Stray light correction for microspectrophotometric determination of intracellular ion concentration.

A s i m p l e c o r r e c t i o n f o r t he e f f e c t of s t r a y l i g h t on a b s o r b a n c e m e a s u r e m e n t s f rom c e l l s f i l l e d w i t h i n d i c a t o r dyes i s d e s c r i b e d . The i n t r a c e l l u l a r i n j e c t i o n o f i n d i c a t o r dyes and t h e m i c r o s p e c t r o p h o t o m e t r i c d e t e c t i o n of changes i n the c o n c e n t r a t i o n of p a r t i c u l a r c y t o p l a s m i c i o n s i s an i m p o r t a n t and i n c r e a s i n g l y p o p u l a r t e c h n i q u e in c e l l u l a r n e u r o s c i e n e e . Three examples a re the use of a r s e n a z o I I I t o measure changes in f r e e c a l c i u m , E r i o c h r o m e Blue to m easu r e changes i n i n t r a c e l l u l a r f r e e magnes ium, and p h e n o l red to measure changes in i n t e r n a l pH ( B r i n l e y e t a l . , 1977; Sca rpa e t a l . , 1978; Ahmed and Connor , 1980) . The u s u a l p r o c e d u r e i s t o measu re the a b s o r b a n c e o f a d y e f i l l e d c e l l a t one o r more w a v e l e n g t h s . G e n e r a l l y , the a b s o r b a n c e change a t one w a v e l e n g t h e s p e c i a l l y s e n s i t i v e to changes in a p a r t i c u l a r i on i s compared to the a b s o r b a n c e a t a second w a v e l e n g t h which i s i n s e n s i t i v e t o v a r i a t i o n s i n i o n i c c o n c e n t r a t i o n . The a b s o l u t e m a g n i t u d e o f d y e r e l a t e d a b s o r b a n c e a t the second w a v e l e n g t h , c a l l e d the i s o s b e s t i c p o i n t , i s u s u a l l y u s e d t o d e t e r m i n e the amount of dye in t he c e l l . T h i s as w e l l as changes in the a b s o r b a n c e a t the f i r s t w a v e l e n g t h are u s e d to q u a n t i f y changes i n t he i n t r a c e l l u l a r c o n c e n t r a t i o n of t he ion o f i n t e r e s t . An o f t e n s e r i o u s l i m i t a t i o n in t h e s e t e c h n i q u e s , t h a t i s f r e q u e n t l y o v e r l o o k e d i n t h e i r a p p l i c a t i o n , i s t h a t s t r a y l i g h t , n o t p a s s i n g t h r o u g h the d y e f i l l e d c e l l bu t s e n s e d by the t r a n s m i t t e d l i g h t d e t e c t o r , w i l l confound b o t h t h e measurement o f t he dye c o n c e n t r a t i o n e s t i m a t e d f rom the i s o s b e s t i c a b s o r b a n c e and the measu remen t o f changes in i o n i c c o n c e n t r a t i o n e s t i m a t e d f rom a b s o r b a n c e changes a t t he i o n s e n s i t i v e w a v e l e n g t h . I o f f e r h e r e a method t o c o r r e c t f o r the e f f e c t o f s t r a y l i g h t on t h e s e a b s o r b a n c e m e a s u r e m e n t s . For r e a s o n s o f c l a r i t y and s i m p l i c i t y , t he d e r i v a t i o n s and c o r r e c t i o n s a r e p r e s e n t e d i n a form i m m e d i a t e l y a p p l i c a b l e to the d e t e c t i o n o f c a l c i u m w i t h a r s e n a z o I I I , 0165-0270/82/0000-0000/~$102.75 © Elsevier Biomedical Press