Setting up the microscope for epifluorescence

The alignment of the light source to achieve an evenly illuminated field is critically important in epifluorescent microscopy 1. Turn on the lamp and allow to warm up. 2. Insert the filter block to be used. 3. Place a white piece of paper on the stage and adjust the light intensity to a suitable level with the slider. Open the condenser diaphragm and the field diaphragm. 4. Adjust the lamp collector focusing until a sharply focused image of the lamp is seen. Centre the image with the lamp...

Purification and preparation of spin labelled vimentin

Spin label compound (O-87500, methyl) methanethiosulfonate MTSL , Toronto Research Chemicals, Toronto, Canada) TCEP (tris-(2-carboxylethyl) phosphine, Molecular Probes, Eugene, OR) 1. Dissolve IBs (from 500 ml of bacterial culture) in 8 ml of 8 M urea (20 mM Tris pH 8, 1 mM EDTA, 8 M urea) and filter through a 0.2 micron filter (Pall Serum Acrodisc, Fisher Scientific). 2. Chromatograph 4 ml of inclusion body solution on a Hi Load 16 60 Superdex 200 column. The column is run with 20 buffer B,...

Setting up the microscope for phase contrast microscopy

The objectives for phase microscopy are different from those used for bright-field only. They include a phase ring and are usually inscribed Ph. Phase contrast objectives can also be used for bright field work. The condenser is also different it contains a series of phase annulae. 2. Kohler illumination is set up as described previously 3. To centre the phase contrast annular diaphragm, start with the objective x10 Ph 1. Set the condenser turret to the appropriate position. 4. Move the...