Light microscopic imaging of living cells Critical parameters: Low light level Speed of data acquisition Quantitative measurements “in vivo” environment

Fluorescence / imaging microscope Xe arc lamp Intensified video camera Filter Wheel Dichroic mirror Video frame grabber I. Optics and image formation Video monitor

Fluorescence / imaging microscope Thermostatized perfusion chamber Manifold Perfusion reservoirs II. Handling / observation of living cells

Metafluor® Software: Fluorescence ratio imaging system Image acquisition via video frame grabber Image processing, analysis and quantification Real time processing (averaging, background subtraction, ratio images) Automation: Control of external devices (filter wheels, monochromators, valves, triggers, etc.) Implemented on Pentium II 333 MHz computer

Some fluorescent dyes that can be used:  Fluorescein-, Rhodamine-based dyes  pH: BCECF, SNARF  Ca 2+ : Fura-2, Fluo-3, Mag-Fura-2, Calcium Green, Rhod-2  Na + : SBFI, SBFO, Sodium Green  K + : PBFI, CD 222  mitochondrial : Rhodamine 123, TMRE  Vesicle release : FM 1-43  Autofluorescence : NAD(P)H  Green Fluorescent Protein (GFP) etc.

Na + homeostasis in astrocytes (simplified model) 3 Na + 1 Glut 1 K + 1 H + Na + K+K+ ATP [Na + ] o =160 mM [Na + ] i  10 mM Na + AKR Glut Kainate AMPA

Structure of SBFI (sodium-binding benzofuran isophthalate)

380nm-ImageControl (ratio image) L-Glu 1mM (ratio image)

Glutamate-evoked intracellular Na + changes

Glutamate: concentration-response analysis

Na + homeostasis in astrocytes (simplified model) 3 Na + 1 Glut 1 K + 1 H + Na + K+K+ ATP [Na + ] o =160 mM [Na + ] i  10 mM Na + AKR Glut Kainate AMPA

Effect of Na + /K + -ATPase inhibition (1)