The Light Imaging Facility (LIF) provides intramural scientists with access to state-of-the-art light imaging equipment and expertise in light imaging techniques. The Core offers training and assistance in a variety of light microscopic techniques including laser scanning confocal microscopy, video microscopy and digital image analysis. A digital image printer and slide maker are available for preparing illustrations for presentations and publications.
Scope of Core Facility
The Light Imaging Facility (LIF) is available to all scientists in the NINDS and, as feasible, to other scientists in the Porter Neuroscience Research Center. New users should contact Carolyn Smith to register and for orientation and training.
Using the Light Imaging Facility
Access: The facility is located in Building 35 (Porter Building). Most of the equipment is on level 2, pods B and C but one of the confocal microscopes is located in the basement (BB520). The LIF also maintains a LSM 510 confocal microscope in Building 49. Facility users will need card key access to Building 35 for entry during evenings and weekends and for entry to Building 49 at any time.
Reservations: Registered users may make reservations to use the LSM 510 confocal microscopes and the Leica STED 3X confocal microscope at the online scheduling website https://facility.ninds.nih.gov/public/facilitylogin.aspx . The digital workstations are available on a first-come first-served basis. Those requesting access to the Zeiss LSM 800 and 880 confocal microscopes may link to the Agendo online scheduler: https://next.cirklo.org/nichd (Instructions for accessing Agendo: AgendoTutorial.doc).
File Storage and Transfer
File storage and transfer: Facility users may store files temporarily on the hard disks of the Facility's computers. A server is available for transferring files between computers, courtesy of the Research Services Branch (RSB). Users are requested to limit their file folders to <100 GB and to delete old files. Be aware that files on the hard disks and server are accessible to others and not secure. Files left on the hard disks or server for longer than six months may be deleted without warning.
To access the LIF server:
- PC Users. In the Start or Search box, type \\nindsdirfs2.ninds.nih.gov/shares
- Mac Users. Select Go/Connect to Server. In the box, type smb://nindsdirfs2.ninds.nih.gov/shares
- Log on using your NIH domain name and password. Select LIF from the list of servers. Then select LIF_vol 1.
Supplies: The Facility maintains a stock of common reagents and expendables such as fluorescent dyes and secondary antibodies, mounting media, coverslips, and LabTek chamber coverglasses. These are provided free of charge for pilot experiments but users are expected to purchase their own supplies for routine use.
Leica SPI STED 3X Super-resolution Confocal Microscope
Location: Porter Building (Bldg. 35) Room 2B912
Laser Lines: White Light Laser 470-670 nm. Depletion lasers: 442, 592, 660 nm.
Detectors: Two hybrid detectors, two standard photomultiplier detectors.
Applications: The Leica STED 3X confocal microscope achieves superior resolution compared to conventional confocal microscopes due to the reduced size of the illuminated spot where fluorescence is generated. The method used in STED 3X reduces the spot to about 50 nm in the horizontal plane and 150 nm in the vertical axis, as compared to 200 nm horizontal and 500 nm vertical for a conventional confocal microscope. The technique requires illuminating the sample with two laser beams that together are sufficiently intense to cause considerable photo-bleaching in the sample. Imaging living samples is feasible, but only for a limited number of time points. The system has both a conventional scanner and a high-speed resonant scanner, useful for imaging rapidly changing specimens, and the "hybrid" detectors provide high sensitivity and dynamic range. The STED technique works well with some but not all conventional fluorophores and fluorescent proteins (see sample preparation protocol).
Sample preparation: 2015 STED_Sample preparation guideAppLetter_EN(1).pdf
Zeiss LSM 880 AiryScan Confocal Microscope
Location: Porter Building (Bldg. 35) Room 2C403
Laser Lines: 405, 488, 561, 633 nm.
Detectors: An AiryScan detector and two standard photomultiplier detectors.
Applications: The Zeiss LSM880 AiryScan Confocal microscope uses a unique 32 channel detector that provides 1.7 fold improved resolution as well as significantly improved signal-to-noise compared to conventional confocal detectors. The high sensitivity of the AiryScan detector makes it possible to detect even very weak fluorescent signals while using low levels of laser illumination. The improved resolution and signal are beneficial for imaging small cellular structures such as synaptic spines, organelles and molecular complexes. However, each color channel must be captured individually and the image acquisition rate is slow. The acquisition time for a stack of three color images from a 3 µm thick sample (16 images with a 1.4 NA objective at 2X zoom) is about 2 min. Acquired images must be processed for optimal resolution in the z-axis.
About the AiryScan Detector: LSM880_Airyscan.pdf
Zeiss LSM 880 Spectral Confocal Microscope
Lation: Porter Building (Bldg. 35) Room 2C401
Laser Lines: 405, 488, 561, 633 nm.
Detectors: Thirty-four channel GaASP spectral detector.
Applications: The Zeiss LSM880 Spectral Confocal Microscope is equipped for multi-channel fluorescent imaging in living samples. The spectral detector together with the software for spectral unmixing makes it possible to image simultaneously multiple different fluorophores, provided that reference spectra for each of the individual fluorophores are available. The spectral detector also can be used for conventional multi-channel fluorescence imaging. The high sensitivity of the detector allows detection of weak fluorescent signals while employing low levels of laser illumination. A heating stage and CO2 incubation system are available for maintaining living samples.
About the Spectral Imaging: Spectral_Imaging.pdf
Zeiss LSM 800 Confocal Microscope
Location: Porter Building (Bldg. 35) Room 2C401.
Laser Lines: 405, 488, 561, 633 nm.
Detectors: Two highly sensitive GaASP detectors.
Applications: The Zeiss LSM800 Confocal microscope is well suited for imaging fixed samples such tissue sections. It can acquire images of up to four fluorophores (blue, green, red and far red) by line-by-line wavelength switching or by frame switching. The acquisition software can collect and stitch tiled images of samples too large to fit within the field of view. It can easily be programmed to capture images of multiple regions on a slide or multi-well plate. The high sensitivity of the detector allows detection of weak fluorescent signals while employing low levels of laser illumination and rapid frame rates.
Four Zeiss LSM 510 Confocal Microscopes
Name/ specifications/ location:
LSM510 405/ three detectors/ 405, 458, 488, 514, 543, 633 nm lasers/ Bldg 35 Rm. 2BC909
LSM510 Meta/ two detectors/ 458; 488, 514, 633 nm lasers/ Bldg. 35 Rm 2C109
LSM510 413/ two detectors/ 405, 458, 488, 514, 543, 633 nm lasers, Bldg. 35 Rm. BB520
LSM510 CF 49/ three detectors405, 458, 488, 514, 561, 633 nm lasers/ Building 49 Rm. 3B68
Applications: The LSM510 confocal microscopes are well suited for imaging fixed samples with blue, green, red and far red fluorophores.
Suggested fluorophore combinations: DAPI/FITC/Cy3/Cy5; DAPI/Alexa 488/555/647; CFP/YFP; EGFP/DsRed or mCherry. Avoid Alexa 594.
Zeiss Lumar Stereo Microscope
Location: Porter Building (Bldg. 35) Room 2B708.
Applications:The Zeiss Lumar stereomicroscope provides exceptionally bright and clear transmitted light and fluorescence images. The 1.5X objective has 30 mm free working distance and magnifies from 12X to 150X. Filter sets are available for UV, cyan, green, yellow, red and far-red fluorescence. A QImaging Micropublisher 5.0 digital color camera is available for photomicroscopy.
GE Typhoon Variable Mode Scanner
Location: Porter Building (Bldg. 35) Room 3A1014.
Laser lines: 457, 477, 488, 532, 633
Applications: The Typhoon 9410 Variable Mode Imager is a versatile multi- purpose laser scanner. For DNA, RNA, and protein samples, choose from: storage phosphor autoradiography, direct blue, green or red excited fluorescence, and chemiluminescence. The appropriate optical components are automatically activated when the scanning mode is selected. Typhoon scans wet or dry gels, blots, mounted and unmounted storage phosphor screens up to 35 x 43 cm. It exhibits outstanding linearity, quantitative accuracy, and extremely low limits of detection. Seven user selectable pixel sizes (10, 25, 50, 100, 200, 500 and 1000 microns) ImageQuant TL v7.0 software.
Epsom V850 Flatbed Scanner
Location: Porter Building (Bldg. 35) Room 2B914.
Applications: High resolution scanner for transparent and reflective originals. Connected to an iMac with Adobe Photoshop software.
- Volocity (Perkin Elmer). 3D image visualization, deconvolution and analysis. Measure volumes, track particles, assess colocalization. File formats: tiff, Zeiss lsm and czi, Leica lif, Nikon and others). 35/2B914.
- Metamorph (Molecular Devices). 2D image analysis. Automated morphometry, track particles, create montages and kymographs. File formtas: tiff, Zeiss lsm. 35/2B914.
- Huygens Image Restoration (Scientific Volume Imaging) and Leica Application Suite. Image restoration by deconvolution. Leica 3D image visualization module. File formats: Leica lif, Zeiss lsm and czi, and others). 35/2B814.
- Zeiss Zen Blue and Zen Black. AiryScan image processing, 3D visualization. File formats: Zeiss lsm and czi. 35/2C403.
- Zeiss LSM510 software. Full software package. 35/2B914.
Short Guide for LSM510: General procedures for operation of LSM 510 written by Tony Collins.
Volocity User Guide: VolocityuserGuide.pdf
Metamorph User Guide: Metamorph-User-Guide-Andrea-contents-April-2013.pdf
Huygens Deconvolution User Guide: HuygensUserGuide.pdf
Fluorescence Microscopy Literature
Basic Confocal Microscopy:
An introduction to the principles of laser scanning microscopy and practical tips for confocal users (C. L. Smith., Current Protocols in Neuroscience; 440 KB).
Confocal Laser Scanning Microscopy:
The theoretical basis of laser scanning confocal microscopy and digital signal processing (Carl Zeiss, Inc.; 864 KB).
AiryScan Detector: nmeth.f.388.pdf
Super-resolution imaging: Super-Resolution.pdf
Principles and Application of Fluorescence Microscopy
Donald Coling, Bechara Kachar
Preparing samples for STED imaging:
Leica STED super resolution overview.pdf
Spectral imaging: Spectral_Imaging.pdf
Freeze substitution for immunofluorescence: Freeze substitution for immunofluorescence
Image Analysis Software
Zeiss LSM Image Browser: Image Browser is a free stripped-down version of the LSM 510 software for PC computers. The Zeiss Image Browser can be downloaded from the LIF server (LIF vol 1/Lif vol 1/Zeiss Image Examiner).
ImageJ: ImageJ is a public domain image processing and analysis program developed by Wayne Rasband in the Research Services Branch (RSB) of the National Institutes of Mental Health (NIMH). The program runs on any platform with Java (including Mac OSX) and can be customized through specialized plugin modules or macros. ImageJ can be downoaded from http://rsb.info.nih.gov/ij/. Manual for ImageJ: Add link to: ImageJ_Manual(1).pdf
Fiji: Fiji is a user-friendly version of ImageJ. It is easy to install, has automatic updates, extensive documentation and lots of plugins for analyzing confocal microscope images and image stacks. Download Fiji: https://imagej.net/Fiji/Downloads
Molecular Expressions: This website is a good source of information about light microscopy, including confocal microscopy. http://www.molecularexpressions.com/primer/index.html