Michael Hooker Microscopy Facility (MHMF.ORG)
Sample preparation, calibration, slides, cover slips, test samples, chambers, etc.
Sample Preparation: Sample preparation is your task since you are the expert for your experiments. However we will discuss the choice of fluorophores/secondary antibodies best suited to the available excitation sources and filters on our resources, and we may be able to give some tips about sample preparation.
Spatial calibration: Spatial calibration is generally unnecessary since the Leica & Zeiss confocal microscope systems are well calibrated out of the factory and generally remain in calibration. However calibration is a good diagnostic of proper machine function. In the x-y plane, calibration is best done with a stage micrometer or haemocytometer. Z-axis calibration and x-y plane calibration can be done by scanning a spherical bead. Calibration should be checked with each wavelength used for your sample in your mounting medium. Co-localization beads are available from Molecular probes, and small samples may be available in the facility. Also a stage micrometers is available.
Slides: Slides are best if glass, which allows good transmission of light for transmitted light mode microscopy. Plastic cover slips should be avoided since they can be inherently fluorescent. Plastic dishes and slide are not suitable for DIC/Nomarski since they polarize light strongly. For confocal avoid Lysine coated slides, which tend to produce reflections when imaging close to the glass surface.
Covers slips: Covers slips should generally be number 1.5 (170 um), although some objectives have a correction ring to set cover slip thickness or are even designed to image with no cover slip (water immersion dipping lenses.) See resource pages for detailed information about the characteristics of the objectives on specific equipment.
|Coverslip number||Thickness range (um)|
|#00||060 - 080|
|#0||080 - 120|
|#1||130 - 170|
|#1.5||160 - 190|
|#2||170 - 250|
|#3||280 - 320|
|#4||380 - 420|
|#5||500 - 600|
For z-series imaging it is important to attach the cover slip is stably attached to the slide. A moving slide as it floats between the mounting media and immersion fluid will cause optical distortions. Use a hardening mounting media, or paint valap on edge of cover slip or use drops of hot dental wax on the corners & edges. Nail polish is not recommended for samples keep for a long time since the acetone will damage dyes and cell structure. Cleaning is important if cells are to be cultured.
Test Samples: Standard test samples are available for training and practice scanning. Buccal Epithelia cells are good test samples for DIC and vital fluorescent dyes are available to make them good DIC and fluorescent test samples.
Live Cells: Correct environment is essential. Consider temperature, gasses, pH buffering and moisture (loss). Especially be careful with Bicarbonate buffering systems, which require equilibration with 5% CO2 and media which contain fluorescent indicators, such as Phenol Red. A potential good media is Ham's F12 or L-15 Liebowitz (phenol red free, part # 21083-027) from Gibco. Phenol Red is mildly fluorescent but does prevent formation of free radicals. Good intracellular pH buffering requires the Bicarbonate & CO2. Artificial buffers such HEPES do not cross cell membranes and hence do not control intracellular pH well. Some cells types are particularly sensitive to lack of good pH control.
Live Cell support: Cells are happiest when adherent, which also aids imaging by cutting down movement. Poly-L-Lysine is a standard (e.g. Sigma). Cells can also be grown in various bio-compatible matrixes such as Matrigel (BD Biosciences). For polarized epithelial cells permeable supports such as Conrning transwells can be used to culture and view cell layers.
Chambers: we are working on getting holders and chambers for live cell imaging under ambient conditions. We are investigating temperature and gas controlled environment chambers. Various chambers for live or fixed cultured cell imaging with single or multiple wells. For best resolution with inverted scopes glass bottom dishes of 170 um thickness (#1.5) should be used. One source of such round dishes can be found here or coverslip style here.
Micro Titer Plates: Dimensions of 24 to 1536 well plates.
Cell Staining: Alexa Fluors
Mounting medium: Mounting medium is available from many sources, and many users mix their own. Ideally refractive index of mounting media should be the same as the slide. If not reflections will be detected when scanning close to the media and slide interface.
Blocking buffers: A simple and inexpensive buffer to reduce background in immunolabeling: 1% cold water fish gelatin (Sigma, stock 20% in H2O), 0.8 % BSA, 2x PBS
Clearing Agents: (similar to mounting media) Increases the transparency of tissues. This permits imaging to deeper layers with less light absorption and scattering.
Immersion Oils e.g. see Cargille Laboratories For simplicity and best optical performance immersion oil is provided at each microscope. Use only Nikon formulated immersion oil for the Nikon scopes, Leica immersion oil for Leica scopes & Zeiss oil for Zeiss scope. Mixing oil will degrade optical performance, so oil from previous viewing should be cleaned off the cover slips. Alcohol and q-tips are provided to help clean off your slides, and are never to be used on objectives. Note: German immersion oils should not be used on Japanese objectives, since they are reputed to remove the anti-reflective coatings.
Immobilizing Live cells/organisms: Try clamped under a cover slip, make spacers with cut cover slips of selected thickness, e.g. #0 for thin space, use depression sides, hydroxy methyl cellulose, carefully cooled in low melting point agar, warmed in heat setting gel, e.g. CyGEL (Biostatus). This list is brief & terse. What actually works best depends on the nature of your sample and the conditions it can tolerate.
We can not possibly list all that is relevant to this page here. However users are invited to submit their favorite reagents, chambers, mounting media listed, etc. here with due credit. Please contact us.
Invitrogen/Molecular probes sells an extensive range of standard test slides, mounting media, cover slip gaskets, fluorescence co-localization beads, not to forget secondary antibody and other conjugates. See also their web site www.invitrogen.com for a more comprehensive catalogue.
Jackson Labs is a good source of secondary antibodies.
Fluorescent Dyes: (This list is intended to be a quick starting point only)
Suggested Fluorescent Dyes for use with Antibody Conjugates:
Copyright 2001-2014 Dr. M. Chua, Program in Molecular Biology & Biotechnology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599
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Last Updated: 2014-07-23