Neuroscience Imaging Center (NIC)

Advanced imaging resources for common use

 OUR MISSION

The goal of the NEUROSCIENCE IMAGING CENTER (NIC) in the Solomon H. Snyder Department of Neuroscience at Johns Hopkins School of Medicine is to provide instrumentation and image processing & visualization software/hardware for analyzing biomolecular localization, dynamics, and inter-molecular interactions with best optical resolution available. This facility also allows users to perform time-lapse and super-resolution imaging of multiple fluorophores in living cells and tissues, and to combine Z-sectioned imaging of fluorescently tagged proteins or ion or voltage indicator dyes with electrophysiological monitoring of electrical activity.

The NEUROSCIENCE IMAGING CENTER promotes interactions among a diverse group of neuroscientists and physiologists at Johns Hopkins University. Such interactions have facilitated the investigation of key issues in basic and clinical neuroscience, as illustrated in our List of Publications of work supported by the NIC.

FINANCIAL SUPPORT AND MEMBERSHIP

The NIC was established through support from the NINDS Core Centers P30 grant and contributions from the JHU School of Medicine, the Department of Neuroscience, and individual users. The NIC is currently supported by the Department of Neuroscience and contributions from individual members. The Director of the NIC is Prof. Dwight Bergles (dbergles@jhmi.edu), and the Manager of the center is Dr. Aleksandr Smirnov (asmirno3@jhmi.edu). Activities in the NIC are supported by the Administrative Coordinator Susan McTeer (smcteer1@jhmi.edu). Instrumentation within the NIC has been purchased using funds contributed by the Kavli Neuroscience Discovery Institute at JHU, the Institute for Basic Biomedical Sciences (IBBS), the Department of Neurology, and individual users. The NIC primarily supports the research activities of JHU SOM faculty who have primary or secondary membership in the Department of Neuroscience.

Applications for use of JHU core facilities are carefully evaluated by NIC personnel and directors to align with the overall mission of the Neuroscience Department. Services are allocated based on instrument availability and other funding-related guidelines. To ensure that access to equipment by department faculty is not unduly limited, we may be unable to grant membership to laboratories whose research falls well outside Neuroscience research goals. However, we are always happy to provide collaborative help with experimental design, approaches for analysis, and in some instances, access to NIC instrumentation for the purpose of performing pilot studies.

An annual NIC membership fee is assessed to member labs the first time they use the NIC Facility. This annual fee is subject for periodic reassessment and is based on PI’s seniority: currently $2000 for Full Professors, $1250 for Associate Professors, and $750 for Assistant Professors. Fees are used to offset the costs of service contracts and to expand Center capabilities. The annual fee provides users with access to all equipment and computing resources located in the NIC (microscopes and image processing workstations). In addition, each user is assessed a one-time basics training fee (currently $200) when using core instrumentation for the first time. Training on using advanced capabilities may be charged at a different hourly rate.

Any questions regarding access to the NIC can be directed to its Manager, Dr. Aleksandr Smirnov, by e-mailing to asmirno3@jhmi.edu or visit this page for more information.

LOCATION

The NIC office is located in Room 1008A, Preclinical Teaching Building, 725 North Wolfe Street, on the Johns Hopkins University, East Baltimore campus.  The phone number is 410-502-7858.

RESEARCH SUPPORT

A great deal of expertise is available to users of the NIC. Dr. Aleksandr Smirnov, PhD (NIC Manager) has experience in single- and multi-photon imaging, super-resolution microscopy, SPIM, SIM, FLIM, FRET, FRAP, FCS, CLEM, immunofluorescence, maintenance of animals for in vivo imaging, and electrophysiological techniques. The NIC Director, Dr. Dwight Bergles, has extensive expertise in imaging and electrophysiological techniques. In addition, the many users of the facility each bring unique knowledge and experience that can be leveraged for the particular experimental needs of investigators.

The first step for all faculty, postdocs, and graduate students who use the NIC is a one-on-one orientation on the relevant piece of equipment with the Center Manager. The individual nature of these sessions allows the Manager to provide training appropriate to the background, expertise, and research question of each individual. Depending on these factors, the orientation may simply cover proper use of the equipment; however, it may also include information on choice of fluorophore, immunohistochemical techniques, and proper imaging parameters, as well as the particular advantages of epifluorescence, confocal, or two-photon microscopy. In addition, because the NIC offers a variety of options for image analysis (e.g. Imaris, ZEN, AutoQuant X3, napari, Neurolucida/Explorer), the Core Manager often works with individuals to help them determine what factors to consider when designing the workflow for quantification of their data and preparation of meta-data for publication/sharing according to a particular Data Management Plan.

The NIC further contributes to educating our community of neuroscientists about imaging and image analysis by organizing demonstrations of equipment and new software releases, facilitating and publicizing seminars of interest to the imaging field, and hosting workshops to promote sharing of information on such topics as in vivo imaging, tissue clearing, and image processing.

CORE SERVICE

Confocal Microscopy

The facility contains four confocal microscope systems that allow high-resolution imaging of labeled cell components in three-dimensional space. 

Zeiss LSM 800 AiryScan.2 / WF

Our Zeiss LSM 800 is typically used for imaging fixed slides, especially when tiling is required, or for the imaging of live tissue.  It consists of an inverted AxioObserver microscope equipped with three GaAsP detectors. These provide enhanced sensitivity compared to traditional PMT detectors, resulting in decreased bleaching and phototoxicity. The LSM800 uses filter-free variable spectral dichroics, allowing the user to define specific emission collection parameters rather than being limited to a particular set of installed emission filters.  Because this system is equipped with 405, 488, 561, and 640 nm lasers, users can collect images from tissue stained with a wide variety of fluorophores ranging from DAPI to far-red.  Wide-field DIC and fluorescence images can also be collected using the CCD camera detector.  The motorized stage, in combination with software features, allows users to perform time-lapse imaging with flexible control of experiment design. The high reproducibility of this stage, combined with software control of acquisition and with post-acquisition processing, facilitates the collection of seamless tiled images.  The LSM800 also includes an AiryScan detector, which uses photon reassignment to provide enhanced sensitivity and spatial resolution (~ 140 nm laterally and 400 nm axially).  This capability is particularly useful for applications that require the resolution of small objects.  Available objectives are 2.5x air, 10x air, 20x air, 40x oil, and 63x oil.  A second 20x air objective is a long working-distance lens which can be used with a wider array of specimen types.  A separate server is available for off-line processing of acquired images.   The full complement of available Zen software from Zeiss is present, allowing users to use this platform to perform a wide array of experiments, including FRET and FRAP.  

  

Zeiss LSM 880 AiryScan.2 / BIG.2 and Chameleon Laser

The Zeiss LSM 880 can be used to image fixed slides, live tissue, or intact mice.  This confocal consists of an upright AxioExaminer.Z1 microscope equipped with three internal detectors - two traditional PMTs and a very sensitive GaAsP detector which reduces the risk of bleaching or phototoxicity. The filter-free spectral detection allows the user to define emission collection parameters, rather than being limited to a particular set of installed emission filters.  Because the 880 is equipped with 405, 458, 488, 514, 561, and 633 nm lasers, users can collect images from tissue stained with a wide variety of fluorophores ranging from DAPI to far-red.  Wide-field DIC images can also be collected using the transmitted light detector.  The motorized stage, in combination with software features, allows users to perform time-lapse imaging with flexible control of experiment design. The height of this motorized stage can be changed to accommodate a variety of sample types, from fixed slides to live mice. The 880 also includes an AiryScan detector, which uses photon reassignment to provide enhanced sensitivity and spatial resolution (~ 140 nm laterally and 400 nm axially).  This capability is particularly useful for applications that require the resolution of small objects.  In addition, the AiryScan detector can be used in a Fast mode to enable rapid collection during time-lapse imaging.  Available objectives are 5x air, 10x air, 25x oil, 40x oil, 63x oil, and 100x oil.  A separate server is available for off-line processing of acquired images.   The full complement of available Zen software from Zeiss is present, allowing users to use this platform to perform a wide array of experiments, including FRET and FRAP.  

In addition to the visible lasers listed above, the 880 confocal is coupled to a near-infrared (IR) tunable pulsed femtosecond Ti:Sapphire laser (Chameleon Ultra II). The IR laser of the two-photon system facilitates imaging of thick specimens, and is capable of imaging fluorescent dyes or proteins in vivo. For two-photon applications on the 880, an additional Zeiss binary GaAsP (BIG.2) detector with band-pass emission filters permits sensitive detection of red or green fluorophores from deep within tissue. This detector is particularly critical for in vivo applications, and has allowed users to monitor cell motility in intact preparations. Because the BIG detector is extremely sensitive to ambient light, we have constructed a light-tight enclosure around this system, so that both microscopes in this room can be used simultaneously. For in vivo imaging, we have three long working distance, high numerical aperture dipping objectives (10x and 20x lenses which are not coverslip-corrected as well as a 20x coverslip collected lens). 40x and 63x dipping lenses are also available.

Zeiss LSM710 confocal / BIG.2 and Chameleon Laser

The Zeiss 710 confocal is most frequently used for imaging live mice or for performing slice imaging, including calcium imaging. It consists of an upright AxioExaminer.Z1 with internal PMTs which have good detection capabilities due to refinements of the light path within the scan head. The filter-free spectral detection allows the user to define emission collection parameters, rather than being limited to a particular set of installed emission filters. Because the 710 is equipped with 458, 488, 514, 543, and 633 nm lasers, users can collect images from tissue stained with a wide variety of fluorophores ranging from GFP to far-red. Wide-field DIC images can also be collected using the transmitted light detector. A variety of custom-made stages are available, permitting the use of a wide range of specimen types. The full complement of available Zen software from Zeiss is present, allowing users to use this platform to perform a wide array of experiments, including FRET and FRAP.

In addition to the visible lasers listed above, the 710 confocal is coupled to a near-infrared (IR) tunable pulsed femtosecond Ti:Sapphire laser (Chameleon Ultra II). The IR laser of the two-photon system facilitates imaging of thick specimens, and is capable of imaging fluorescent dyes or proteins in vivo. For two-photon applications on the 710, an additional Zeiss binary GaAsP (BIG.2) detector with band-pass emission filters permits sensitive detection of red or green fluorophores from deep within tissue. This detector is particularly critical for in vivo applications, and has allowed users to monitor cell motility in intact preparations. Because the BIG detector is extremely sensitive to ambient light, we have constructed a light-tight enclosure around this system, so that both microscopes in this room can be used simultaneously. For in vivo imaging, we have three long working distance, high-NA dipping objectives (10x and 20x not coverslip-corrected, and 20x coverslip collected). 40x and 63x dipping lenses are also available.

Thorlabs Bergamo II Multi-photon Microscope and Discovery Laser

The creation of transgenic animals expressing multiple fluorophores, including red-shifted fluorophores, is an important development in the field of in vivo imaging. In order to take advantage of these advances, researchers need access to two-photon lasers that supply adequate power at long wavelengths. Our Discovery NX laser with Total Power Control  provides high average power across a large tuning range (660 nm to 1320 nm) as well as a second high-power fixed output at 1040 nm. The Discovery is integrated into a Bergamo II multiphoton microscope, which has been provided by Thorlabs as part of our strategic partnership with this company. 

Additional Equipment for Confocal Imaging

Custom, interchangeable temperature-controlled stages are available for use in live cell/tissue imaging experiments. Tissue superfusion is achieved using a gravity-fed system that passes through computer-controlled solenoid valves. Solutions can be oxygenated and, if necessary, heated using an in-line heater or a peltier chamber heater, depending on the application.

Support for in vivo applications is also available, including isoflurane vaporizers, anesthesia induction chambers, and temperature-controlled warming pads.

A piezoelectric focus drive is available when rapid z-focus control is required. Piezoelectric positioning drives provide higher focusing speed (~10 vs 100 msec) and better resolution (~1 vs. 100 nm) than stepper motor-drives. This is especially advantageous for applications such as tracking mitochondrial movement and assessing process motility of labeled cells in vivo, where the ability to assess temporal changes is limited by the time required to construct high-quality z-stack images.

Electrophysiological Components

Whole cell current and voltage clamp recordings can be performed using an Axon Instruments Multiclamp 700B amplifier. Acquisition is controlled by a Digidata 1322A digital-to-analog converter, and a Pentium 4 PC computer running pClamp10 and Origin analysis software. Additional equipment such as a secondary amplifier (Brownlee), a timer/stimulator (A.M.P.I), black-and-white monitor, camera controller, and halogen power supply are positioned in a moveable rack with castors, allowing this system to be moved into position near whichever microscope best suits an investigator's needs.

Epifluorescence Imaging

Zeiss Cell Observer

The Zeiss Cell Observer system consists of an AxioObserver inverted microscope equipped with fluorescent and transmitted light, an Axiocam MRm, and filter sets compatible with DAPI, GFP, Cy3, and Cy5. Available objectives are 5x, 10x, 20x, 40x, and 63x. This microscope is often used for long-term imaging of cells because it is equipped with an environmental chamber to permit control of temperature, humidity, and CO2. A motorized stage facilitates the identification and imaging of multiple regions of interest, and the Experiment Designer software module permits flexible design of experiments and provides various post-processing capabilities.

The system can also be used to image fixed slides. One feature that is useful to many labs is the motorized stage, which supports acquisition of tiled images and allows users to capture large regions of interest at high magnification. Post-processing features are available to remove line artifacts that are often apparent in such tiled images.

Keyence All-in-One BZ-X710 Microscope

The Keyence is a benchtop widefield/SIM inverted microscope that is used primarily to image fixed slides.  It can also be used with an on-stage incubator within which temperature, humidity, and CO2 can be controlled, providing the option of performing long-term imaging of live cells. Both epi- and transmitted-light illuminations are available, and fluorescence filter sets are compatible with DAPI, GFP, TRITC, TxRed and Cy5.  In addition to the very sensitive monochrome camera, the Keyence contains a color camera that is used for imaging specimens containing cresyl violet or similar dyes (for H&E staining). A motorized stage facilitates the identification and imaging of multiple regions of interest and also allows the collection of tiled & Z-stacked images. This microscope also has the ability to use structured-light illumination (SIM) to improve sectioning in 3D.

Zeiss Stereo Zoom Fluorescence Microscope

Our Zeiss AxioZoom microscope provides the advantages of a stereomicroscope - zoom optics and long working distance - with the high resolution of a light microscope. The microscope has two high numerical aperture zooms lenses which, combined with the motorized zoom, allow quick and easy switching through the entire zoom range of 7x-260x.

The microscope is equipped with both fluorescence excitation and transmitted-light sources. Investigators can view and image large samples with excellent brightness and resolution without the need for tiling and stitching. In addition, the long working distance means that the microscope can aid in the dissection of small tissue samples. Finally, this microscope is also used to perform in vivo imaging of transgenic mice in awake state.

Image Analysis

Imaris for Neuroscientists

The Center has two dedicated image analysis workstations each running Imaris (Andor) software, which is designed to provide automated quantification of fluorescent three-dimensional images.  Imaris can perform isosurface rendering, object detection/counting, filament tracing/particle tracking, and quantitative co-localization; these operations can be used to extract a wide array of quantitative information from images.  Additionally, by interfacing with Matlab, ImageJ or Python, the ImarisXT module allows users to develop their own task-oriented algorithms, or to use analysis modules developed by others. The Imaris suite of programs is compatible with most standard formats, include Tiff series, BMP series, and images collected with Zeiss, Leica, and Olympus acquisition software. Our service contract with Andor ensures that we always have access to the most recent updates and improvements to the program; furthermore, this contract provides access to a knowledgeable representative who can help with determining the most efficient workflow for image quantification on an individualized basis.  

Neurolucida / Explorer

Additional image analysis capabilities are provided by our Neurolucida Neuron Tracing system (MBF Biosciences). This system offers a variety of drawing/analysis options, including cell tracing/morphology quantification, 3D brain mapping, and serial section reconstruction. The software is paired with a Zeiss AxioImager microscope equipped with transmitted light and a motorized stage, to allow tracing directly from slides. Alternatively, image files collected on another system (including confocals) can be imported into the Neurolucida software. The analysis capabilities of Neurolucida complement those which can be achieved using the Imaris suite of programs, because the latter is geared almost entirely toward analysis of fluorescent images, whereas Neurolucida excels at reconstruction and analysis of brightfield images (e.g., DAB labeled cells). Because this system includes a color camera, it also provides a route for obtaining color images of brightfield samples, such as LacZ.

Autoquant X3

Autoquant by Media Cybernetics is a deconvolution software package that produces extremely high-quality results for both widefield, 2P and confocal images.

Zeiss ZEN Desk

Both the LSM800 and the LSM880 Airyscan.2 confocal computers are directly connected to servers which provide the entire complement of post-acquisition processing available in the Zen software from Zeiss. This enables processing to be done off-line, freeing time on the confocals, especially for super-resolution image acquisition.

Stereotaxic Surgery Station

The Angle2 stereotaxic system (Leica) consists of a software-assisted stereotaxic instrument that facilitates the accurate targeting of brain regions by using software combined with transponders that detect the position of the pipette carrier. Advantages include an on-screen atlas to facilitate identification of targets, the ability to use an angled approach without the need for path-of-approach calculation, and the ability to automatically correct for head tilt.

APPLICATION FOR SERVICE

Researchers wishing to use the NIC must first complete the Application for Service and forward to the Center Manager, Dr. Aleksandr Smirnov (asmirno3@jhmi.edu). Once approved, arrangements for an orientation session will be made. Access to the Core will not be granted without an approved application and all users must complete an orientation session.

EQUIPMENT SIGNUP

Procedures for reserving equipment will be reviewed in an orientation session required prior to equipment access. Upon completion of the orientation session and a preliminary training period, equipment is also available on a walk-in basis. Links to publicly shared calendars are below.

 

Contact Information

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