1076
JOHN W. KELLY4 . u ~GUNNAR SVENSSON
Vol. 62
ABSORPTION MEA1SUREMENT WITH h LENS-CUVETTE I N SATURATED SOLUTIOKS OF A METACHROMATIC DYE BY JOHN W. KELLY’AND GUNNAR SVENSSON Institute for Cell Research, Medical Nobel Institute, Karolinska Institutet, Stockholm Receiued Aprzl
4 , 1868
A lens-cuvette was used in a scanning and recording microspectrophotometer (Caspersson) to measure the absorbance of saturated aqueous solutions of toluidine blue. The cuvette is a plane-convex spherical lens resting convex face down on an optical flat plate, presenting layer thicknesses of 0.004 cm. t o less than 0.0001 cm. The absorption spectrum of toluidine blue a t the highest concentrations (0.82-1.04 X IO-‘ M ) resembles closely the well-known metachromatic spectra of dilute solutions of the dye in the presence of certain anionic polyelectrolytes or of the dye bound a t selected sites in cells and tissues. This finding supports the theory of Schubert and Levine on metachromasy in solution. It suggests that application of this theory t o metachromatically-stained solid substrates may be realized as the limited spectral analyses of these substrates are extended.
Special technical difficulties arise in securing thin layers of known dimensions for absorption measurement in highly concentrated solutions. Down to about 0.01 cm. thickness, cells are calibrated against solutions in standard cells by the Bouguer-Lambert 1aw12but “below say 0.005 cm. it becomes almost impossible to calibrate the thickness of an absorption cell pre~isely.”~For certain infrared spectral analyses, where cells of the order of 0.001 cm. thickness are required, authors sometimes explicitly state that accurate cell dimensions cannot be q ~ o t e d . The ~ well designed variable cell of Adams and Katz5 illustrates problems typical of a multi-component assembly. Such cells are difficult t o fill, empty and clean without disassembly so that it is not always possible to calibrate them by the Bouguer-Lambert law. Calibration may sometimes be effected by counting fringes, requiring special polishing for flat and parallel windows. A lens-cuvette devised by Svenssono permits absorption measurements in layers of any thickness from a fraction of a micron to about 40 p. The “cuvette” consists of a plane-convex spherical lens of quartz or glass resting convex face down on a plane-parallel plate of the same material. A drop of solution is drawn by capillarity between lens and plate and sealed with a drop of immiscible liquid. Layer thickness is determined by the distance of t,he measuring spot from the lens center. Main features of the lens-cuvett’e are: (1) elimination of all filling and cleaning difficulties; (2) easy nianipulat,ion of 0.1 pl. solution volumes; (3) meamrement of solutions with absorbances of 0.1-5 per p ; (4) over-all measurement error of k 1%. In addition to general analytical uses for the lenscuvette, the device bears a close dimensional relation t o certain problems in the microspectrophotometry of biological objects. In such objects, the absorbing layer (t,issue section or cell) is rarely thicker than 10 p and the concentration of absorb(1) J o h n Simon Guggenheim Fellow. 1967-1958. Permanent address: Medical College of Virginia, Richmond, Va. (2) L. F. Epstein, F. Karush and E. Rabinowitch. J . O p t . S O C .Am., S 1 , 77 (1941). (3) L. Michaelis, Cold Spring Harbor Symp. Quant. B i d . , 12, 131 (1947). (4) E. K. Plyler, Disc. Faradau Soc., 9, 1.00 (1950). See also papers of Brown and Sheppard and of Gore for discussion and references to other absorption cells. ( 5 ) R . b1. AdaniR and ,I. .J. K a t s , J . O p l . So?. A m . , 46, 895 (lSS6). (I;) G . Svensson. E a p . Cell Research 9, 428 (1955); .Ilikrockirn. Acta, 1-6, 1\45 (19M).
ing material may be well above the range accessible to standard solution measurement. Model systems of naturally-occurring compounds with selective absorption, such as nucleic acids, proteins or pigments, may be studied a t high concentrations in the lens-cuvette. Applied chromogens, particularly dyes, may also be examined a t concentration levels approaching or equalling those obtaining in solid, stained substrates. A special study of the thiazine dye, toluidine blue, was made in the lens-cuvette. This dye is the most widely used of metachromatic dyes for the histochemical detection of strongly acidic polyelectrolytes, such as heparin or chondroitin sulfate. General features of metachromatic reactions are reviewed elsewhere.’ The useful quality of metachromatic dyes is their marked susceptibility to color changes under the influence of appropriate substrates, called chromotropes. This quality is a t least circumstantially related to the extreme deviations from Beer’s law displayed by these dyes in the absence of any chromotrope. Setting forth a new theory of metachromasy in solution, Schubert and Levines question the prevailing view that seems to require actual binding of dye t o a substrate. Important evidence was found by these authors suggesting that the spectral changes of a metachromatic dye as it becomes highly concentrated are similar to the changes occurring when a chromotrope is added to a dilute solution of the dye. This evidence came from a study of dyes a t the highest concentrations a t which measurement could be made, limited only by the absorption cells. The main object of this investigation was to obtain information for a comparison between the spectrum of toluidine blue in solution a t maximum concentration and the spectra measured in the microspectrophotometer when the dye is bound in selected tissue or cellular sites. For n solution in the lens-cuvette, the limiting factor is the solubility of the absorbing compound. Experimental and Results Dyes.-Two samples of toluidine blue 0 , C. I. 925, were used. Sample 1 was obtained from the Hartman-Leddon Company, Philadelphia, and sample 2 was obtained from (7) J. W. Kelly, in “Protoplasmatologia,” ed. by L. V. Heilbrunn a n d F. Weber, Springer-Verlag, Vienna, 1956, pp. 1-98: M. Sohnbort and D. Hamerman, J . Histochem. and Cytorhem., 4, 159 (1950). (8) M. Schubert a n d A. Levine, J . Am. Chem. Sac., 77,4197 (195.5).
Sept)., 1968
A
1.2 --
1.0
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0.8
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0.G
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T
0
A
/O
0.2
Unfiltered, lens r = 5 0 mm Filtered, lens r =IOOmm
-1 I
0
I
I
1
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C
10 15 Cell depth, p. Fig. 1,-Absorbance of a saturated solution of toluidine blue (sample 2 ) measured a t two wave lengths in the lens-cuvette. The slope of a line, or mean value of A/t, is used to calculate molar absorbance, C X .
0
5
seal the dye solutions in the cuvette. The main principles of measuring in the cuvette are reviewed here; full details with descriptive figures are presented elsewhere.6 The perpendicular distance from a plane tangent to a spherical surface is t = xa/2r (for t
