NIBIB Advanced Imaging & Microscopy (AIM) Trans-NIH Shared Resource

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 theAIM 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.

AIM is a Trans-NIH shared resource open to all NIH investigators. For ll inquiries about AIM collaboration, please contact AIM directly. Please do not place a request through CREx.