High Spatial and Temporal Resolution Imaging Core (HSTRI)

Center of Biomedical Research Excellence

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

Dr. Normand Leblanc

Core Director

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

Microscopy and Imaging Specialist

To Be Named

Instruments Supported by the Core

Laser scanning confocal microscope
The HSTRI Core manages a FluoView FV1000 laser-scanning confocal imaging system running on Windows 7-based FV10-ASW Ver.4.2a acquisition and imaging software. The microscope is located in room L-304D of the Center for Molecular Medicine (CMM). The system comprises an inverted Olympus IX81 microscope, which has four filter-based fluorescence photomultiplier tube (PMT) detectors and one transmitted differential interference contrast (DIC) detector. This setup allows for up to four fluorescent dyes to be imaged simultaneously; it also has an additional channel for transmitted light or DIC imaging. The microscope has five objectives: 10x (UPlanSApo, NA 0.40, WD 3.10 mm), 20x (UPlanSApo, NA 0.75, WD 0.60 mm), 40x oil-immersion (UPlanFL N, NA 1.30, WD 0.20 mm), 60x oil-immersion (PlanApo N, NA 1.42, WD 0.15 mm) and 100x oil-immersion (UPlanSApo100XO, NA 1.40, WD 0.13 mm) objectives that are also equipped for DIC optics. The four existing lasers on the system provide six excitation lines for confocal illumination: 405 nm (diode laser), 458 nm (argon laser), 488 nM (argon laser), 515 nm (argon laser), 543 nm (HeNe laser), and 635 nm (diode laser). Images can be obtained using line-by-line or frame-by-frame scanning. This system is capable of capturing different fluorescence signals simultaneously or sequentially, depending on the need of the experimenter. The pinhole size can be adjusted from 50 µm to 800 µm in 5-µm steps. Images can be obtained with a resolution ranging from 64 x 64 pixels to 4096 x 4096 pixels. Images can be captured individually, as a time series, or as a Z-series (3D stack). This system is also connected to a mercury lamp source for epifluorescence illumination, allowing visualization of fluorescence using four filter cubes: Ex BP360-370/Em 420-460, for DAPI (4',6-diamidino-2-phenylindole); Ex BP400-440/Em LP475, for CFP (cyan fluorescent protein); Ex BP470-495/Em BP510-550, for GFP (green fluorescent protein); and Ex 530-550/Em LP575, for Cy5 (cyanine 5). The system is driven by a computer equipped with a comprehensive analysis package. The microscope was recently upgraded with the Globals for Images software package (SimsFCS) developed by the Laboratory of Fluorescence Dynamics at the University of California, Irvine. This allows the confocal system to be used for fluorescence correlation spectroscopy as well as raster image correlation spectroscopy (RICS).

Spinning-Disc Confocal Microscope
A spinning-disc confocal imaging system consisting of an Olympus IX71 microscope equipped with 20x, 40x, 60x, and 100x objectives is housed room 8B of the Manville Health Sciences Building. The illumination and detection system of this microscope consist of an Andor iXon DU-897 cooled CCD camera, Yokogawa CSU-21 Nipkow spinning-disk confocal scanner unit, and a laser launch with 408-, 488-, and 538-nm solid-state lasers and integrated acousto-optic tunable filter (AOTF). System control and data acquisition are provided by Andor iQ 2.0 software running on a Windows 7-based PC. The microscope system is equipped with a voltage clamp station that includes an Axopatch 200B amplifier, Digidata 1320 A/D converters, and a Sutter micromanipulator for dual patch clamp and confocal imaging. 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 used for image analysis.

• Total internal reflection fluorescence microscope (TIRFM)
An inverted Olympus IX71 epifluorescence microscope equipped with an Olympus CellTIRF system and analysis software housed in room 9F of the Manville Health Sciences Building is also supported by the HSTRI Core. In addition to standard 20x and 40x fluorescence objectives, the TIRFM system includes 60x (NA=1.45) and 100X (NA=1.45) 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 four TTL-shutter-controlled laser lines (445, 488, 543 and 633 nm). Images are acquired with IPLab 4.0.8 software running on a Windows 7-based PC. The system is also equipped with a patch clamp workstation comprising an Axopatch 200A patch-clamp amplifier, a MHW-3 Narishige micromanipulator, a TMC (series 63-543) vibration-free isolation table, a GWINSTEK digital storage oscilloscope (model GDS-2102), and an AD/DA Digidata 1322A acquisition system (Molecular Devices Corp.) running on a separate Windows 7-based PC. This configuration allows for simultaneous recording of membrane currents or voltage, and TIRFM imaging. The system is equipped with a state-of-the-art perfusion system for live cell imaging during physiological experiments. Finally, a Dual-View (Optical Insights, LLC) system for simultaneous recording of multiple fluorophores or dual FRET and TIRFM imaging is available and could be installed upon demand.

• Super-resolution microscope
The HSTRI Core manages a state-of-the-art Leica DMi8/SR GSD-3D super-resolution microscope system. The microscope is housed in room 8E of the Manville Health 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-, 523-, and 647-nm lasers.

• Two-photon microscope
The HSTRI Core also supports a Thorlabs Bergamo II Series two-photon microscope with selected features from models B248 and B264 is housed in room 5 of the Manville Health Sciences Building. The Bergamo system is a custom-built, two-photon microscope equipped with a broadly tunable Ti-sapphire laser (~4 W average power, 670-1080 nm, ~80 MHz, 140 fs pulse width). This model is a rotating microscope composed of a Primary Galvo-Resonant Scanner (8 kHz) for high-speed imaging and a secondary galvo-galvo scanner for user-defined region of interest (ROI) shapes and photostimulation patterns. Using this system, it is possible to perform epifluorescence functional imaging with video frame rates of 30 F/s at 512 x 512 pixel resolution and 400 F/s at 512 x 32 pixel resolution, with simultaneous targeted photostimulation or compound uncaging. This configuration incorporates a Dodt illumination module that can perform Dodt-contrast, laser-scanning and wide-field imaging; this module can be installed and removed for switching between in vitro and in vivo experiments. The detector assembly incorporates a green bandpass emission filter (525-540 nm) as well as an orange/red bandpass emission filter (605-670 nm) and two GaAsP PMT detectors. The microscope is also equipped with a piezo objective positioner that allows volume imaging.

• Förster Resonance Energy Transfer (FRET) microscope systems
The HSTRI Core is responsible for the management of two fluorescence microscopy workstations located in rooms L-306B and L-306C in CMM that are equipped for conducting live cell imaging of Fluorescence (or Förster) Resonance Energy Transfer (FRET) responses. Each microscope system includes a vibration isolation table, an Olympus IX70 or IX71 inverted microscope equipped with a 40x1.3 NA water immersion objective, a Hamamatsu Orca D2 dual-chip camera or Hamamatsu Orca ER camera with Optical Insights Dual-View Micro-Imager and a filter cassette for simultaneous measurement of fluorescence emissions from cyan and yellow fluorescent proteins, a Sutter DG4 light source with 300 W Xenon arc lamp and fiber optic light guide used for excitation, and SimplePCI image acquisition and analysis software running on a Windows 7-based PC.

• Ca2+ Imaging and cell shortening system
The HSTRI also administers one workstation located in room L-306C in CMM that allows for simultaneous measurements of Ca2+ transients and myocyte shortening. This system consists of an inverted Nikon TE2000 microscope equipped with an IonOptix Ca2+ and contractility system that includes a Hyperswitch light source, MyoCam-S CCD camera, cell-framing adapter, photomultiplier sub-system, fluorescence system interface, and a Windows 7-based PC with IonWizard data acquisition and analysis software.

• Lattice LightSheet Microscope
Soon to Come

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