Studies of dispersed beta cells have been used to infer their behavior in the intact pancreatic islet. When dispersed, beta cells exhibit multiple metabolic glucose-response populations with different insulin secretion properties. This has led to a model for glucose-dependent insulin secretion from the islet based on a step-wise recruitment of individual beta cells. However, previously reported synchronous and uniform Ca2+ activity and electrical responses indicate that beta cell behavior within intact islets is more uniform. Therefore, uncertainty remains whether beta cell metabolic heterogeneity is functionally important in intact islets. We have used two-photon excitation microscopy to measure and compare the glucose-induced NAD(P)H autofluorescence response in dispersed beta cells and within intact islets. Over 90% of beta cells in intact islets responded to glucose with significantly elevated NAD(P)H levels, compared with less than 70% of dispersed beta cells. In addition, all responding beta cells within intact islets exhibited a sigmoidal glucose dose response behavior with inflection points of approximately 8 mm glucose. These results suggest that beta cell heterogeneity may be functionally less important in the intact islet than has been predicted from studies of dispersed beta cells and support the role of glucokinase as the rate-limiting enzyme in the beta cell glucose response.
Two-photon excitation fluorescence microscopy provides attractive
advantages over confocal microscopy for three-dimensionally
resolved fluorescence imaging. Two-photon excitation arises
from the simultaneous absorption of two photons in a single
quantitized event whose probability is proportional to the square
of the instantaneous intensity. For example, two red photons
(∼700 nm) can cause the transition to an excited electronic
state normally reached by absorption in the ultraviolet (∼350
nm). In the fluorescence experiments described here, the final
excited state is the same singlet state that is populated during
a conventional fluorescence experiment. Thus, the fluorophore
exhibits the same emission properties (e.g., wavelength shifts,
environmental sensitivity) used in typical biological microscopy
studies. Three properties of two-photon excitation give this
method its advantage over conventional optical sectioning
microscopies: (1) the excitation is limited to the focal volume,
thus providing inherent three-dimensional resolution and minimizing
photobleaching and photodamage; (2) the two-photon technique
allows imaging of UV fluorophores with only conventional visible
light optics; (3) red light is far less damaging to most living
cells and tissues than UV light and permits deeper sectioning,
because both absorbance and scattering are reduced. Many cell
biological applications of two-photon excitation microscopy
have been successfully realized, demonstrating the wide ranging
power of this technique.
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