Simultaneous multiple wavelength fluorescence video microscopy shows Ca2+ regulation of pH in living cells

Stephen J. Morris; Diane M. Beatty; Bibie M. Chronwall

doi:10.1117/12.182714

17 August 1994 Simultaneous multiple wavelength fluorescence video microscopy shows Ca²⁺ regulation of pH in living cells

Stephen J. Morris, Diane M. Beatty, Bibie M. Chronwall

Author Affiliations +

Proceedings Volume 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV; (1994) https://doi.org/10.1117/12.182714
Event: OE/LASE '94, 1994, Los Angeles, CA, United States

Abstract

We have designed an epifluorescence video microscope for simultaneous dual excitation of indo 1 (for [Ca²⁺]_i) at 350 nm and SNARF 1 (for pH_i) at 540 nm. The microscope will simultaneously capture all four emission images at 405, 475, 575, and 640 nm from the two ratio dyes at video frame or field rates. Popular dyes for measuring [Ca²⁺]_i, such as indo 1 and fura 2, have pH- dependent Kd's; thus changes in pH_i can be misinterpreted as changes in [Ca²⁺]_i. For any pixel (or region of interest), we use the pH value to generate the appropriate Kd. The corrected Kd is then used to calculate a corrected calcium value. Using the imaging system, we show that, for the peptide secreting melanotropes of the pituitary intermediate lobe, changes in intracellular calcium ([Ca²PLU)]_i) produce changes in intracellular pH (pH_i). Melanotropes grown in primary explant culture and double-loaded with indo-1 acetoxy methyl ester (AM) and SNARF-1 AM, were examined for Ca²⁺/pH interactions. Following experimentation, cells were positively identified by (beta) -endorphin fluorescence immunohistochemistry. K-induced depolarization of melanotropes produced increases in [Ca²PLU)]_i due to activation of L-type Ca-channels. Ca²⁺ entry was closely coupled to reductions in pH_i. Effects were dependent upon entry of extracellular Ca²⁺ rather than release from intracellular stores. The close association between increases in intracellular Ca²⁺ and H⁺ suggest that the pH_i changes are due to release of H⁺ upon binding of Ca²⁺ to intracellular buffers. Although the pH drop is `passive,' it will be sensed by all cytoplasmic components. Thus it represents a second messenger pathway, akin to the generation of cAMP or inositol trisphosphate, which cannot fail to influence numerous pH-dependent cell activities.

Citation Download Citation

Stephen J. Morris, Diane M. Beatty, and Bibie M. Chronwall "Simultaneous multiple wavelength fluorescence video microscopy shows Ca²⁺ regulation of pH in living cells", Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); https://doi.org/10.1117/12.182714

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available