July 2008 Jonathan Sommer

Optical Mode Image  Has two modes to view samples Optical mode (white light) and Fluorescence mode  Depending on your sample either mode maybe useful to you.

 Most importantly please respect the equipment it’s expensive!

 New Olympus IX70 Inverted Epi flourescence Research Microscope (Price: $22,500.00) The retail price on this unit from Olympus was $28,221.00

Fluorescence Mode Image

   Optical mode uses a Tungsten Halogen lamp to illuminate specimen Fluorescence mode uses a Mercury Xenon Arc Lamp, with specific filters to select excitation and emission wavelengths Microscope has two ports, the first for a CCD camera and the seconded compatible with SLR camera systems

   Currently on the microscope we have 10x and 40x The microscope has built in 1.5x, 10x->15x 40x->60x Also available is a 100x oil immersion objective for micron and sub micron sized substrates of interest

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Filters are labeled and directional 520 nm 60 nm  Filter cubes inside of a spinning turret located below objective stage  Carefully slide turret out from the right-hand side  Use specialty key (lug nut) to remove retainer and then remove the filter  ALWAYS WEAR WHITE GLOVES!!!

  Know your Abs. and Emission regions for your sample Most of our filters are wide and narrow band pass, 60nm and 30nm respectively. Fluorescence Filters

   First uncover the microscope, turn on the PC/moniter and sign the log book.

Then turn on the mercury xenon arc lamp (fluorescence) by turning the power on the olympus box (white) located to the left of the microscope The tungsten halogen lamp (optical) is turned on by the toggle switch located on the rear on the microscope by the base

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Gently remove Filter turret from microscope Remove lid and using screw driver remove filter cube Put on white gloves, and change filters Replace filter cube lid, and then replace filter turret on microscope

SLR C-Port    New CCD uses USB connection, focuses really fast, use traverses and PC monitor Old CCD focuses really slow, higher resolution images For color images in optical mode, you can use Digital SLR camera to get color images of your sample

   Focusing takes practice and mostly patients If photo bleaching is a problem, use shutters to block the light Weak or to bright, adjust the exposure time, if its still to bright consider using a neutral density filter

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CCD gives you black and white image Free software on web ImageJ Has variety of common dye colors to stain images, record your filter sets in your notebook!!

ImageJ download http://rsbweb.nih.gov/ij/download.html

  Cleaning Optical Elements - The first rule in fluorescence microscopy (and all other forms of microscopy) is to keep the optical elements completely free of dust, dirt, oil, solvents, and any other contaminants. The microscope should be kept in a low vibration smoke-free room that is clean as possible and has minimal disturbance of the circulated air. Use a dust cover on the microscope when not in use and keep all accessories in air-tight containers. Avoid using corrosive solvents to clean any part of the microscope, and use only diluted soapy water to clean non-optical surfaces. Objectives should be kept clean using the following tips: Never drag anything across the lens surface with a high degree of pressure, including lens paper, to avoid the possibility of introducing very fine scratches onto the surface.

 Clean the lens with a solvent designed for optical surfaces or, in an emergency, absolute ethanol. Avoid other solvents because they might react with optical coatings on the glass.  Dust the lens surface with compressed gas prior to cleaning with a solvent to remove loose particles.   Soak a Q-tip with lens cleaner or ethanol and very gently wipe it over the lens several times, turning the cotton tip before each pass. Blot excess solvent with lens tissue and allow the lens to dry thoroughly. Repeat this procedure. Oil-immersion lenses can be cleaned with solvent as described above. Never use oil with an objective that is intended to be used dry, because cleaning a dry objective can be much more difficult than one designed specifically for oil.

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Olympus has a great website!

 http://www.olympusmicro.com/primer/index.html

 http://www.olympusmicro.com/primer/java/fluore scence/opticalpaths/index.html

 http://www.olympusmicro.com/primer/java/fluore scence/exciteemit/index.html