Femtosecond pulse width control in microscopy by two-photon absorption autocorrelation

A technique for both the measurement and the control of the pulse width at the focal point of a high-NA lens, based on two-photon absorption interferometric autocorrelation, is presented. The technique is applied to measuring the pulse broadening induced on a pulse propagating through a high-NA lens system for several objectives. It is known that the pulse width may increase by up to ?50% of its original value by propagation through an objective which has wide field compensation for spherical and chromatic aberrations. The two-photon absorption autocorrelation technique allows adjustment of the actual pulse width in the focus of a high-NA lens through pre-chirp compensation. The pulse width is shown to be almost independent of penetration depth into the sample, while the amplitude of the autocorrelation signal shows a strong decrease with depth. The ability to both measure and control the actual pulse width under strong focusing conditions is of direct importance to, among others, two-photon absorption imaging approaches.     

Two-photon excited lifetime imaging of autofluorescence in cells during UV A and NIR photostress

By monitoring coenzyme autofluorescence modifications. as an indicator of cell damage. the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UV A radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser. focused through highnumerical- aperture microscope optics. provided diffractionlimited mlcrobeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (_T-mapping). Upon the onset of UV or NIR exposure. Chinese hamster ovary cells exhibited blue/green autofluorescence witq a mean lifetime of 2·2 ns. which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 m W. 300 J cm^-2 ) resulted in oxidative stress correlated with increased autofluorescence intensity. onset of nuclear fluorescence. and a fluorescence lifetime decrease. The cellular response to femtosecond NIR micro beams depended significantly on peak power. Peak powers above a threshold value of about 0·5kW (average power: 6mW). 0·55kW (7mW) and 0·8kW (lOmW) at 730nm. 760nm and 800nm. respectively. resulted in the onset of short-lived luminescence with higher intensity (100x) than the intracellular NAD(P)H fluorescence. This luminescence. accompanied by destruction of cellular morphology. was localized and occurred in the mitochondrial region. In contrast. beams at a power of less than 0·5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.     

Destruction of polymer growth substrates for cell cultures in two-photon microscopy

The choice of the growth substrate for cell cultures used in fluorescence microscopy is guided by several factors including the type of cells studied and the type of microscopy used. Usually, cells can be cultured on either polymer or glass substrates. One type of polymer, termed Aclar, presents several attractive features: the adhesive properties are better than those of glass, the optical properties are comparable to those of glass, it is biochemically inert, unbreakable, flexible and has a high surface tension, convenient for seeding cells on the cover slip. However, here we show that when imaging with two-photon microscopy, which is based on a femtosecond pulsed laser source, local damage of the Aclar substrate occurs, starting at an average intensity of 10⁵ W cm⁻² at the focal point and for exposure times insufficient to cause cell damage. This leads to the appearance of gas bubbles on cultures plated on Aclar cover slips, which perturb the imaging. By contrast, this phenomenon does not occur on borosilicate cover slips, probably because of their different physical (thermal conductivity, absorbance, melting point) and material homogeneity properties. Thus, for cell culture applications using pulsed lasers with high intensities, the use of glass is preferable to Aclar. The results also reveal that substrates can be more susceptible to thermal damage than the cells themselves.