The requirement to functionally probe biological structures, with ever increasing selectivity and three-dimensional resolution is a frontier area in microscopy. Non-linear optics has a unique potential in this regard with numerous studies focused on the potential of three-dimensional imaging with super-resolution. In this paper we demonstrate that non-linear optical phenomena, such as second harmonic (SH) generation, which is very sensitive to the membrane potential, can be locally enhanced by complexing or approaching a SH generating molecular probe to a nanoantenna of a silver or gold nanoparticle. This gives complexes with gigantic optical non-linearities. These contrast enhancing non-linear optical complexes have the potential to be directed selectively to specific nanometric regions in cells in order to report on alterations on the structure and the function in such regions while overcoming the inherent inefficiency of non-linear optical interactions.

A 10-μm thick electrophoretically sedimented film of bacteriorhodopsin (bR) was used, for the first time, to accurately measure width of 120-fs light pulses produced by a mode-locked Ti: sapphire laser. No Maker fringes structure was observed in the second harmonic generation pattern. The results show that thin films of bacteriorhodopsin have characteristics that are of considerable practical importance in femtosecond pulse characterization.

Submicrometer-size thermocouples at the tip of gold-coated glass micropipettes containing a platinum core were produced and tested. The response time of such thermocouples measured with different techniques appeared to be not bigger than a few microseconds. The calculations indicate that the spatial selectivity of this new class of thermocouple devices can be less than 2 μm along the pipette and less than 50 nm across the pipette. The suitability of this thermocouple for light intensity measurements with micrometer spatial resolution is demonstrated by measuring the focused beam of an argon-ion laser. In addition, such thermocouples are intrinsically suitable for applications in scanned probe microscopies. All these unique advantages make the pipette thermocouples a new and promising sensor in a variety of applications.

The nonlinear optical properties of the bacteriorhodopsin chromophore in the bR568 and K states are investigated by second harmonic generation. The comparison of amplitudes and phases of the second-order nonlinear optical polarizabilities of the retinal chromophore in the two states has revealed a noticeable increase of the induced dipole of the retinal as a result of the bR568 --> K transition. The results have been explained in terms of recent theoretical understandings of the nonlinear optical properties of polyenes. Within the context of these understandings we have discussed the molecular origins of the light-induced color changes and the possible mechanism of photon energy storage observed in this protein.

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.

The nonlinear optical phenomenon of second harmonic generation is shown to have intrinsic sensitivity to the voltage across a biological membrane. Our results demonstrate that this second order nonlinear optical process can be used to monitor membrane voltage with excellent signal to noise and other crucial advantages. These advantages suggest extensive use of this novel approach as an important new tool in elucidating membrane potential changes in biological systems. For this first demonstration of the effect we use a chiral styryl dye which exhibits gigantic second harmonic signals. Possible mechanisms of the voltage dependence of the second harmonic signal are discussed.