D. B. Brewer

Optical properties of amyloid stained by Congo red: history and mechanisms.

Amyloid stained by Congo red has striking optical properties that generally have been poorly described and inadequately explained, although they can be understood from principles of physical optics. Molecules of Congo red are orientated on amyloid fibrils, and so the dye becomes dichroic and birefringent. The birefringence varies with wavelength in accordance with a fundamental property of all light-transmitting materials called anomalous dispersion of the refractive index around an absorption peak. The combination of this and absorption of light, with modification by any additional birefringence in the optical system, explains the various colours that can be seen in Congo red-stained amyloid between crossed polariser and analyser, and also when the polariser and analyser are progressively uncrossed. These are called anomalous colours.

Physical basis of colors seen in Congo red-stained amyloid in polarized light

Amyloid stained by Congo red is traditionally said to show apple-green birefringence in polarized light, although in practice various colors may be seen between accurately crossed polarizing filters, called polarizer and analyzer. Other colors are seen as the polarizer and analyzer are uncrossed and sometimes when the slide is rotated. Previously, there has been no satisfactory explanation of these properties. Birefringence means that a material has two refractive indices, depending on its orientation in polarized light. Birefringence can change linearly polarized light to elliptically polarized, which allows light to pass a crossed analyzer. The birefringence of orientated Congo red varied with wavelength and was maximal near its absorption peak, changing from negative (slow axis of transmission perpendicular to smears or amyloid fibrils) on the shortwave side of the peak to positive (slow axis parallel) on the longwave side. This was explained by a property of any light-absorbing substance called anomalous dispersion of the refractive index around an absorption peak. Negative birefringence gave transmission of blue, positive gave yellow, and the mixture was perceived as green. This explains how green occurs in ideal conditions. Additional or strain birefringence in the optical system, such as in glass slides, partly or completely eliminated blue or yellow, giving yellow/green or yellow, and blue/green or blue, which are commonly seen in practice and in illustrations. With uncrossing of polarizer or analyzer, birefringent effects declined and dichroic effects appeared, giving progressive changes from green to red as the plane of polarization approached the absorbing axis and from green to colorless in the opposite way. This asymmetry of effects is useful to pathologists as a confirmation of amyloid. Rather than showing ‘apple-green birefringence in polarized light’ as often reported, Congo red-stained amyloid, when examined between crossed polarizer and analyzer, should more accurately be said to show anomalous colors.

Max Schultze (1865), G. Bizzozero (1882) and the discovery of the platelet

Max Schultze published the first accurate and convincing description of platelets as part of a study devoted mainly to the white blood cells in 1865. He recognised them as a normal constituent of the blood and ‘enthusiastically recommended’ them as an object for further study by ‘those concerned with the in‐depth study of the blood of humans’. In 1882, Bizzozero demonstrated the value of this recommendation in his much more comprehensive study. He observed them microscopically in the circulating blood of living animals and in the blood removed from the blood vessels. In well‐planned experiments, he showed that they were the first component of the blood to adhere to damaged blood vessel walls in vivo and, in vitro, that they were the first components of the blood to adhere to threads that subsequently became covered with fibrin.

A simple technique for the demonstration of urinary casts.

(RECEIVED FOR PUBLICATIONMARCH 27,1953) Duringrecent investigations intothenature of hyaline castswe becameimpressed again bythe well-known difficulty ofdetecting themunder ordinary conditions ofmicroscopic observation andillumination. Theycan,ofcourse, berendered moreobvious byracking downthesubstage condenser orbyusing phase-contrast microscopy, but boththese techniques require acertain amountofjudgment anddonotyield anything like asstriking andobvious aresult asthemethoddescribed here. Moreover, this methodreadily reveals thepresence ofsmall numbers ofcasts andtheyareeasily seen inarapid survey ofthewholeareaofthecoverslip withthelowest powerofthemicroscope. Themethodconsists ofnegative staining ofthe deposit withindian ink.Onedropofthecentrifuged urinary deposit ismixedwithonedropof indian ink(Mandarin Black, WinsorandNewton) _ onaslide. A coverslip isplaced onthemixture andpressed firmly downon totheslide. The excess fluid iswipedfromtheedgesofthecoverslip andtheslide examined underthemicroscope. Anycasts present areimmediately obvious aslong, _

Inside Lab Invest

Many pathologists loathe the thought of ordering a Congo red stain to identify amyloid in routine tissue sections. For some of us, the challenge lies in determining what hue really represents significant “apple green” among a spectrum of colors that frequently includes nonspecific autofluorescence. This is true whether controls or diagnostic samples are being evaluated. For others, the problem is the sometimes onerous search to find functional polarizers, which often disappear if not attached to the microscope. Regardless of how pathologists view this very common diagnostic method, most of us do not have a complete understanding of how it works, and this may add to our general disdain of the method. Help is on the way. In this issue, Dr. Howie and colleagues provide an erudite and lucid explanation of the physical basis for colors observed in Congo-red-stained tissue sections under polarizing illumination. The carefully written, very informative report has enough background and definitions that the reader doesn’t need to be a rocket scientist to fully understand it. The authors report that the spectrum of colors (not just apple doi:10.1038/labinvest.2008.8 8 88 23 231 8 8