Electron Microscopy

The Electron Microscopy Unit provides instrumentation, training, and services for: immuno electron microscopy, electron tomography, and specimen preparation, including cryo-techniques.

Micro Analytical Immunochemistry

The Micro Analytical Immunochemistry Unit employs a variety of techniques to isolate, identify and quantitate proteins, peptides, lipids, antibodies and other biological molecules of interest. The unit currently has a range of active projects using MALDI-TOF analysis and imaging, single and multiplex ELISA, and ICP-OES spectroscopy.

Microfabrication and Microfluidics

The Microfabrication and Microfluidics Unit specializes in rapid design, fabrication and characterization of microfluidic devices, including single or multilayer templates, microfabrication in silicon/glass, PDMS, thermoplastics, and agarose. Capabilities also include structured surface modification, such as microcontact printing, and plasma treatment of PDMS devices for irreversible bonding or for surface activation, as well as finite element modeling. The unit also houses a 3D extrusion bioprinter and a rheometer for measuring viscoelastic properties of materials from 4 to 50 degrees Celsius.

Quantitative Methods for Macromolecular Interactions

The Quantitative Methods for Macromolecular Interactions (QMMI) Unit specializes in the following: biophysical characterization of individual macromolecules and their interactions, measurement of solution-phase molecular weight, sedimentation coefficients, and translational diffusion coefficients, hydrodynamic radii, and overall asymmetry of macromolecules or their assemblies, establishment of the stoichiometry of complexes, and insight into secondary structure or changes in structure upon ligand binding.

Scanning Probe Microscopy

The Scanning Probe Microscopy Unit specializes in nanoscale imaging of molecular complexes, lipid bilayers, cells and tissues, molecular recognition, protein unfolding, force spectroscopy, high resolution viscoelastic property mapping, correlated AFM and fluorescence microscopy, and mathematical modeling, image analysis, finite element analysis.

Established Technologies

MIF provides intellectual, technical and material support for investigators using the MIF imaging facilities. MIF preclinical imaging resources include magnetic resonance imaging, micro-computed tomography, high frequency ultrasound with photoacoustic imaging, and optical bioluminescence and fluorescence imaging. MIF does NOT have PET imaging or an irradiator.

Resources include:

• 9.4T Animal MRI System, includes cryoprobe option
• 7T/20cm Animal MRI System
• 4.7T/40cm Animal MRI System
• 14T Vertical MRI Microimaging System
• Micro CT Imaging: in-vivo and ex-vivo
• Bioluminescence/Fluorescence Imaging
• High Frequency Ultrasound with Photoacoustic Imaging
• DEXA Body Composition scanner

Services not offered:

• PET imaging
• Microscopy / Histology
• Two-photon imaging
• Laser capture microdissection

Established Technologies

NMRF is responsible for the following activities:

• Provide intellectual, technical and material support for investigators using its imaging and computer resources.
• Manage the NMR Center wide computer network.
• Provide support for NMR Center Steering Committee activities

NMRF imaging resource includes a state-of-the-art 7.0 T human MRI system and other resources required for anatomical, functional and spectroscopy studies.

NMRF core personnel includes a Director (Dr. Alan Koretsky), a Technical Director, an MRI Physicist, an MRI Technologist, a Network/Systems Administrator and a program assistant.

Established Technologies

The Pathology Service offers a Mouse Phenotyping Service with comprehensive pathologic analysis for genetically engineered mice. The Service provides standardized gross and histopathologic analyses, including serum chemistries and hematology. The workup is designed to evaluate the expected and unexpected phenotypes of the gene manipulations. Analysis is recommended to be performed on age-matched mice to include wild type, homozygote and heterozygote mice. This service is offered by a fee for service mechanism and the current charge is $515/mouse.

The Pathology Service, Division of Veterinary Resources, ORS provides transmission electron microscopic analysis of diagnostic case materials originating from the Pathology Diagnostic Service. EM services are also provided to intramural research scientists on a time available basis. Inquiries may be made to Ms. Patricia Zerfas at 301-496-4464 or by email at zerfasp@ors.od.nih.gov.

The Pathology Service, Division of Veterinary Resources, ORS offers collaborative research pathology support on a time available basis.

User Guidelines

Preliminary and final pathology reports are provided to the principal investigator, institute veterinarian and facility veterinarian.

A pathology submission form should be completed which is available online at http://dvrapps.ors.od.nih.gov/DVR20/SubmissionForms.aspx

The Advanced Imaging & Microscopy (AIM) Resource is a trans-NIH shared resource that houses, operates, disseminates, and improves non-commercial, prototype optical imaging systems developed at the NIH. The facilities at AIM are available for use by the entire NIH intramural research community.
While we welcome users with any size imaging project, AIM specializes in large, yearlong (or longer), collaborative research efforts with NIH labs – providing sample preparation, microscope operation and development, and data processing/analysis as needed. We take care of the imaging and processing so that you can focus on the biology.

Contact Details
Harshad Vishwasrao, Ph.D.
Phone: 301-496-2552
Email: harshad.vishwasrao@nih.gov

Established Technologies

Dualview Inverted Selective Plane Illumination Microscope (DISPIM):

The DISPIM is a dual light sheet microscope that acquires two orthogonal views of a sample. These two views can then be fused and joint deconvolved to generate a single volume with high isotropic resolution. Like other light sheet microscopes, the DISPIM provides rapid volume imaging with a low photodose and is therefore ideally suited for long term or high repetition rate imaging in samples where phototoxicity/photobleaching can be a problem. Unlike conventional light sheet microscopes, the joint deconvolution process provides good resolution in all directions.
Resolution: 330nm in X, Y, Z
Speed: 200Hz framerate, 1-5Hz volumetric
Channels:
GFP or similar 488nm excitation, 500-550 emission
mCherry or similar: 561nm excitation, 570-650 emission
Maximum field of view (1 z-stack): 300 microns x 300 microns x 4 millimeters
Strengths:
Similar to other light sheet approaches, and in contrast to confocal microscopy, the only part of the specimen being illuminated is the plane being imaged. This dramatically reduces the photodose to the sample. DISPIM is ideal for imaging experiments in which photobleaching or phototoxicity is a critical issue – for example long time series requiring repeated imaging of the same sample.
This is the fastest microscope currently available at AIM.
Limitations:
Depending on how much scattering and absorption occur in a sample, the depth to which we can acquire high quality images is typically limited to 50-100microns. The microscope itself however can accommodate much larger samples.
Resolution is adequate for tracking cells and larger subcellular organelles.
Reference:
Wu et al. Nature Biotechnology 31(11): 1032-1038, Nov 2013
For more information, please visit the AIM DISPIM site.

Instant Structured Illumination Microscope (ISIM)/ Total Internal Reflection Fluorescence SIM (TIRF-SIM):

The ISIM is a fluorescence microscope that combines rapid image acquisition (100Hz) with a resolution roughly double that of a confocal microscope in all three dimensions. Unlike other structured illumination microscopes, the resolution is enhanced via hardware in a single frame acquisition rather than computationally processing multiple images, effectively making this an ‘instantaneous’ structured illumination microscope. Recently, the Shroff Lab has extended the capability of this microscope to rapid, super-resolution TIRF.
Donating Lab: Hari Shroff (NIBIB)
Resolution: 140nm in X, Y. 350nm in Z
Speed: Framerate up to 100Hz
Channels:
GFP or similar 488nm excitation, 500-550 emission
mCherry or similar: 561nm excitation, 570-650 emission
Maximum field of view (1 z-stack): 150 × 150 × 500 microns
Strengths:
Spatial resolution superior to that of a confocal microscope at substantially higher speeds.
Spatial and temporal resolution also available in TIRF.
Limitations:
Best for thin samples (<30 microns). Thicker samples induce progressively worse background and optical aberration.
Requires bright/photostable dyes for repeated imaging.
Reference:
York et al. Nature Methods 10, 1122-1126 (2013)
For more information, please visit the AIM TIRF-SIM site.

Interferometric Photoactivated Localization Microscope (IPALM):

Capable of unsurpassed optical resolution, this system is a clone of the original prototype developed at the Janelia Research Campus but offers 3 color capability. IPALM combines photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry providing <20 nm resolution in all three dimensions. This microscope is currently located in the Taraska Lab and will move to AIM once the facility is operational.
Donating Lab: Justin Taraska (NHLBI)
Resolution: 20nm in X, Y. 10nm in Z.
Channels: 405 nm, 488 nm, 561 nm and 633 nm excitation laser lines.
Strengths:
Highest resolution optical microscope currently available.
Provides unparalleled axial resolution.
Limitations:
Difficult sample preparation protocol.
Sample thickness is restricted, and only sub-volumes of mammalian cells can be imaged.
For more information, please visit the AIM IPALM site.

High throughput single molecule imaging microscope:

The high throughput single molecule microscope was designed to image large numbers of cell samples in a multi-well format. This microscope can routinely image up to 8 coverslips and 10,000+ cells per acquisition. An included analysis pipeline automatically segments images to identify nuclei, cell bodies, and puncta (RNAs, receptors, etc.) and generates statistics on these puncta (number/cell, co-expression, co-localization, etc.). This microscope is currently located in the Larson Lab and will move to AIM once the facility is operational.
Donating Lab: Daniel Larson (NCI)
Channels:
Seven color Lumencor excitation bands: violet, blue, cyan, teal, green, yellow and red)
Four detection channels: DAPI, GFP, Cy3, Cy5 (or similar)
Strengths:
User friendly with automated analysis pipeline. Seven color bands for excitation.
Limitations:
Standard widefield optics yields standard widefield resolution.
Does not have TIRF capability.
For more information, please visit the AIM site.

Single molecule TIRF/Inclined illumination Microscope:

This single molecule detection microscope is based on widefield illumination with the flexibility of tuning between TIRF and inclined illumination for different applications – enabling localization-based imaging techniques including single molecule tracking, PALM and dSTORM. The TIRF illumination enables the monitoring of single molecule interactions on the glass coverslips or the imaging of biomolecules in a very thin layer in live cells (mostly on the basal plasma membrane). With the live cell environment system, it also allows single molecule tracking in live cells.
Donating Lab: Hari Shroff (NIBIB)
Resolution: 20 nm in XY and 50 nm in Z after localization
Channels: 405 nm, 488 nm, 561 nm and 633 nm excitation laser lines
Strengths:
Single molecule detection sensitivity in vitro and in vivo
TIRF or inclined illumination provides better signal to noise compared to normal widefield illumination.
Limitations:
Currently no 3D PALM and dSTORM capabilities, these may be added in the future.
For more information, please visit the AIM site.

Microscope	Description	Spatial Resolution	Speed (frames/s)	Photodose	Sample
Instant structured illumination microscope (ISIM)	Fast super-resolution	140nm lateral 350nm axial	100	High	Live or fixed. Small, thin samples like cells, organoids.
Dual view Inverted Selective Plane Illumination Microscope (DISPIM)	Fast, gentle, high resolution light sheet for live samples	330nm isotropic	100	Low	Live or fixed. Cells, up to medium size, transparent samples like organoids, C. elegans, zebrafish.
Cleared Tissue DISPIM	Fast multi-view lightsheet for large, cleared tissue samples	500nm isotropic	100	Low	Fixed and cleared. Up to 5mm thick slabs of tissue, up to 2cm x 3cm lateral size.
Single Molecule TIRF/Inclined Illumination Microscope	Variable inclination illumination single molecule imaging. Including STORM.	20 nm lateral 50-60 nm axial	100	High	Live or fixed. Small, thin samples like cells.
Interferometric Photoactivated Localization Microscope (IPALM)	Localization microscope with unprecedented lateral and axial resolution.	20 nm lateral 10nm axial	Not suitable for live cells	High	Fixed and specially mounted. Small and thin samples like cells, bacteria.

This table describes the spatial resolution, speed, and sample suitability of the five microscopes currently being operated by the Advanced Imaging and Microscopy (AIM) Resource.

User Guidelines

The CSC was initiated in the incubator space of the Common Fund and has transitioned to the intramural program of the National Heart, Lung, and Blood Institute. It is dedicated to the production of new imaging probes for the intramural NIH research community. CSC is administered by the National Heart, Lung, and Blood Institute for the benefit of the entire community of intramural NIH scientists, and is already working on projects for requesting scientists from more than a dozen NIH Institutes. Nearly all of these imaging probes are not commercially available, nor are they viable commercial products and most are new compositions-of-matter. CSC serves scientists from the intramural community who would otherwise be limited in their investigations of promising new imaging probes by a lack of synthetic chemistry or radiochemistry capabilities.

The CSC was born from the realization that imaging technologies will be crucial in basic, translational and clinical research in the 21st century, and that the synthetic chemistry required to reliably produce imaging probes lies at the heart of research within imaging technologies. To this end the CSC has recruited the equipment and expertise to concurrently synthesize multiple types of imaging probes for bioscientists with diverse research interests, encompassing all imaging modalities, including optical, radionuclide, ultrasound, and magnetic resonance. The CSC embodies an exciting new approach to apply and combine chemistry and imaging sciences toward specific problems in biology and medical sciences, and will be a truly interdisciplinary effort aimed at maximizing returns from the revolutionary new discoveries being described in modern imaging. A significant part of the CSC will also be directed, independently, to the discovery of new imaging approaches and compositions. The CSC has been set up in a newly refurbished modern laboratory in Rockville, MD and houses scientific staff, mostly chemists, who have interests and expertise in one of more aspects of molecular imaging.

The CSC is generating known and novel imaging probes for targeting receptors, cells and tissues, and for preclinical in vivo evaluations by its intramural collaborators. Many such interesting agents have been described in the scientific literature, but are often not explored further due to lack of a reliable supply of reagent, and one aspect of the CSC’s mission is to rectify this situation. CSC-supplied reagents will not be limited to one imaging modality, but will include the flexible application of diverse technologies. Also, the CSC will seek to develop novel state-of-the-art imaging probes in collaboration with biological and biomedical intramural scientists who can provide or suggest suitable targeting agent/receptor pairs.