High Spatial and Temporal Resolution Imaging Core (HSTRI)

Under the supervision of Dr. Normand Leblanc, Ph.D., the HSTRI Core aims to establish, maintain, and operate a facility providing state-of-the-art high spatial and temporal resolution imaging methodologies for studying cellular structure, signaling pathways, and function in health and disease. Recent advances in microscopy have produced a new generation of commercially available instruments and molecular tools that enable biomedical researchers to investigate the relationships between structure and function in biological systems with unprecedented spatial and temporal resolution, near or below the diffraction limit. These new technologies allow visualization of the ultrastructure of cells and how it impacts localized signaling pathways. Training and advising investigators in the use of the new technologies as well as educating them about the scientific possibilities offered by these technologies, is a crucial part of maximizing the Core's impact on cardiovascular as well as other areas of research.

Director and Staff

Core Director

Normand Leblanc, Ph.D.
Email: nleblanc@unr.edu
Phone: (775) 784-1420

Microscopy and Imaging Specialist

Peter Blair, Ph.D.
Email: pblair@med.unr.edu
Phone: (775) 291-8305

Instruments Supported by the Core

A. Microscope Systems

Leica Stellaris 8 Confocal Microscope 

The HSTRI Core manages a Leica Stellaris X8 confocal microscope housed in room 47 of the Savitt Medical Sciences Building. It is composed of a Leica DMi8 inverted fluorescence microscope equipped with Adaptive Focus Control & Closed Loop Focus (20 nm re-positioning accuracy), OKO Labs opaque microscope enclosure environmental control with active mixing CO2, Humidity and Heat. It comprises a LED3 light engine for eyepiece visualization with filter cubes (DAPI, CFP, GFP, Rhod LP) and a scanning XY Stage. The system is equipped with Assay Editor for plate screening, super Z high speed / high precision Z galvo stage for XYZ, XYZT, XZY and XZYT high speed imaging. The system has four objectives: PL APO 10x/0.40 CS2 (Air) - 2.74 mm FWD; PL APO 20x/0.75 IMM CORR CS2 (H2O, Silicone, Glycerol, Oil) - 660 µm FWD; PL APO 93x/1.30 STED motorized correction collar (Glycerol) - 300 µm FWD; PL APO 100x/1.40 STED (Oil) 130 µm FWD; and PL APO 86x/1.20 W motorized correction collar (Water). The system is also equipped with the White Light Supercontinuum Laser Extreme (440 nm-790 nm) and a 405 nm Diode - 50 mW CW, a high performance 8 channel crystal Based Acousto optical beam splitter with 10 µs switch time, a Tandem Scanner FOV + 8K Resonant Point Scanner with prism based spectral imaging (extended red range 410 nm - 850 nm). Light detection is accomplished via Power HyD Detectors including 2 Spectral HyD S (high QE/PDU, highest dynamic range, high light level OK, photon counting, spectral, FLIM), 2 Spectral HyD X (high QE/PDU, high dynamic range, photon counting, spectral, ideal for FLIM), 1 Spectral HyD R (red optimized high QE/PDE 720 nm - 850 nm, high dynamic range, photon counting, spectral, ideal for FLIM), and a PMT based brightfield detector. The Stellaris X8 is also capable of performing Lightning with Expert Mode (spectral, multi-channel super-resolution imaging: 120 nm XY & ~200 nm Z resolution at 1.4 NA; works with all detectors and all objectives), and 3D Tau STED nanoscopy with 592 nm / 660 nm / 775 nm depletion lasers (93x & 100x STED WHITE objectives (sub 50 nm XY resolution; sub 130 nm Z resolution; multi-channel, multi- color standard, gated or Tau STED, and Spectral Fluorescence Lifetime Imaging (FLIM; FALCON Fast FLIM, FLIM FRET, Phasor Analysis; Tau sense average photon arrival time with gating, contrast and separation). The system is controlled by LAS X Software running on a high-performance HP Z6G4 computer workstation comprising 2 HD monitors, 192 GB RAM / 6 TB HDD storage, Nvidia Quadro RTX 6000 graphics with 24 GB memory, 4608 CUDA cores and 2 Thunderbolt ports (TB3).

Leica Stellaris 5 Confocal Microscope

The Leica Stellaris 5 confocal system consists of a DMi8 inverted automated fluorescence microscope, a computer table, a high-end Windows 10-based computer (HP CUDA Workstation) and a 38" high-resolution curved monitor, an optical table & air compressor for vibration-free imaging, 6 objectives (HC PL FLUOTAR 5x/NA 0.15, Obj. HC PL APO 10x/NA 0.40 CS2, HCX PL FL L 20x/NA 0.40, HC PL APO 20x/NA 0.75 CS2, HC PL APO 40x/NA 1.30 Oil CS2, and HC PL APO 63x/NA 1.40 Oil CS2), 6 solid-state diode lasers (405, 448, 488, 514, 561, and 638 nm) for multicolor imaging, a fast laser scanning head (tunable speed large FOV 22 mm linear, cooled point scanner), motorized DIC and phase contrast, and 4 Peltier-cooled SuperResolution HyD S High PDE SiPM Detectors allowing for true photon counting, power counting & analog modes, and BF imaging. As for the Stellaris X8 microscope, the system also has Adaptive Focus Control with 20 nm repositioning accuracy. It displays high temporal (131 FPS, 512 x 16 x 4 Spectral + 1 BF for 655 FPS total) and spatial (8K x 8K) resolution. LAS X Software controls this instrument and is capable of 3D GPU-based visualization, online linear unmixing, live data mode, FRET, FRAP and FLIP imaging modes.

Olympus-Based Spinning Disc and TIRF Confocal Microscope

A spinning disc confocal and TIRF imaging system is built around an Olympus IX83 microscope, allowing seamless switching between confocal and TIRF imaging modalities. The microscope is equipped with 20x, 40x, 60x, and 100x objectives and is housed in room 45 of the Savitt Medical Sciences Building. The system contains an automated XY stage for multi-position acquisition, as well as a TrueFocus Zero drift automatic focus. The illumination system of this microscope consists of an Andor Integrated Laser Engine (ILE) and Borealis with 405-, 488-, 538-, and 640-nm solid-state lasers and integrated acousto-optic tunable filter (AOTF). The spinning disc confocal side consists of a Yokogawa CSU-21 Nipkow spinning-disk confocal scanner unit and an iXon Ultra897 cooled EMCCD camera. The TIRF side contains a 2-line motorized Olympus IX3 CellTIRF Illuminator and an iXon DU-897 cooled EMCCD camera. An Oko-Touch stage-top incubator provides temperature and CO2 regulation for long-term imaging of live cells. System control and data acquisition are provided by CellSens 3 software running on a Windows 10-based PC. This microscope is also equipped with a Rapp Optics flash photolysis system. SparkAn, a powerful Ca2+-image data analysis software package provided by the laboratory of Drs. Adrian Bonev and Mark Nelson of the University of Vermont, is available for image analysis.

4-Line Olympus Total Internal Reflection Fluorescence (TIRFM) and FRET Microscope System Combined with a Patch Clamp Workstation

An inverted Olympus IX71 epifluorescence microscope equipped with the Olympus CellTIRF system and CellTIRF control software (v. 1.4) managed by the HSTRI core is housed in room 47 of the Savitt Medical Sciences Building. In addition to standard 20x and 40x fluorescence objectives, the TIRFM system includes 60x (NA=1.45) and 100x (NA=1.49) oil immersion TIRF objectives, a fast, highly sensitive, charged-coupled ImagEM Enhanced C9100-13 Hamamatsu digital video camera (refresh rate ≈ 32 Hz at full 512 x 512 pixels without binning), and the 4-line Versalase-CellTIRF laser launch merge module housing three TTL-shutter-controlled diode lasers (Versa 50 mW 488 nm, 50 mW 561 nm and 140 mV 637 nm). The system is also equipped with a TTL-shutter-controlled Coherent 40 mV 445 nm laser and a DV2 Dual View beam splitter (Optical Insights, LLC) allowing for CFP/YFP FRET imaging in either standard epifluorescence or TIRF imaging mode. Images are acquired using Visiview software (Visitron Systems) with a high-end desktop computer (HP SB EliteDesk 800) running under Windows 10 operating system. The microscope is also coupled to patch clamp workstation that is composed of an Axopatch 200A patch clamp amplifier (Axon Instruments, Inc.), an MHW-3 Narishige micromanipulator, a TMC (series 63-543) vibration-free isolation table and TMC Faraday cage, a GWINSTEK digital storage oscilloscope (model GDS-2102), and an AD/DA Digidata 1322A acquisition system and PClamp software (v. 9; Axon Instruments, Inc.) running on a separate Windows 7-based desktop computer. This configuration allows for simultaneous recording of membrane currents or voltage, and TIRFM/FRET imaging. Finally, the system has a state-of-the-art perfusion system to allow for rapid solution changes during physiological experiments.

Leica DMi8/SR GSD-3D Super-Resolution Microscope

The HSTRI Core manages a Leica DMi8/SR GSD-3D super-resolution microscope system. The microscope is housed in room 49 of the Savitt Medical Sciences Building. The GSD-3D system uses ground-state depletion with individual molecule return (GSDIM) to produce super-resolution images of biological samples. This technique is also referred to as direct stochastic optical reconstruction microscopy (dSTORM). In conventional fluorescence microscopy, delocalized π electrons in a fluorescent dye are transferred from a ground state (S0) to an excited state (S1). As they oscillate back to the ground state, they emit fluorescent light. This occurs very rapidly, on the order of nanoseconds. The GSDIM technique lowers the number of electrons that are involved in this oscillation cycle by switching most of the fluorescent dye to a triplet state (T1). Single molecules return spontaneously from the triplet state to an excitable ground state and fluoresce at a very slow rate, on the order of milliseconds. As a result of this process, individual fluorophore molecules periodically ‘flash' within the sample and are detected by a sensitive camera. An algorithm can determine the exact position of single fluorophores. Positional information for all fluorophores is collected in several thousand separate images collected over several minutes; their coordinates are then used to compute a super-resolution GSDIM image. The GSD-3D system offers lateral resolution of 20 nm and axial resolution of 50 nm, providing advanced three-dimensional (3D) localization of cellular structures. GSDIM allows fluorophores commonly used for conventional immunolabeling protocols (e.g., Alexa-488) to be used for super-resolution imaging, a significant advantage over other platforms. The GDS-3D system is coupled to an inverted microscope (DMI6000B; Leica). Images are obtained using 100X (NA 1.47) or 160X HCX Plan-Apochromat (NA 1.47) oil immersion lenses and an electron microscopy charge-coupled device (EMCCD) camera (iXon3 897; Andor Technology). Samples are excited with 500 mW 488 nm, 523 nm, and 647 nm lasers.

3i Lattice LightSheet Microscope

The HSTRI Core manages a custom-built 3i Lattice LightSheet Microscope that is capable of 3D live cell imaging. The system is located in room 50 of the Savitt Medical Sciences Building. Its main advantages are that the microscope can break the diffraction limit with Structured Illumination Microscopy (SIM), and is ideal for single molecule imaging and photomanipulation. Lightsheet thickness is 0.4 µm at 50 µm length. Bessel beam lattice sheet illumination is accomplished via cylindrical lenses and high-speed SLM for multicolor imaging, annular mask array for various lightsheets, galvo mirrors to control lattice movement in x and z, cameras in image and Fourier space to inspect the lattice and annular mask, 25x/1.1NA 28x/0.71NA water immersion detection objectives, and piezo x,y translation stages and piezo imaging objective control. Images are acquired using a high-speed high-resolution 2Kx2K Hamamatsu ORCA-Flash4.0 V3 sCMOS camera. Samples are excited with 405, 488, 560, and 642 nm lasers. Image acquisition and analysis are performed by 3i SlideBook software (v. 6) running under Windows 10 on a powerful desktop computer comprised of Dual 16-Core Xeon Gold 2.9GHz processors, 128GB RAM, 8GB NVIDIA Quadro RTX4000 workstation graphics card, 512GB OS SSD, 8TB Fast Acquisition Drive, and 20TB additional storage.

B. Software for Advanced Image Analysis

Imaris Software

The HSTRI Core purchased one fixed and one floating license of the advanced imaging analysis software package Imaris (v. 9.7; Oxford Instruments). The fixed license is associated with a single high-end Windows 10-based computer workstation located in room 52 of the Savitt Medical Sciences building. The computer housing the fixed license is a PowerServe Uniti G4000 Storage Server (PSSC Labs) which is custom-configured and has the following characteristics: it has 2U rackmount chassis with redundant power supply, 16 x Total Xeon Scalable Processor Cores/2.1 GHz, 2 x Intel Xeon Scalable Processors, 32 Cores with hyperthreading enabled, 256 Gb high performance 2933 MHz DDR4 ECC registered memory, 10.6+GBperProcessorCore, 2 x 480 GB solid state SATA lll (SSD) Enterprise hard drive for operating system, 6 x 1 TB SATA lll 7200 RPM Enterprise hard drive for data storage, a GeForce RTX-2080Ti /11Gb/GDDR6 memory/graphics Card, and 2 x 10/100/1000/10000 10 GigE SFP + network ports. The floating license can be accessed anywhere within the UNR Medical School by trained users, allowing them to analyze their images on their own computer. Imaris is designed for motion detection (average and instantaneous speed, acceleration, track length, cell cycle duration), cell biology (cell volume, number of vesicles per cell, distance to membrane, vesicles per nucleus, distance to cell center, spot or particle detection (count, position (x, y and z), distance between spots, distance to surface object, speed) and surface (area, volume, intensity, elipcity, sphericity) analysis. The two licenses comprise several specialized Imaris packages designed for specific tasks: 1) Imaris XBT/Colocalization module; 2) Imaris ClearView Deconvolution module; and 3) Imaris Neuroscientist module.

C. Sample Preparation Equipment for Histology, Immunohistochemistry and Immunocytochemistry

Leica CM 1950 Cryostat for Sectioning Frozen Tissues 

A Microtome for Paraffin Block Sectioning of Tissues

Both are located in room 48 of the Savitt Medical Sciences Building.

Services Provided

• Technical support and training of core users
• Development and troubleshooting of methods and standard imaging procedures including assistance with advanced image analysis techniques
• Optimization of histology sample preparation for imaging such as sectioning and staining for fixed and live cell imaging
• Education of core users with novel microscopy and imaging techniques surfacing on the horizon

Citation

For project leaders, project leader mentors, any personnel funded by COBRE:This publication [or presentation or poster] was made possible by a grant from the National Institute of General Medical Sciences (P20GM130459) from the National Institutes of Health.
Research reported in this publication utilized the Transgenic Animal Genotyping and Phenotyping Core and the High Spatial and Temporal Imaging Core facilities of the University of Nevada, Reno, supported by the National Institute of Neural Medical Sciences of the National Institutes of Health (P20GM130459 Sub#5451 and P20GM130459 Sub#5452).

For Core users who are not funded by COBRE directly:Research reported in this publication utilized the Transgenic Animal Genotyping and Phenotyping Core and the High Spatial and Temporal Imaging Core facilities of the University of Nevada, Reno, supported by the National Institute of Neural Medical Sciences of the National Institutes of Health (P20GM130459 Sub#5451 and P20GM130459 Sub#5452).