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Dictionary for microscopy and imaging
Don't know what that microscopy/imaging term means? Check it in the Photonics dictionary.
Atlases and databases
3D MRI digital atlas database of adult C57BL/6J mouse brain is part of the Mouse Phenome Database (MPD) project.
The Allen Institute for Brain Science has 3D Allen Brain Atlas that contains information on 3000 genes active in mouse brain.
Brainmaps is an interactive digital brain atlas of human, mouse, cat, and rhesus monkey brains.
The Online Bioinformatics Resources Collection (OBRC) contains annotations and links for 1936 open source bioinformatics databases and software tools.
3D embryo anatomy atlas is part of the Edinburgh mouse atlas project.
For gene expression atlas of the mouse brain, see NINDS Gensat.
EMAGE is a database of gene expression patterns during mouse embryo development, part of the Edinburgh mouse atlas project
Solunetti has a histology section that contains description of different tissues as well as some images of histological sections. The site is currently under development.
Digital Morphology (part of the National Science Foundation Digital Libraries Initiative) has high-resolution X-ray computed tomography-generated digital images of various species, also 2D and 3D visualization and anatomy description.
WebMicroscope is a web site managed by the Universities of Helsinki and Turku. Contains e.g. atlas of breast histopathology, Department of Pathology slide meetings and other slide seminars.
For example Invitrogen's Molecular Probes provides several probes for actin labeling. See Probes for Actin by Molecular Probes.
Alexa Fluor Dyes
Invitrogen's Molecular Probes fluorescent dyes and conjugates. For more information, see the informative Molecular Probes pdf file on Alexa Fluor Dyes (their spectra, conjugates, applications etc.).
Primary antibodies (signal transduction molecules): Cell Signaling.
Secondary antibodies: Jackson ImmunoResearch Europe.
Primary and secondary antibodies: Molecular Probes.
See Microscopy techniques - Fluorescence imaging - Bleaching below. Wright Cell Imaging Facility at Toronto Western Research Institute has published a summary of mounting media and antifade reagents.
Attachment of cells and tissues
Sometimes a special adhesive is needed for attachment of cell and tissues on glass or plastic. Poly-L-lysine, collagen, fibronectin, and silane are often used. BD Cell-Tak Cell and Tissue Adhesive can also be used.
See Nexcelom web site for Cellattice micro-ruled coverslips.
Karl Hecht Assistent sells coverslips that are 0.170 +/- 0.01 mm thick.
If you do not want to use DAPI (blue) staining for DNA, you can also use e.g. SYTOX Green (green), 7-AAD (red), or To-PRO-3 (far red) dyes, all available from Invitrogen's Molecular Probes. For a far red dye, you can also try Biostatus Draq5.
For a comparison of DNA stains in living cells, see Martin et al. (Cytometry 67A, 1 (Sept), 45-52, 2007).
If you need protocols for DNA staining, check the Biomedical Imaging and Analysis Facility (University of Western Australia) pdf.
You can label DNA or RNA probes e.g. by using Invitrogen's Molecular Probes ChromaTide labeled nucleotides or ARES DNA labeling kits in which aminoallyl dUTP is enzymatically incorporated into DNA by various methods.
Hope-fixative does not contain formaldehyde and it leads to permanent fixation without crosslinks. RNA and DNA can be isolated from HOPE-fixed tissue.
For information about perfusion fixation, see the article by Scouten et al in Microscopy Today (p. 26).
Refractive index of the immersion oil used should be as close to the refractive index of the specimen. Use the immersion oil suggested by the microscope manufacturer. E.g. Cargille offers several immersion oils of various refractive indexes.
Rowiak offers new, non-contact laser microtome for slicing tissues without embedding or other preprocessing steps.
Wright Cell Imaging Facility at Toronto Western Research Institute has published a summary of mounting media and antifade reagents.
Organ labeling (fluorescent)
Molecular Probes provides Organelle Lights fluorescent proteins that are target various organelles and come in a variety of colors. Pre-packaged viral particles are used to deliver the Organelle Lights proteins into living cells.
Self-assembling fragments of GFP can be used to tag and detect proteins. See: Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein (Nat. Biotechnol. 23, 102-107, 2005) by Stéphanie Cabantous, Thomas C Terwilliger & Geoffrey S Waldo.
You can immobilize non-adherent cells using Biostatus CyGel, which is a liquid when cold and a gel when it is warm. CyGel is compatible with live cells and optically clear with low autofluorescence.
Whole cell staining (fluorescent)
Short instructions how to use Axiovert 200 can be found here.
Depth of field
See Zeiss web site.
Depth of focus
See Zeiss web site.
See Zeiss publication Microscopy from the very beginning by Dr. H. G. Kapitza, p. 36. Also information on eyepiece reticles and micrometers.
Field of view
See Zeiss web site.
The microscopy room microscopes have Chroma's filters. On Chroma web site you can select either a filter or a fluorochrome and see the spectra as well as recommendations which filter to use for a particular fluorochrome. You can also check Molecular Probes' table for spectral characteristics and recommended bandpass filter sets for their dyes.
E.g. LSM 510 Meta uses Zeiss filter sets.
See also Zeiss guidelines of how to select a proper filter set.
Have to be selected according to the objective used. Oil objectives have higher numerical aperture (NA) than dry (air) objectives and, thus, better resolution. For thick (>10-15 micrometers) samples, spherical aberration becomes more important than NA for determining image quality. For that reason, water or glycerol are used between the objective and cover glass.
Safety sheet for Zeiss Immersion oil 518F (for fluorescent samples)
Safety sheet for Zeiss Immersion oil 518N (for non-fluorescent samples)
Safety sheet for Zeiss Immersol W (for water objectives)
Safety sheet for Zeiss Glycerin Immersion Fluid (for live-cell imaging with glycerin objectives)
Provides optimized illumination of your specimen - should always be done before using a microscope with transmitted light! See Zeiss instructions how to do it. For more detailed information on Koehler illumination, see Molecular Expressions web page on Köhler Microscope Illumination. Excellent, short article explaining the concept of Koehler illumination is written by Jan Hinsch (Leica Microsystems).
Magnification (of the microscope)
See Zeiss publication Microscopy from the very beginning by Dr. H. G. Kapitza, p. 3 and 6.
See the nice introduction to multiphoton microscopy by the Laboratory for Optical and Computational Instumentation (LOCI).
Numerical aperture (of an objective)
See Zeiss publication Microscopy from the very beginning by Dr. H. G. Kapitza, p. 4.
See Zeiss instructions of how to select the best objective for your imaging.
See Zeiss publication Microscopy from the very beginning by Dr. H. G. Kapitza, p. 5-6. To calculate resolution both at sample and camera, go to Institut Pasteur PFID Microscope resolution calculator page.
Sampling frequency (rate)
You must ensure that the optical resolution of the microscope matches the digital resolution of the camera. Nyquist theory tells that the sampling frequency (= number of pixels in digital imaging) should be more than twice the maximum frequency component of the signal (= minimum distance between two objects that can still be distinguished). You can calculate that by first determining the optical resolution (in nm) of your imaging system (in xy if using single z plane; both in xy and z if taking z-stacks). Then use Nyquist theory for determining the camera resolution (in pixels) that you should be using. To calculate optimal sampling frequency, go to Institut Pasteur PFID Microscope resolution calculator page.
Stallion HSI live cell imaging platform (Zeiss / Intelligent Imaging Innovations (3i))
Molecular Probes has a new product called Image-iT FX signal enhancer (Cat.NO 136933) which is supposed to improve fluorescence signal-to-noise ratio by eliminating nonspecific background signal.
Can be reduced by using antifade products, see e.g. Molecular Probes ProLond and SlowFade.
Zeiss interactive fluorophore database allows you to superimpose the excitation and emission spectra of multiple fluorophores on a normalized axis. Use this site if you are concerned about spectral overlap of your fluorophores.
See EAMNET teaching module on calcium imaging by M. Cruz and S. Castel.
See the links on the LSM 510 Meta page.
M. Stanley's article in Microscopy Today (p. 64) features some important aspects of protecting your vision when using fluorescence microscope.
FLIP (fluorescence loss inr photobeaching)
See an informative EAMNET teaching module on FRAP by S. Terjung and R. Pepperkok, also covers FLIP..
See Fluorescent Spectra, a large database by Carl Boswell and George McNamara with more than 350 fluorescent proteins listed; Fluorescence Spectra Viewer by Molecular Probes (if the visit the page for the first time, you first have to select Finland, then continue, to get to the Viewer); interactive fluorophore database by Zeiss that allows you to superimpose the excitation and emission spectra of multiple fluorophores; Curv-o-matic by Omega (an interactive database of fluorophore absorption and emission spectra, along with filter set recommendations and accompanying filter spectral curves). On Chroma's web site there is also spectral information.
FRAP (fluorescence recovery after photobeaching)
See an informative EAMNET teaching module on FRAP by S. Terjung and R. Pepperkok.
FRET (fluorescence resonance energy transfer)
Visit FRETImaging.org for FRET resources and FRET mailing list.. See also Molecular Probes handbook (note1.2) for a short description of FRET. Follow this link for a pdf file of "Correction of emission crosstalk for FRET sensors" by T.-L. Chew and R. L. Chisholm. There is also an informative EAMNET teaching module on FRAP by S. Terjung and R. Pepperkok.
If you have difficulties seeing GFP, it is worth trying GFP antibodies (e.g. Roche monoclonal). Use of antibodies often enhances GFP signal significantly. Also for some other fluorescent proteins there are specific antibodies available.
DIC (differential interference contrast)
Used for thick, unstained specimen. For more information, see Zeiss publication Microscopy from the very beginning by Dr. H. G. Kapitza, p. 23.
Good general overview of phase contrast is available e.g. on the Molecular Expressions web site. Phase contrast is used for viewing thin, unstained specimen.
Live cell imaging
For best results, use 0.17 mm glass bottom culture dishes, available from e.g. MatTek, Nunc, Ibidi, or BD Falcon. E&K Scientific sells reusable flexiPerm silicon inserts in single, 4-, 8-, and 12-chamber formats. World Precision Instruments has cover-glass bottom dishes called FluoroDish that have good UV transmission and that are also available with black walls for fluorescence imaging.
Stovall Flow Cell culture chambers are designed for imaging biofilms in vivo.
Digital Image processing and analysis
2D gel image analysis
Ludesi offers online pay-per-image 2D gel image analysis.
Computational imaging can be used for methamatical modeling of molecules and cells. Free Virtual Cell software (from National Resource for Cell Analysis and Modeling) allows you to do simulations e.g. for FRAP data and membrane diffusion.
Douglas W. Cromey, SWEHSC Cellular Imaging Core, has a good introductory web page on digital imaging (explaining pixels, resolution, bit-depth, RGB/CYMK, file formats). See also: Figures.
Figures (preparing for a manuscript)
Be careful of how you manipulate your images - it is best to always keep the original data unchanged and do the manipulations using copies of your images. Also, be aware of the requirements scientific journals have for the figures. For example, see the new guidelines for preparaing figures for the Science and Journal of Cell Biology (see Editorial Policies section). In fact, JCB has a specially trained editor who is looking for signs for inappropriate manipulation of images!
Douglas W. Cromey, SWEHSC Cellular Imaging Core, has a good web site on digital imaging ethics, with guidelines. Another good reference: M. Rossner and K. M. Yamada (2004) What's in a picture? The temptation of image manipulation. J. Cell Biol. 166 (1), 11-15.
Gamma curve is the shape of a line connecting the input and output values responsible for generating an image. Gamma correction means editing an image's gamma curve. This correction is nonlinear which is useful for correcting the nonlinearity of computer CRT monitor displays. For an overview of gamma, see gamma correction page on Olympus web site.
The input is an image; the output is data. In other words, you extract information from your image.
The input is an image; the output is a modified image. Mainly used before image analysis for enhancing certain features.
See the paper by Vazquez et al. (Current Biology 11: 1227-1239, 2001).
AxioVision is a Zeiss imaging software that can be used to aquire images with AxioCam CCD cameras. In Axiovert (Axiovert 200), we use AxioVision 3.1. For instructions, see Zeiss guide to AxioVision 3.
Need to create pseudocolors in ImageJ? Article by J. Sheffield in Microscopy Today (p. 52) will give you detailed instructions.
For a collection of ImageJ plugins, macros, and instructions, visit MBF ImageJ for Microscopy by McMaster Biophotonics Facility.
LSM Toolbox by P. Pirrotte and J. Mutterer lets you work with Zeiss lsm files, preserving metadata.
Importing data in Excel
When data tables are imported via text file format, there is a danger of misinterpreting numerical information. This is especially the case in CellInsight, since the international settings in the computer are American, versus our Finnish ones. See the guide for correct importing of data tables.
Douglas W. Cromey, SWEHSC Cellular Imaging Core, has a good collection of links to Photoshop tutorials on his web site. He has also some tips for highlighting text and overlaying confocal images in Photoshop. Especially you should check his article "Potentially the most dangerous dialog box in Adobe Photoshop", which deals with image size and resolution.
If you want to merge red/green images in Photoshop, check Dr. Paul Rigby's (BCMRC) instructions.
Books that can be borrowed from BIU
Adobe Photoshop CS2 User Guide
AxioVision User's Guide
Image-Pro Plus: Start-Up Guide, Reference Guide, Auto-Pro Reference
LSM 510 Meta manual
Page updated 05.06.2013