By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.

High-resolution nonlinear optical imaging of live cells by second harmonic generation

Summary The imaging performance in single-photon (l-p) and two­ photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of.the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled •. giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.

Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy

We report on two-photon fluorescence excitation (TPE) action
cross sections for five widely used molecular
fluorophores. Measurements were performed by use of ultrashort
(∼100-fs) Ti:sapphire pulsed excitation over the range 690–960
nm. TPE spectra were obtained by comparison with a fluorescein
calibration standard. Large cross sections were found for the
cyanine reagent Cy 3 (∼140 GM) and for Rhodamine 6G (∼150
GM), both at 700 nm [1 GM = 10-50
(cm4 s)/photon]. Several fluorophores show
interesting and desirable blue shifts with respect to twice the
one-photon absorption wavelength. Fluorophore fluorescence
intensities showed no significant departure (±4%) from quadratic
illumination power dependence, indicating genuine two-photon
processes. Implications of these measurements for two-photon
laser-scanning microscopy are discussed.

Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0–50 microm) and at deeper depths (100–150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

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Jörg Martini

Papers

Multifocal multiphoton microscopy : new detection methods and biological applications

Multifocal Multiphoton Microscopy