of the metal halide lamp, out of the beam path of the laser. Furthermore, the emission filters are designed for enhanced blocking in the UV range. The net result is that one can uncage and image coincidentally, without mechanical switching of filters or shutters.