Department II Eilers » Research

Live imaging of adipose tissue

Obesity is frequently associated with a chronic low-grade inflammation within adipose tissue (AT), characterized by a significant increase in macrophages and, to lesser extent, other immune cells, such as T-cells, B-cells, mast cells and neutrophils. The spatial and temporal activation of these cells as well as their accumulation in the AT seems to be tightly linked to so-called crown-like structures (CLS). CLS are accumulations of adipose tissue macrophages (ATMs) around dead adipocytes and are thought to reflect a scavenger response. At present, data on the life cycle of CLS are missing. In a fruitful cooperation with Drs. Ingo Bechmann and Martin Gericke from the Institute of Anatomy we perform long-term imaging of ATMs, adipocytes and CLS within live AT explants in order to better understand the cellular events underlying AT physiology and pathophysiology.


Quantitative deep-tissue Ca2+ imaging

We employ ultra-fast, time-correlated single-photon counting (TCSCP) instrumentation in combination with two-photon microscopy (TPM) to record the fluorescence lifetime of Ca2+-sensitive indicator dyes in the depth of intact tissue. For technical reasons, TPM does not allow simple excitation or emission ratioing of dyes such as fura-2 or indo-1. Our fluorescence lifetime imaging (FLIM) approach overcomes this long-standing limitation of TPM by allowing fully quantitative deep-tissue Ca2+ imaging. A variety of high-, mid- and low-affinity Ca2+ indicators are suitable for Ca2+ FLIM, which allow to study a broad spectrum of Ca2+ signaling events.


'Silent' signal integration in spines and dendrites

In addition to electrical signals, neurons generate short-lived and highly localized second messenger signals, such as dendritic Ca2+ transients. These signals seem to be of utmost importance for neuronal developmant as well as for the cellular processes that underly learning and memory formation. We analyse neuronal Ca2+ signalling and its specific targets on the molecular level. Since Ca2+ signals last only a fraction of a second and may occurre in compartments as small as a femtoliter (10-15 l), we apply high-resolution microscopic techniques such as confocal and two-photon microscopy. Combining our measurements with numerical simulations of the interactions of channels, buffers, pumps and diffusion, we hope to be able to decipher the functional consequences of dendritic Ca2+ signalling.


Chloride homeostasis in immature neurons

The neurotransmitter GABA plays an important role in the postnatal development of the central nervous system. Being the predominant inhibitory transmitter in the adult, GABA is the most important excitatory transmitter in immature brain. This inverse action is thought to be due to an elevated intracellular chloride concentration in immature neurons.

Using fluorescence lifetime imaging (FLIM) we try to establish a method to quantify the chloride concentration in individual cells in intact brain tissue. In FLIM, individual photons, emitted from a chloride-sensitive indicator dye excited with a brief laser pulse, are counted on a nanosecond (10-9 s) timescale. The measured fluorescence decay allows to directly quantify the chloride concentration. After establishing our FLIM-based approach, we hope to be able to monitor the cellular mechanisms that govern GABA-mediated maturation of the brain.


Mobility of proteins in neuronal synapses and dendrites

Using fluorescence recovery after photobleaching (FRAP) we quantify the mobility of endogenous proteins in the neuronal cytosol. FRAP allows the determination of the specific diffusion coefficients as well as the analysis of protein-protein-interactions in submicrometer large cellular compartments. We aim at identifying low-affinity, fast protein interactions in the vicinity of synapses.