NDLI: Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

Please wait, while we are loading the content...

Year: 2012 Issue: 70

Tissue engineering of the intestine in a murine model.

In vivo imaging systems (IVIS) detection of a neuro-invasive encephalitic virus.

Generation and recovery of ß-cell spheroids from step-growth PEG-peptide hydrogels.

Methods for intravenous self administration in a mouse model.

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

Spatial multiobjective optimization of agricultural conservation practices using a SWAT model and an evolutionary algorithm.

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Simultaneous intracellular recording of a lumbar motoneuron and the force produced by its motor unit in the adult mouse in vivo.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

An in vitro preparation for eliciting and recording feeding motor programs with physiological movements in Aplysia californica.

Quantitative measurement of the immune response and sleep in Drosophila.

Surgical technique for spinal cord delivery of therapies: demonstration of procedure in gottingen minipigs.

Preparation and pathogen inactivation of double dose buffy coat platelet products using the INTERCEPT blood system.

Procedure for decellularization of porcine heart by retrograde coronary perfusion.

Manual drainage of the zebrafish embryonic brain ventricles.

Targeted labeling of neurons in a specific functional micro-domain of the neocortex by combining intrinsic signal and two-photon imaging.

A novel Bayesian change-point algorithm for genome-wide analysis of diverse ChIPseq data types.

Assaying DNA damage in hippocampal neurons using the comet assay.

Recurrent herpetic stromal keratitis in mice, a model for studying human HSK.

Visualization of endoplasmic reticulum localized mRNAs in mammalian cells.

Optimized staining and proliferation modeling methods for cell division monitoring using cell tracking dyes.

Tactile conditioning and movement analysis of antennal sampling strategies in honey bees (Apis mellifera L.).

Rearing and injection of Manduca sexta larvae to assess bacterial virulence.

Monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy.

The measurement and treatment of suppression in amblyopia.

Simultaneous electroencephalography, real-time measurement of lactate concentration and optogenetic manipulation of neuronal activity in the rodent cerebral cortex.

The green monster process for the generation of yeast strains carrying multiple gene deletions.

Portable Intermodal Preferential Looking (IPL): investigating language comprehension in typically developing toddlers and young children with autism.

Using the BLT humanized mouse as a stem cell based gene therapy tumor model.

The insect Galleria mellonella as a powerful infection model to investigate bacterial pathogenesis.

Glycan profiling of plant cell wall polymers using microarrays.

Chromosome replicating timing combined with fluorescent in situ hybridization.

Year: 2012 Issue: 69

Year: 2012 Issue: 68

Year: 2012 Issue: 67

Year: 2012 Issue: 66

Year: 2012 Issue: 65

Year: 2012 Issue: 64

Year: 2012 Issue: 63

Year: 2012 Issue: 62

Year: 2012 Issue: 61

Year: 2012 Issue: 60

Year: 2012 Issue: 59

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Watanabe, Shigeki Richards, Jackson Hollopeter, Gunther Hobson, Robert J. Davis, Wayne M. Jorgensen, Erik M.
Description	Author Affiliation: Watanabe S ( Department of Biology, Howard Hughes Medical Institute, University of Utah.)
Abstract	Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/3995
Journal	Journal of Visualized Experiments
Issue Number	70
Language	English
Publisher	MyJove Corp.
Publisher Date	2012-12-03
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Microscopy, Electron Microscopy, Fluorescence Nanotechnology Proteins Chemistry Animals Caenorhabditis Elegans Caenorhabditis Elegans Proteins Freezing Research Support, Non-u.s. Gov't Video-audio Media
Content Type	Text
Resource Type	Article

Central Library (ISO-9001:2015 Certified)
Indian Institute of Technology Kharagpur
Kharagpur, West Bengal, India | PIN - 721302

See location in the Map
03222 282435
Mail: support@ndl.gov.in

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in