TY - JOUR
T1 - Real-time imaging of exocytotic mucin release and swelling in Calu-3 cells using acridine orange
AU - Shumilov, Dmytro
AU - Popov, Alexander
AU - Fudala, Rafal
AU - Akopova, Irina
AU - Gryczynski, Ignacy
AU - Borejdo, Julian
AU - Gryczynski, Zygmunt
AU - Grygorczyk, Ryszard
N1 - Funding Information:
This study was supported by the Cystic Fibrosis Canada (RG), the National Institutes of Health (USA): NIH-5R21CA149897-02 and R01EB12003 (Z.G.) and NIH-HL090786 (J.B.).
PY - 2014/3/15
Y1 - 2014/3/15
N2 - Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200μM) AO solutions presented absorption maximum at 494nm, emission maximum at 525nm and only ~1.76ns fluorescence lifetime. By contrast at high concentrations (4-30mM) favoring formation of AO aggregates, a very different absorption with maximum at ~440nm, dramatically red-shifted emission with maximum at 630nm, and over 10-fold increased fluorescence lifetime (~20ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98±0.07×10-7cm2/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was ~10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications.
AB - Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200μM) AO solutions presented absorption maximum at 494nm, emission maximum at 525nm and only ~1.76ns fluorescence lifetime. By contrast at high concentrations (4-30mM) favoring formation of AO aggregates, a very different absorption with maximum at ~440nm, dramatically red-shifted emission with maximum at 630nm, and over 10-fold increased fluorescence lifetime (~20ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98±0.07×10-7cm2/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was ~10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications.
KW - Acridine orange
KW - Cystic fibrosis
KW - Fluorescence lifetime
KW - Mucin granules
KW - Spectral shift
KW - Total internal reflection fluorescence
UR - http://www.scopus.com/inward/record.url?scp=84896490974&partnerID=8YFLogxK
U2 - 10.1016/j.ymeth.2013.09.004
DO - 10.1016/j.ymeth.2013.09.004
M3 - Article
C2 - 24055436
AN - SCOPUS:84896490974
SN - 1046-2023
VL - 66
SP - 312
EP - 324
JO - Methods
JF - Methods
IS - 2
ER -