Measuring serotonin distribution in live cells with three-photon excitation

Science

Volumn 275, Issue 5299, 1997, Pages 530-532

  Maiti, S ^a   Shear, Jason B ^a,b   Williams, R M ^a   Zipfel, W R ^a   Webb, Watt W ^a  

^a Department of Materials Science and Engineering   (United States)

^b UNIVERSITY OF TEXAS AT AUSTIN   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

SEROTONIN;

ABSORPTION SPECTROSCOPY; ARTICLE; CELL; CELLULAR DISTRIBUTION; FLUORESCENCE; HUMAN; NONHUMAN; PHOTON; PRIORITY JOURNAL;

ANIMALS; CELL SURVIVAL; CYTOPLASMIC GRANULES; DOPAMINE; LASERS; MICROSCOPY, FLUORESCENCE; PHOTOCHEMISTRY; PHOTONS; RATS; SEROTONIN; TRYPTOPHAN; TUMOR CELLS, CULTURED; ULTRAVIOLET RAYS;

EID: 0031025151     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5299.530     Document Type: Article

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22. The cellular background fluorescence, present both in serotonin-loaded and control cells, maps the distribution of Trp-containing proteins (and possibly other cellular material possessing similar spectral characteristics) in these cells. The nuclei, which presumably contain a lower concentration of such molecules, are seen as darker regions.
23. The cells remain capable of exocytosis after exposure to the infrared radiation used in generating a three-dimensional image (9). In contrast, we have thus far been unable to observe exocytosis after exposure to 450-nm radiation required for two-photon excited fluorescence imaging of the granules, indicating the presence of possible wavelength-dependent photochemical mechanisms that make 3PE less damaging. Infrared illumination caused some visible (>400 nm) wavelength fluorescence from the serotonin granules. Whether this originates from indolic degradation products suggested in the literature [C. Lambert et al., Biochim. Biophys. Acta 993, 12 (1989)] is currently under investigation ( J. B. Shear, C. Xu, R. M. Williams, S. Maiti, W. W. Webb, unpublished data).
24. The cells remain capable of exocytosis after exposure to the infrared radiation used in generating a three-dimensional image (9). In contrast, we have thus far been unable to observe exocytosis after exposure to 450-nm radiation required for two-photon excited fluorescence imaging of the granules, indicating the presence of possible wavelength-dependent photochemical mechanisms that make 3PE less damaging. Infrared illumination caused some visible (>400 nm) wavelength fluorescence from the serotonin granules. Whether this originates from indolic degradation products suggested in the literature [C. Lambert et al., Biochim. Biophys. Acta 993, 12 (1989)] is currently under investigation ( J. B. Shear, C. Xu, R. M. Williams, S. Maiti, W. W. Webb, unpublished data).
25. i) (3) where Δz is the extent of the granule along the z direction and the sum is taken over all horizontal planes intersected by the granule.
26. We have measured fluorescence as a function of serotonin concentration (from solutions buffered at ∼pH 6) under conditions identical to imaging. The fluorescence increases approximately linearly at lower (≲50 mM) concentrations, but the slope decreases at higher concentrations {presumably as a result of quenching by energy transfer to neighboring molecules [T. Förster, Ann. Phys. (Leipzig) 2, 55 (1948), translated by R. S. Knox, University of Rochester]}. The intensity peaks at ∼250 mM and decreases thereafter. A priori, the observed level of granular fluorescence may also represent a concentration in this "inverted" region, but a time-dependent study of serotonin loading in these cells (9) rules out this possibility. Also, serotonin fluorescence is pH-sensitive, and the vesicle interiors are believed to be acidic [ M. B. De Young et al., Arch. Biochem. Biophys. 254 222 (1987) ]. We infer from measurements of fluorescence as a function of pH (9) that our estimate (made at pH 6) should be accurate to ±25% for vesicular pH between 5 and 7. Nevertheless, unknown factors in the vesicle lumen may introduce larger errors.
27. We have measured fluorescence as a function of serotonin concentration (from solutions buffered at ∼pH 6) under conditions identical to imaging. The fluorescence increases approximately linearly at lower (≲50 mM) concentrations, but the slope decreases at higher concentrations {presumably as a result of quenching by energy transfer to neighboring molecules [T. Förster, Ann. Phys. (Leipzig) 2, 55 (1948), translated by R. S. Knox, University of Rochester]}. The intensity peaks at ∼250 mM and decreases thereafter. A priori, the observed level of granular fluorescence may also represent a concentration in this "inverted" region, but a time-dependent study of serotonin loading in these cells (9) rules out this possibility. Also, serotonin fluorescence is pH-sensitive, and the vesicle interiors are believed to be acidic [ M. B. De Young et al., Arch. Biochem. Biophys. 254 222 (1987) ]. We infer from measurements of fluorescence as a function of pH (9) that our estimate (made at pH 6) should be accurate to ±25% for vesicular pH between 5 and 7. Nevertheless, unknown factors in the vesicle lumen may introduce larger errors.
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29. Serotonin, dopamine, and Trp were obtained from Sigma (certified >99% pure by thin-layer chromatography) and used without further purification. Samples at 5 mM concentration were prepared in 25 mM Mops buffer (pH 7.0) and measured at room temperature with excitation by a mode-locked 76-MHz titanium:sapphire laser source (MIRA 900, Coherent). The laser beam was passed through a Pockel's cell-based noise controller (LASS II, Conoptics) and a longpass dichroic reflector (400 DCLP, Omega) and finally focused into the sample by a microscope objective [1.25 numerical aperture (NA), oil immersion, Zeiss Neofluar]. The sample was contained in a 0.9 mm by 0.9 mm square glass capillary tube (number 8290, VitroDynamics; uniform transmission between 300 and 700 nm) with 170-(μm-thick walls. The UV fluorescence was collected by the same objective and reflected by the UV dichroic onto a Hamamatsu HC125-02 photon-counting module, after passing through a series of UV bandpass filters (UG11 colored glass filter from Schott and interference filters from Corion). The output of the detector was transmitted to a Stanford Research Systems SR400 photon counter, which provided the fluorescence count rate. The pulsewidth at each wavelength (160 to 220 fs) was measured by an intensity autocorrelator (FR103, Femtochrome), and the average power was measured by a thermopile power meter (Molectron).
30. -9 above 400 nm) and detected with a photomultiplier tube-amplifier module (HC125-02, Hamamatsu).
31. -9 above 400 nm) and detected with a photomultiplier tube-amplifier module (HC125-02, Hamamatsu).
32. -9 above 400 nm) and detected with a photomultiplier tube-amplifier module (HC125-02, Hamamatsu).
33. This work was carried out in the Developmental Resource for Biophysical Imaging and Optoelectronics with funding provided by NSF (grant DIR8800278) and NIH (grants RR07719 and RR04224). We gratefully acknowledge the NIH Parallel Processing Resource for Biomedical Scientists and the use of the IBM SP2 supercomputer at the Cornell Theory Center. We also thank Coherent and Spectra-Physics for loaning us Ti:sapphire mode-locked laser systems. J.B.S. is an NSF postdoctoral fellow.