Leica TCS SP8 Resonant-scanning confocal/multiphoton microscope is configured for both multiphoton and confocal imaging on an upright stand. Confocal fluorescence excitation at 405, 448, 488, 552 and 633 nm with multiphoton fluorescence excitation provided by a Spectra-Physics MaiTai DeepSee titanium-sapphire laser, providing tunable illuminations from 690 to 1040nm. The system is configured with 4 internal (descanned) PMTs and 4 external (non-descanned) detectors – 2 PMTs and 2 HyD hybrid detectors. The SP8 MP includes both standard galvanometric scanning for imaging at up to 1800 Hz and resonant scanning for imaging up to 8000 Hz, supporting full frame imaging at up to 30 fps. The automated stage supports automatic time lapse studies of multiple fields and tile acquisitions.
Leica TCS SP8 Dive confocal/multiphoton microscope has confocal capabilities equivalent to those of the system described above, but is mounted on an inverted stand. This system is also equipped with several features that extend our capabilities for intravital microscopy. First, it is equipped with two IR lasers, a Spectraphysics MaiTai DeepSee laser and a Spectraphysics Insight laser system that extends multiphoton excitation out to 1300 nm, improving intravital microscopy of red fluorescent proteins. The two-laser system also facilitates simultaneous excitation of multiple, spectrally separated probes. Second, this system is configured with 4 spectral detectors on the non-descanned pathway (2 PMTs and 2 HyD detectors), improving detection and distinction of multiple probes. Finally, this system is equipped with a two channel 2-photon ISS Fast FLIM fluorescence lifetime system and a motorized stage supporting tile acquisitions and time-lapse multiple filed studies.
Olympus Fluoview 1000 MPE confocal/multiphoton microscopesystem supports both confocal and multiphoton microscopy. Fluorescence emissions may be collected either with internal (descanned) detectors (one photomultiplier tube and two spectral detectors) or with a 4-channel external (non-descanned) detector module. Confocal fluorescence excitation is provided at 405, 488, 515, 559 and 635 nm and multiphoton fluorescence excitation is provided by a Spectraphysics DeepSee tunable titanium-sapphire laser, providing tunable illuminations from 710 to 990nm. The system is mounted on an IX-81 inverted microscope stand and is equipped with an automated stage, supporting automatic time course studies of multiple fields and tile acquisitions.
Nikon live cell imaging system is configured for high-speed imaging of living samples. Built around an inverted Nikon Eclipse Ti stand, it supports both confocal and widefield imaging. Confocal microscopy is based upon a Yokogawa CSU-X1 spinning disk, with laser illumination provided at 442, 488, 514 and 561 nm via an Agilent launch with images collected using a Photometrics Prime 95B Scientific CMOS. Widefield epifluorescence microscopy can be conducted using a Xenon lamp source, with optics suitable for excitation from 340 nm to 700 nm, with images collected by a Hamamatsu Orca Flash 4.0LT Scientific CMOS. It is equipped with a Perfect Focus system, a motorized stage that supports automatic time course studies of multiple fields and a stage-top incubator for maintaining a heated and CO2 dependent environment to ensure stable long term imaging of cultured cells.
Keyence BZ-X810 microscope system is an automated high-content imaging system with advanced automated image collection and analysis capabilities, supporting a variety of cellular and biochemical assays of living or fixed specimens mounted in dishes, flasks, multi-well plates, and microscope slides or chambers. System supports imaging in fluorescence, brightfield, and oblique illumination with a highly sensitive, cooled CCD camera and a built-in darkroom, generating publication-quality images. It is equipped with DAPI, GFP, RFP, Cy5, and Cy7 epi-filter cubes, 4x/0.1, 10x/0.3, and 20x/0.75 objectives, automated stage, and a stage-top environmental incubation system supporting time-lapse imaging of live cells. Automated quantification can be performed with the Keyence analysis application augmented with three additional module licenses (Hybrid Cell Count, Time-lapse, Advanced Observation).
Akoya Phenocycler-Fusion multiplex fluorescence imaging system is an integrated high-speed imaging coupled with automated fluidics control covering a full workflow from staining to analysis. It supports multiplexed fluorescence imaging of 40+ probes in the same sample, using a proprietary process of repeated sequential imaging and stripping of fluorescently-labeled DNA-barcode probes. The Fusion microscope is also capable of high throughput slide scanning with filters for Brightfield and the proprietary Opal dyes between 440 nm and 780 nm.
Molecular Devices IXM XLS microscope system is an automated high-content imaging system with advanced automated image collection and analysis capabilities, supporting a variety of cellular and biochemical assays of living or fixed specimens mounted in dishes or multi-well plates.
ISS Alba Fast FLIM system is a high-speed fluorescence imaging system that supports frequency-domain-based fluorescence lifetime imaging, fluorescence correlation microscopy and PIE. It is mounted on an Olympus IX71 microscope stand equipped with Olympus 10x/0.4, 20x/0.75, 40x/1.3 oil, and 60x/1.2W objective lenses coupled to a piezo stepper motor. The ISS FastFLIM system is coupled to the Alba LCSM. The lasers (10mW 405nm and 5mW 448nm diode lasers, and a 200 mW Supercontinuum White Light Laser) are modulated by the FastFLIM system at frequencies of 10 MHz or higher, and measurements can be analyzed using multiple digital harmonics. FRET FLIM measurements can be acquired for cyan, green or yellow donor probes, and the donor/acceptor signals are detected with two identical APDs. The APDs are capable of single-molecule detection, and the system is equipped with the ISS Alba FCS module. This enables one channel FCS or two channel cross-correlation FCS (FCCS). The ISS VistaVision firmware is also optimized for pulsed interleaved excitation (PIE) that enables crosstalk-free measurements of FCCS and measurements for FRET-FLIM from two independent probes simultaneously. The IS VistaVision software is used for phasor analysis of the FLIM measurements. The system is equipped with a stage-top environmental control system maintaining live specimen at 37º C and 5% CO2.
Image analysis, data management and storage - Four computer workstations, each with multicore Xeon processors, up to 256 GB video RAM, and up to 128 GB of system memory run a variety of image processing software (Metamorph, ImageJ, Autoquant, Imaris, Halo, Fluorender and the CODEX software suite) as well as Adobe Photoshop and Premiere to support preparation of publication-quality figures and videos. All microscopy systems and computers are connected to a 1000Base-TX Ethernet switch. The ICBM is also supported by the supercomputing infrastructure provided by Indiana University.