Cellular and Molecular Imaging Core

Cellular and Molecular Imaging Core

Two researchers in a lab wearing protective gear and working with equipment The mission of the Cellular and Molecular Imaging (CMI) Core facility of the Integrative Neuroscience COBRE center is to provide infrastructure and resources necessary to perform cellular and molecular imaging of biomolecules as well as media preparation to further research at the University of Nevada, Reno (UNR). The Core facility is located on the third floor of the Fleischmann Lifesciences building in the Biology Department of the College of Science.

The CMI Core facility provides access to state-of-the-art instruments including a new Scientifica HyperScope two-photon imaging system for deep tissue scanning in live brains and whole animals, a Leica SP8 confocal microscope system for high-performance quantitative imaging and analysis of tissues and cells, a GE Phosphoimager for Northern blotting, a BioRad ChemiDoc for Western blotting, two BioRad Real-Time PCR instruments for precise and quantitative PCRs, a QIAgility for automated PCR set-up, a GeneSys 180 UV/Vis spectrophotometer, and a Quibit 4 fluorometer.

Further capabilities of the core includes media preparation instruments and resources for husbandry of Drosophila and C. elegans used as model organisms.

Core director

Graduate assistant

Services provided to users

  • Technical support, assistance and training of core users
  • Open and immediate access to basic molecular imaging instruments
  • Hourly fees for confocal and two-photon imaging through iLab scheduling
  • Support for the Neuro-CMB graduate seminar series to invite external speakers

The equipment supported by the Cellular and Molecular Core includes:

  • Scientifica HyperScope two-photon microscope

    Two-photon microscopy has become an indispensable tool for cellular neuroscience, allowing high-resolution fluorescent imaging of cells in vitro and in vivo living brain and whole animals. The CMI core contains a Scientifica Hyperscope two-photon platform housed in room FA 318 of the Fleischmann Life Sciences building. The HyperScope system is a custom-build, two-photon microscope equipped with a Mai-Tai eHPDS broadly tunable Ti-sapphire laser (690-1040 nm), multiple objectives (16x, 20x and 40x), and stands on a Thorlabs breadboard and anti-vibration table. With the Scientifica SciCam Pro CCD camera, it is possible to perform functional imaging of video frame rates of 24 fps (full resolution) and 40 fps (binned 2 x 2). The detector assembly includes one red-shifted PMT and one GaAsP PMT modules, and epifluorescence imaging with filter sets for GFP (470/40x extinction, 525/50m emission) and mCherry (560/40x extinction, 630/75m emission). The scan mirrors in the imaging path can be arranged in multiple configurations depending on your needs. With up to 3 mirrors in series, you can choose between galvo/galvo (GG), resonant/galvo (RG), or resonant/galvo/galvo (RGG) arrangements. The microscope is fitted with an ultra-smooth Z-axis drive to ensure vibration free movement, with the Z-axis being controlled with software for acquiring z-stacks with a minimum step size of 0.1 mm. The addition of a Piezo z-axis objective positioner allows volume imaging up to 400 mm. The microscope can be easily configured and switched between in vitro and in vivo applications by removing the substage optics and riser block. The system is enclosed in a black-out cage to prevent light and noise pollution of biological samples.

    This platform includes an award winning Scientifica SciScan acquisition software, allowing users to integrate SciScan easily with custom-written applications, such as LabView and ScanImage. The ScanImage software interface (Vidrio Technologies) used by Scientifica was developed by HHMI Janelia Farm specifically for neuroscience applications, and is considered to be the most advanced software for two-photon laser scanning on the market.

     

    Key unique features of the Scientifica HyperScope two-photon include:

    • SliceScope upright microscope with motorized frame sitting on top of a Thorlabs breadboard with tapped holes on top of a TMC Cleantop Optical Table
    • Motorised positioning of objective and condenser in Z-axis, controlled by PatchPad
    • Motorized Movable Base Plate for use with riser block and in vitro applications
    • HyperScope Scan Path including up to three scanning mirrors
    • HyperScope X-Galvo module
    • HyerpScope Resonant X module (8 kHz resonant galvanometer)
    • Pockel Cell (680-1100 nm) with 500:1 extinction ratio
    • Workstation and data acquisition, which enables resonant imaging (with SciScan) and RG or RGG system for resonant and/or galvo imaging using ScanImage
    • Detectors: 1 red-shifted PMT module, and 1 multi-photon GaAsP PMT module
    • Objectives include: 16x (CFI75 LWD 16xW N.A. 0.80, W.D. 3.0 mm), 20x (XLUMPLFLM 20xW, N.A. 1.0, WD 2.0 mm, water immersion), 40x (LUMPPLFLN 40xW, N.A. 0.8, W.D. 3.3mm, water immersion)
    • The system is compatible with RMS, M25, M27 and M32 threaded objectives allowing users to easily interchange objectives
    • Filter sets: GFP (470/40x, 495LP, 525/50m), mCherry (560/40x, 585LP, 630/75m)
    • SciCam Pro CCD camera with 24 fps at full resolution (40 fps binned 2 x 2)
    • 15x eye-piece with FOV of 14.5mm and tiltable nose piece
    • Piezo Objective Z-drive allows fast acquisition of volumes up to 400 microns travel for large and small objectives
    • Laser: Mai-Tai eHP DS-244 Ti-sapphire oscillator (690 to 1040 nm, >2.4W average power, ~80 MHz repetition rate, <70 fs pulse width)
  • Leica SP8 TCS confocal microscope

    Computer next to a confocal microscope

    The Leica SP8 scanning confocal microscope allows high-performance quantitative imaging and analysis of tissues and cells. The CMI core houses an upright confocal microscope in room 324 of the Fleischmann Life Sciences building, and is capable of simultaneous collection from up to 3 detection channels (two PMTs, one HyD) using multiple laser lines (diode 405, blue 488, green 552, red 638 nm). The confocal is configured with a conventional tandem scanner (8 kHz), a super Z-galvo stage type S, multiple objectives (10x, 20x, 25x, 40x, 63x), and stands on an anti-vibration table. The confocal is connected to a computer workstation with 30-inch monitor and Leica LAS software, which provides high-performance cellular and imaging analysis.

    Key features of the Leica SP8 confocal include:

    • DM 6000 upright with EL 6000 fluorescence
    • Super Z Galvo stage, Type S
    • Objectives: 10x (HC PL FLUOTAR 10x/0.30), 20x (HC PL APO 20x/0.7 IMM CORR CS2), 25x (HCX IRAPO L 25x/0.95 W 0.17), 40x (HC PL APO 40x/1.30 Oil CS2), 63x (HC PL APO 63x/1.40 OIL CS2)
    • Tandem resonant scanner 8 kHz
    • 2 PMT SP confocal detectors for imaging
    • 1 HyD SP GaAsP detector for gated imaging
    • Lasers: 405 nm DMOD Compact, Port UV/405, Blue 488 nm, Green 552 nm, Red 638 nm
    • LAS AF SP8 software
  • GE Typhoon FLA-7000 Phosphoimager

    Typhoon Phosphoimager laser scannerThe Typhoon Phosphoimager is a fast and versatile laser scanner for sensitive and quantitative measurements of radio-isotropic-labeled samples. The Typhoon is housed in the CMI core in room 312 of the Fleischmann Life Sciences building, and is a non-confocal variable mode laser scanner offering high-speed and resolution for precise quantitation of proteins, nucleic acids and other biomolecules. The scanner comprises "filmless" autoradiography with storage phosphor screens. The laser can generate 16-bit images at up to 25 μm pixel resolution to give precise quantitation in gels and blots, with a high speed to scan gels of up to 240 x 250 mm in less than 2 min. Two phosphor screens with a size of 200 X 250 mm are available for autoradiography. The scanner can detect a broad range of isotopes in radiolabeled samples. Directly adjacent to the phosphoimager system, there is a PC computer with a software package that allows on-site quantitative and qualitative analysis of images and data.

    Key features of the Typhoon imager include:

    • Applications: radioisotopic labels by storage phosphor, chemifluorescent Western blots, and single fluorescence, as well as digitization of colorimetric stains (e.g., Coomassie™ Blue and silver-stained gels)
    • Resolution: 16-bit images are generated at up to 25 µm pixel resolution
    • Speed: 24 x 25 cm gel can be scanned in less than two minutes at 100 µm resolution
    • Four lasers: blue (473 nm), green (532 nm), red (635 nm), and red (650 nm)
    • Radioisotopes: 3H, 14C, 32P, 33P, 35S
    • Max scanning area: phosphorimaging: 20 × 40 cm, fluorescence: 24 × 40 cm
  • Biorad Chemidoc Imager and Turbo-Blotter

    The Chemidoc is an easy, high-performance gel and blot imaging for accurate quantification of protein samples in gels and membranes. This Chemidoc imager in the CMI core is housed in room 312 of the Fleischmann Life Sciences building, and streamlines Western blotting in a five-step approach by combining traditional blotting techniques with innovative tools to allow improved band quantitation and quick assessment of electrophoresis results and blot transfer quality prior to western blotting. The Chemidoc enables rapid western blot results in as little as 15 min with efficient protein transfer in 3 min, and 5 min stain-free gel imaging. The imbeded software includes all the tools for quantitative and qualitative image and data analysis. Images taken on the ChemiDoc can also directly be print.

    Key features of the ChemiDoc system include:

    • Electrophoresis applications: protein including stain free and DNA gel imaging
    • Blotting applications: identify and quantify proteins, DNA, protein-ligand interactions, etc. with chemiluminescence or colorimetric detection
    • Internal computer with Image Lab Touch software
    • Automatic recognition of application-specific tray and adjustment of imaging parameters and software options
    • Illumination sources: Trans-UV, 302 nm excitation, Epi-white, Trans-white (requires White sample tray provided), Trans-blue 450-490 nm excitation (requires Blue sample tray provided)
    • Detection: chemiluminescence filter 590/110 nm standard emission filter for protein and DNA gel and blot imaging.
    • Detector: cooled CCD camera, 6 megapixels
    • Exports data to a USB drive as SCN, TIFF, JPEG files

     The Trans-Blot Turbo transfer system housed in the CMI core in room 314 (Tissue Culture) combines traditional blotting techniques with modern filter paper and buffers allowing rapid transfer of proteins from gels to membranes with minimal preparation time. The system integrates a high amperage power supply that directs current between a built-in platinized titanium anode and a stainless steel cathode.

    Key features of the Turbo-Blotter include:

    • Two cassettes with rapid transfer of 1-4 mini or 1-2 midi gels in 3 min in a single run
  • Integra Mediaclave with Dose-It Pump and AirClean flow workstation

    Mediaclave 10 equipmentThis media autoclave in the CMI core is housed in room 313 of the Fleischmann Life Sciences building, and allows the rapid and gentle preparation of 1-10 liter culture medium. Precise controlling of temperature, time and pressure during the sterilization process guarantee constant high quality. The mediaclave can be used for the preparation and sterilization of numerous types of culture media, including media for  C. elegans research. The wide adding port facilitates safe addition of supplements prior to dispensing. The mediaclave is connected to a peristaltic pump to fill petri dishes and vials.

    Key features of the Mediaclave include:

    • Rapid and gentle preparation of 1-10 liter culture medium
    • Large and strong magnetic stirrer with adjustable speed and reversing rotation direction ensures homogenous media preparation over a wide viscosity range
    • Pt-1000 temperature probe and microprocessor control of process parameters.
    • Fast heating and rapid cooling
    • Peristaltic pump DOSE-IT housed in a AirClean vertical flow workstation can be used to fill containers of unusual volumes or shapes (i.e. petri dishes, bottles, flasks)
  • CFX96 Touch Real-Time PCR

    CFX96 Touch Real-Time PCR equipmentTwo real-time PCRs in the CMI core are housed in room 312 of the Fleischmann Life Sciences building combines advanced optical technology with precise thermal control to deliver sensitive, reliable detection, quick set-up runs and monitoring of amplification traces in real-time on the integrated LCD touch screen. Directly adjacent to the two qPCR detection systems, there is a PC computer containing easy to use software for on-site analysis of qPCR data.

    Key features of the CFX96 include:

    • Up to 5-target multiplexing with sample volumes as low as 10 μl
    • Thermal gradient feature
    • Visualize all run data at once and export only the data you need in the format you want
  • QIAGEN QIAgility automated PCR set-up

    The QIAgility is housed in the CMI core in room 312 of the Fleischmann Life Sciences building, and is a compact benchtop instrument that enables rapid, high-precision setup of PCR experiments with ready-to-use software for ease and convenience. The software provides step-by-step guidance for worktable setup and automatic calculation of all mixes, eliminating the need to program pipetting steps. Just select the kit and cycler, define your targets and start the run. For added flexibility the QIAgility is operated via a laptop computer.

    Key features of the QIAgility include:

    • Highly suited for laboratories performing PCR and RT-PCR reactions with applications in gene expression, cloning, genotyping and more
    • The instrument uses 50 µl and 200 µl Conductive Filtered Tips for liquid-level sensing
    • Accessories included: 2 x Adapter, 96 x 0.2 ml PCR Tubes 1 x Adapter, Flip-Cap 32 x 1.5 ml Tapered Base 1 x Adapter, 72 x 0.1 ml Strip Tubes 1 x Master Mix Block, 5 x 5 ml 1 x Master Mix Block, 1 x 5 ml, 4 x 2 ml 1 x Master Mix Block, 1 x 5 ml, 4 x 1.5 ml 1 x Reagent Block, 16 x 0.2 ml / 2 ml Flat / 1.5 ml Tapered Tubes 4 x Tip Rack Holder for 96-Tip Rack.
    • HEPA filter: preinstalled dual-cartridge 3M Air-Mate HEPA filter system provides positive pressure clean air within the closed hood
    • Preinstalled UV lamp for workable decontimation
    • Operating temperature, 15–30 °C (59–86°F)
  • ThermoScientific GeneSys 180 UV/VIS spectrophotometer

    This spectrophotometer is housed in the CMI core in room 312 of the Fleischmann Life Sciences building, and performs high-throughput quantitative UV-Vis measurements where a double beam is required as a reference cell position. Optimized for usability and performance, this system features a high-resolution color touchscreen, and optional Wi-Fi networking. The compartment is a 8-position cell changer (standard).

    Key features of the GeneSys 180 UV/VIS include:

    • Double Beam/Reference Cell
    • Standard 8-position cell changer with switchable turret
    • Room light resistance for 'open-lid' operation
    • Accommodates cells up to 100 mm pathlength
    • Exports data to a USB drive or directly to your computer via a USB cable
  • Quibit 4 Fluorometer

    The Qubit 4 Fluorometer in the CMI core is housed in room 314 (Tissue Culture) accurately measures DNA, RNA, and protein quantity. The Qubit 4 Fluorometer and RNA IQ Assay Kit can work together to accurately distinguish intact from degraded RNA in just two easy steps. For all Qubit assays, the concentration or quality of the target molecule in the sample is reported by a fluorescent dye that emits a signal only when bound to the target, which minimizes the effects of contaminants on the result. The easy-to-use touch-screen menus make it easy to select and run the assays you need, with results displayed in just a few seconds.

    Key features of the Qubit 4 Fluorometer include:

    • Quantitation of DNA, RNA, and protein in less than three seconds per sample
    • Measures intact RNA in less than 5 seconds per sample
    • High levels of accuracy using only 1–20 μL of sample, even with very dilute samples.
    • Stores results from up to 1000 samples
    • Large 5.7-inch, state-of-the-art color touch screen for easy workflow navigation
    • Graphical display indicates when samples are in the extended range or out of range
    • Exports data to a USB drive or directly to your computer via a USB cable

Information for new and existing users

The CMI Core facility maintains a student assistant on staff to provide basic training and technical support in order to assist researchers with all of our equipment and software. For information about the core, please contact the core director Alexander M. van der Linden avanderlinden@unr.edu.

For confocal and two-photon imaging:

  • University clients: If you already have an account, log into iLab Solutions with your NetID and go to the Schedule Equipment tabs. If you are a new user, please contact Alexander van der Linden.
  • Non-University clients: Please contact Alexander van der Linden.
  • Visit the iLab Help Site or UNR iLab system for further guidance using iLab Solutions.
  • Core facility recharge rates: The core currently uses hourly fees for the confocal and two-photon (future) microscopes through iLab. Rates for University clients (as of 07/01/2019) is $10/hour for the confocal platform and $30/hour for the two-photon platform (in progress)

For molecular imaging and media preparation:

  • University clients: Free and direct open access to all core instruments. Users are required to sign-in their name, email and lab each time they use equipment in the core.
  • Non-University clients: Please contact Alexander van der Linden at avanderlinden@unr.edu.