Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 µm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized.
High-speed two-photon imaging based on multi-foci excitation requires the use of spatially resolved detectors, such as charge coupled device (CCD) cameras, instead of single channel photomultiplier tube (PMT). The performance of systems based on both a PMT and a CCD in turbid medium was evaluated by measuring the image point spread function (PSF) and the image contrast as a function of depth and scattering coefficient with single point scanning. We found no significant change in the full-width at half maximum of the point spread function (PSF) for depth up to 100 μm. However, the CCD lost contrast significantly faster as a function of depth and increase scattering. This discrepancy is resolved by measuring a low amplitude but broad tail in the PSF distribution. The tail of the PSF distribution can be up to 200 μm in diameter. We further evaluate scattering effects in the imaging of GFP neurons in a mouse brain slice.
Two-photon scanning microscopy has been successfully applied in studying tissue structures and biochemistry with subcellular spatial resolution. We developed a 16-channel two-photon scanning microscope with high-performance single photon counting readout electronics. The apparatus incorporates a mode-locked Ti:Sapphire laser, a scanning microscope, a spectrograph with multi-anode PMT (16 channels with ~ 7 to 10 nm spectral resolution each), and a 16-channel photon counting card (PhCC) with integrated high-speed data link (payload 1.2 Gbps). Each PhCC detection channel features single-photon sensitivity and 100 MHz photon counting bandwidth. The multi-color single-photon counting detection scheme allows ultra-sensitive measurements in a broad spectrum of biomedical applications. We present spectroscopic studies of ex vivo tissue autofluorescence.
Non-invasive optical diagnosis of cellular and extracellular structure and biochemistry in thick tissue is becoming a reality with the maturation of the two-photon imaging. Today, the slow imaging speed of typical two-photon microscopes is a major hurdle in realizing their clinical potential. We have developed a high-speed two-photon microscope optimized for acquiring 3-D tissue images in real time. The scanning speed improvement of this system is obtained by the use of an air bearing polygonal mirror. The maximum achievable scanning rate is 40 microseconds per line, which is about 100 times faster than conventional scanning microscopes. High-resolution fluorescence images were recorded in real-time by an intensified CCD camera. Using this instrument, we have monitored the movements of protozoas and mapped the collagen/elastin fiber structures in excised human skin.
We report the implementation of intensity modulated diode lasers in frequency-domain pump-probe studies, diode lasers are compact, stable, and economical units that require little maintenance. In our study, a 365 nm diode laser is used as the excitation source and the output of a 680 nm unit induces stimulated emission from excited state fluorophores. By modulating the intensities of the two diode lasers at slightly different frequencies, and detecting the fluorescence signal at the cross-correlation frequency, both time-resolved and high spatial resolution imaging can be achieved. The laser diodes are modulated in the 100 MHz cross-correlation signal has been used for time-resolved imaging of fluorescent microspheres and mouse fibroblasts labeled with nucleic acid stains TOTO-3. These results demonstrate and feasibility of using intensity modulated diode lasers for frequency-domain, pump-probe studies.
A new versatile system for the measurement of time-resolved fluorescence emission spectra of biomolecules is presented. Frequency doubling and tripling of a Ti:Sapphire laser allows excitation over a wide wavelength range. The influence of increasing the spectral resolution on the time resolution has been investigated. System performance can be optimized for best resolution in the spectral or time domain, respectively. System performance can be optimized for best resolution in the spectral or time domain, respectively. The currently achieved temporal resolution is 6 psec, and the best spectral resolution is 3 nm. Long fluorescence decays can be resolved with optimal time resolution by way of taking into account the flyback of the streak camera. With the system described, the core complex ((alpha) (beta) )3APCLC8.9 of the phycobilisome from the photosynthetic cyanobacteria Mastigocladus laminosus has been analyzed. Lifetime analysis clearly demonstrated the influence of the linker polypeptide on the phycobiliprotein complex and the identity of native and reconstituted complex.