The biomechanical response of tissues serves as a valuable marker in the prediction of disease and in understanding the related behavior of the body under various disease and age states. Alterations in the macroscopic biomechanical response of diseased tissues are well documented; however, a thorough understanding of the microstructural events that lead to these changes is poorly understood. In this article we introduce a novel microbiaxial optomechanical device that allows two-photon imaging techniques to be coupled with macromechanical stimulation in hydrated planar tissue specimens. This allows that the mechanical response of the microstructure can be quantified and related to the macroscopic response of the same tissue sample. This occurs without the need to fix tissue in strain states that could introduce a change in the microstructural configuration. We demonstrate the passive realignment of fibrous proteins under various types of loading, which demonstrates the ability of tissue microstructure to reinforce itself in periods of high stress. In addition, the collagen and elastin response of tissue during viscoelastic behavior is reported showing interstitial fluid movement and fiber realignment potentially responsible for the temporal behavior. We also demonstrate that nonhomogeneities in fiber strain exist over biaxial regions of assumed homogeneity.

Gaining insight into how the nervous system functions is a challenge for scientists, particularly because the static morphology of the brain and the cells within tell little about how they actually work. Fixed specimens can provide critical structural information, but the jump to functional neurobiology in living cells is obviated with these preparations. In order to grasp the complexity of neuronal activity, it is necessary to observe the brain in action, from the level of subcellular signaling to the whole organism. Recent advances in nonlinear microscopy have given rise to a new era for biological research. In particular, the introduction of multiphoton excitation has drastically improved the depth and speed to which we can probe brain function. In order to better appreciate recent contributions of multiphoton microscopy to our current and future understanding of biological systems, an historical awareness of past microscopy applications is useful.

A new technique for determining cellular viscosity has been developed based on two-photon excitation and fluorescence correlation spectroscopy. We have applied this method to map intracellular diffusion coefficient of small molecules at selected cytoplasmic locations of mouse fibroblast cells. The cytoplasmic diffusion rate is one tenth of that in an aqueous environment.

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons (∼700 nm) can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet (∼350 nm). In the fluorescence experiments described here, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g., wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. Three properties of two-photon excitation give this method its advantage over conventional optical sectioning microscopies: (1) the excitation is limited to the focal volume, thus providing inherent three-dimensional resolution and minimizing photobleaching and photodamage; (2) the two-photon technique allows imaging of UV fluorophores with only conventional visible light optics; (3) red light is far less damaging to most living cells and tissues than UV light and permits deeper sectioning, because both absorbance and scattering are reduced. Many cell biological applications of two-photon excitation microscopy have been successfully realized, demonstrating the wide ranging power of this technique.