The violet light microscope; a method for visual estimation of heme in living cells.

By M. H. F. WILKINS, M.A., PH.D. AND S. DE CARVALHO, M.D. H EME COMPOUNDS have relatively sharp absorption bands ins the visible spectrum w’hich have beers much used to identify heme enzymes ins tissue sections, e.g. the classical work of Keilins.tm These bands are, however, not sufficiently strong for consvensienst estimations of the small amounts of heme in a single cell. The Soret band, at the extreme violet end of the visible spectrum, is often as much as 10 times higher thans the longer w’avelensgth hands referred to above. As a result, it has beets possible to estimate heme in insdividual blood cells by measurinsg the Soret bansd absorption.2’4 For qualitative estimation of heme the complications of photographic or photoelectric recordinig are not tsecessary, and simple visual observations with the microscope of the absorption w-ithin cells of violet light ins the Soret band w-ill suffice. It is isot likely that such ais obvious technsic is new, but, as we have founsd tso examples of its havinsg been used or its potentialities recognsised,* a description of the method may he useful. The mains technical problem is to obtains a very bright. source of violet light; tunsgsten filament lamps are not very bright, but mercury arcs emit a comsvenient bright linse at 4047 A light. If a monochromator is available, a cheap medium high pressure lamp is adequate (e.g. Osira 125 watt), hut most w’orkers will probably prefer to use a light filter, ansd thens a brighter amsd more expemisive lamp such as a compact-source high pressure mercury lamp will be nsecessary (e.g. B.T.H. 250 watt compact source). Onse filter solutions of iodine ins carbons tetrachloride s ’ill suffice although copper nsitrate ins water is sometimes added.5’ 6 It is most importanst that. t.he illuminsations of the microscope be very carefully arransged amid that iso light be lost. A good achromatic consdensser should be used. With a monsochromator, ams image of the lamp is focused on the insput slit h)y a lenss; the output slit is focused by the condenser oni the object; ansd a lens is placed nsear the output slit to focus ams image of the prism of the monsochromator on the consdensser diaphragm. With the compact source lamp and filter, a lens of about 4 inches focal lensgth should be used to focus a magnsified image of the sosmrce on the coisdenser diaphragm, a distatsce of 2 or more feet betw’een lamp amid microscope beinsg nsecessary. Ans apochromatic objective is desirable as achromatic objectives may require comssiderahle tube lensgth adjustmenst whets imsed w’ith violet light. The microscopist’s eye may require a few minutes to become accustomed to violet light. For photomicrography, the additions of a light filter of sodium nitrite ins w’ater