Specimen Chambers for Live Cell Microscopy
For live cell microscopy, cells must be grown in dishes that are amenable to imaging. Our live cell microscopes are inverted, the objective lens is below the sample and we image though the bottom of a dish. Therefore, cells must be plated on a glass coverslip bottom dish. The coverslip should be #1.5, which indicates a thickness of 170 micrometers. The coverslip can be coated to assist the cells in adhering (i.e. with poly‐D‐lysine, collagen, fibronectin). Consult the literature in your field for the appropriate coating. Additionally, the microscope must be equipped with the right adaptor so the dish will fit onto the stage. Below is a list of dishes that will work on our specific microscope stages. Plastic bottom dishes do NOT work because they are too thick to image though, plastic coverslips are not recommend for several reasons, including their inherent fluorescence.
Nikon A1R and Perkin Elmer Spinning Disk
Disposable:
MatTek : 35mm disposable plastic dishes
http://glass‐bottom‐dishes.com/pages/
In Vitro Scientific : 35mm disposable plastic dishes
http://www.invitrosci.com/product_detail.php?product_id=31
Lab‐Tek: Lab‐Tek II Chambered Coverglass (note 8 well configuration only the middle 4 wells are accessible for imaging)
http://www.nuncbrand.com/en/page.aspx?ID=235
Reusable:
Invitrogen Attofluor chambers : Reusable 35mm metal dishes used in combination with 25mm round coverslips (available to demo from ICI, please inquire)
http://www.invitrogen.com/1/1/4930‐attofluor‐cell‐chamber‐microscopy.html
25 mm round #1.5 coverslips: Available from many vendors
NeuVitro: http://www.neuvitro.com/german‐coverslip‐25mm‐diameter‐circular.htm
Warner Instruments: http://www.warneronline.com/product_info.cfm?id=11
Olympus Cell^R TIRF
Bioptechs FCS2 chamber : This chamber warms cells to 37 and can provide 5% CO2. An additional feature is optional perfusable laminar flow over the cells.
http://www.bioptechs.com/Products/FCS2/fcs2.html Cells should be plated on
40 mm # 1.5 coverslips Bioptics (cat# 40‐1313‐0319)
(All dishes listed above will fit on the TIRF microscope stage, but cells can only be imaged at room temperature, and will not be warmed. The FCS2 chamber must be used if temperature and/or CO2 control is desired)
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Maintaining Living Cells on the Microscope
Control of the environment is one of the most critical factors for successful live‐cell imaging experiments. Aspects of the environment that can be controlled include the physical parameters of the chamber in which the cells are grown and imaged, temperature control, atmospheric conditions (gas mixture and humidity), nutritional supplements, growth medium buffering (pH), and osmolarity of the culture medium. Cells should be maintained in a way that promotes normal growth and function. Cell Imaging Media
Cells are typically grown and maintained in a cell incubator at 37°C with 5% CO2. The pH value of NaHCO3 buffered media depends on the CO2 content of the incubator atmosphere. When the CO2 supply to an incubator fails, media becomes alkaline and cells are adversely affected and may die. Cells can be imaged in their normal growth medium only if 5% CO2 is supplied to the dish on the microscope stage. This is possible on all of our live cell microscopes.The pH indicator phenol red can interfere with collection and interpretation of weak fluorescent signals. All media used for microscopy should be free of phenol red.
Instead of providing CO2, an alternate approach is to use HEPES buffered medium. HEPES buffer does not require a controlled atmosphere. Buffer strength for cell culture applications is usually in the range of 10 to 25 mM. While most cell types do well in HEPES buffered medium, cell viability should be evaluated by the researcher prior to use for live cell imaging. Do not image live cells in PBS. For further information, consult the literature in your field. Depending on cell type or if extended time lapse imaging (many hours to days) is necessary you may need to use CO2 and your usual media (without phenol red) on the stage.
500ml Cell Imaging Media (CIM)
Hanks' BBS 4.9g (from Sigma H1387)
10mM HEPES (= 1.19g)
No Vitamins
OPTIONAL: Amino acids (all concentration as in regular media except Tyr and Trp)
Dissolve in MiliQ
pH to 7.4
filter sterilize
CIM can be supplemented with 1% ‐ 5% fetal bovine serum (FBS).
Warm CIM to 37°C. When ready to image, remove the cell growth medium, wash, and add CIM.
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Quick Guide to ICI Live Cell Microscopes
Nikon A1R Point Scanning Confocal
Multipoint stage
Photobleaching/photoactivation/photoconversion
z‐stacks
perfect focus
DIC
37 degrees (microscope enclosed in environmental box)
optional CO2; humidified
Laser lines: 404, 457, 476, 488, 514, 568, 638
Objectives : 10x 0.3NA; 20x 0.75NA multi‐immersion; 40x 1.3NA Oil; 60x 1.4NA Oil; 100x 1.49NA Oil
Common Fluorophores: GFP, mCherry, CFP, YFP, PAGFP, Dendra2
Perkin Elmer Spinning Disk Confocal
Multipoint stage
z‐stacks
Phase Contrast
37 degrees (microscope enclosed in environmental box)
optional humidified CO2
Laser lines: 488, 568, 647
Objectives: 5x ; 10x ; 20x; 40x NA 1.3 Oil; 63xNA 1.4 Oil; 100x NA 1.4 Oil
Camera: Hamamatsu Orca‐ER
Common Fluorophores: GFP, mCherry, DRAQ5 (far red)
Olympus Cell^R TIRF
Bioptics FCS2 + Objective heater
Laser lines: 488 and 561
Objective: 60x 1.49NA TIRF objective
Camera: Hamamatsu Orca‐ER
Common Fluorophores: GFP, mCherry
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References
Live Cell Microscopy
Frigault et al. Live‐cell microscopy ‐ tips and tools. J Cell Sci (2009) vol. 122 (Pt 6) pp. 753‐67
Hoppe et al. Live cell fluorescence microscopy to study microbial pathogenesis. Cell Microbiol (2009) vol. 11 (4) pp. 540‐50
Bakstad et al. Quantitative measurement of single cell dynamics. Curr Opin Biotechnol (2012) vol. 23 (1) pp. 103‐9
Marcus AI Visualization of spindle behavior using confocal microscopy. Methods Mol Med. 2007;137:125‐37.
Fluorescent Proteins
Shaner et al. A guide to choosing fluorescent proteins. Nat Methods (2005) vol. 2 (12) pp. 905‐9
Datta and Patterson. Optical highlighter molecules in neurobiology. Curr Opin Neurobiol (2012) vol. 22 (1) pp. 111‐20
TIRF
Mattheyses et al. Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci (2010) vol. 123 (Pt 21) pp. 3621‐8
Axelrod. Total internal reflection fluorescence microscopy in cell biology. Meth Enzymol (2003) vol. 361 pp. 1‐33
FRAP
Chen et al. Methods to measure the lateral diffusion of membrane lipids and proteins. Methods (2006) vol. 39 (2) pp. 147‐53
Snapp et al. Measuring protein mobility by photobleaching GFP chimeras in living cells. Curr Protoc Cell Biol (2003) vol. Chapter 21 pp. Unit 21.1
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