We have developed a new fluorescence microscope that addresses the spectral and speed limitations of current light microscopy instrumentation. In the present device, interference and neutral density filters normally used for fluorescence excitation and detection are replaced by acousto-optic tunable filters (AOTFs). Improvements are described, including the use of a dispersing prism in conjunction with the imaging AOTF and an oblique-illumination excitation scheme, which together enable the AOTF microscope to produce images comparable to those obtained with conventional fluorescence instruments. The superior speed and spectral versatility of the AOTF microscope are demonstrated by a ratio image pair acquired in 3.5 ms and a micro-spectral absorbance measurement of hemoglobin through a cranial window in a living mouse.
A newly developed microscope using acousto-optic tunable filters (AOTFs) was used to generate in vivo hemoglobin saturation (SO2) and oxygen tension (PO2) maps in the cerebral cortex of mice. SO2 maps were generated from the spectral analysis of reflected absorbance images collected at different wavelengths, and PO2 maps were generated from the phosphorescence lifetimes of an injected palladium-porphyrin compound using a frequency-domain measurement. As the inspiratory O2 was stepped from hypoxia (10% O2), through normoxia (21% O2), to hyperoxia (60% O2), measured SO2 and PO2 levels rose accordingly and predictably throughout. A plot of SO2 versus PO2 in different arterial and venous regions of the pial vessels conformed to the sigmoidal shape of the oxygen-hemoglobin dissociation curve, providing further validation of the two mapping procedures. The study demonstrates the versatility of the AOTF microscope for in vivo physiologic investigation, allowing for the generation of nearly simultaneous SO2 and PO2 maps in the cerebral cortex, and the frequency-domain detection of phosphorescence lifetimes. This class of study opens up exciting new possibilities for investigating the dynamics of hemoglobin and O2 binding during functional activation of neuronal tissues.
The nature of presynaptic calcium (Ca 2ϩ ) signals that initiate neurotransmitter release makes these signals difficult to study, in part because of the small size of specialized active zones within most nerve terminals. Using the frog motor nerve terminal, which contains especially large active zones, we show that increases in intracellular Ca 2ϩ concentration within 1 msec of action potential invasion are attributable to Ca 2ϩ entry through N-type Ca 2ϩ channels and are not uniformly distributed throughout active zone regions. Furthermore, changes in the location and magnitude of Ca 2ϩ signals recorded before and after experimental manipulations (-conotoxin GVIA, diaminopyridine, and lowered extracellular Ca 2ϩ ) support the hypothesis that there is a remarkably low probability of a single Ca 2ϩ channel opening within an active zone after an action potential. The trial-to-trial variability observed in the spatial distribution of presynaptic Ca 2ϩ entry also supports this conclusion, which differs from the conclusions of previous work in other synapses.
We report a new source of femtosecond light pulses which is broadly tunable in the infrared. A singly resonant optical parametric oscillator based on a thin crystal of KTiOPO4 is pumped by intracavity femtosecond pulses at 620 nm from a standard colliding-pulse passively mode-locked dye laser. Oscillation results in stable, continuous outputs of femtosecond pulses at 108 Hz repetition rate and milliwatt average power levels in both signal and idler beams. Here we demonstrate tuning from 820 to 920 nm and 1.90 to 2.54 μm with a single set of mirrors. With multiple sets of mirrors, continuously tunable outputs from ∼0.72 to ∼4.5 μm should be possible, making this a uniquely versatile femtosecond laser source.
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