Cell-cell fusion induced by the hemagglutinin (HA) spike-glycoprotein of influenza virus has been studied extensively using transfected cells expressing the protein. The initial fusion event is the formation of a small pore connecting the two cells. Its appearance has been measured by sudden changes in capacitance by whole cell patch clamp and more recently by the sudden change in the fluorescence of a possibly potential-sensitive fluorophore. Using a new design of fluorescence video microscopy capable of capturing multiple images at differing wavelengths of light simultaneously at video rates, this fluorescence event is shown to follow exponential kinetics, with a life time, τ, of approximately 350 ms.

A dual excitation microfluorimeter is described for measuring rapidly changing, intracellular calcium signals. A spinning sector wheel is used in conjunction with a beam masking device to provide rapid, efficient switching between the 2 excitation wavelengths. Exposure intervals as short as 120 μs can be achieved, yielding ratio samples at a rate of 6 kHz. Emission photons are collected using a photomultiplier tube operating in counting mode. When tested using FURA-2 as the calcium reporting dye, throughput noise in the system is demonstrated to be due to the statistical fluctuation inherent in photon counting. An example of the operation of the system, using a guinea pig cardiac myocyte, demonstrates that sufficient ratio data may be acquires to fully characterize the fastest components of the intracellular calcium signal.

Optical tweezers, or the single-beam optical gradient force trap, is becoming a major tool in biology for noninvasive micromanipulation on an optical microscope. The principles and practical aspects that influence construction are presented in an introductory primer. Quantitative theories are also reviewed but have yet to supplant user calibration. Various biological applications are summarized, including recent quantitative force and displacement measurements. Finally, tantalizing developments for new, nonimaging microscopy techniques based on optical tweezers are included.

We have used confocal laser scanning microscopy (CLSM), high-voltage electron microscopy (HVEM), and intracellular recording techniques to study volume changes in cultured Aplysia pacemaker neurons. Hyper- and hypo-tonic artificial sea water (ASW) decreased the pacemaker frequency and led to depolarization and hyperpolarization, respectively. However, when negative or positive current was injected into neurons in normal ASW, the frequency decreased with hyperpolarization but increased with depolarization. This suggests that the membrane potential is not the only factor underlying the reduction of the pacemaker activity.

Changes in cell volume were monitored with a CLSM and paralleled progressive changes in osmolarity. The neurons swelled and shrank nonuniformly, and, although an optical section through the middle of the cell was monitored every 4 s for as long as 14 min, a regulatory volume decrease or increase was never observed, indicating an osmometer-like behavior. The time course of shrinkage was faster than swelling after returning to control ASW after a hypotonic shock, reflecting a possible mechanical stress on the cytoskeleton.

Thick sections observed in the HVEM confirmed that membrane infoldings were present in our cultured Aplysia neurons. We hypothesize that a change in length of the latter in shrunken and swollen neurons could provide an explanation on the ultrastructural level for the increase and decrease in membrane surface area observed by CLSM. We conclude that by combining a confocal microscope with an electrophysiological set-up, three-dimensional morphology and physiological properties can be studied in living cells in real-time. This approach provides the means to correlate cell volume-related alterations and physiology.

A new class of gold immunoprobes has been developed with substantially different characteristics from the usual colloidal gold probes. Instead of chunks of gold metal (colloidal gold) just adsorbed to immunoglobulins by ionic or hydrophobic interactions, well-defined gold compounds are covalently attached to antibodies. Gold, along with several other metals, is known to form organo-cluster compounds with multiple metal atoms. The first gold cluster immunoprobe developed was undecagold (Au11), containing 11 gold atoms in an 0.8-nm sphere (Figure 1), covalently attached to Fab′ antibody fragments (Hainfeld, 1987). A more recent development has been the synthesis of a larger 1.4-nm gold cluster (Hainfeld et al, 1991; Hainfeld and Furuya, 1992).