Biomedical subjects

Christof Buehler

Publications and source records attributed to Christof Buehler.

3 recordsLinked to original sources

PubMed2005

Single-photon counting multicolor multiphoton fluorescence microscope.

We present a multicolor multiphoton fluorescence microscope with single-photon counting sensitivity. The system integrates a standard multiphoton fluorescence microscope, an optical grating spectrograph operating in the UV-Vis wavelength region, and a 16-anode photomultiplier tube (PMT). The major technical innovation is in the development of a multichannel photon counting card (mC-PhCC) for direct signal collection from multi-anode PMTs. The electronic design of the mC-PhCC employs a high-throughput, fully-parallel, single-photon counting scheme along with a high-speed electrical or fiber-optical link interface to the data acquisition computer. There is no electronic crosstalk among the detection channels of the mC-PhCC. The collected signal remains linear up to an incident photon rate of 10(8) counts per second. The high-speed data interface offers ample bandwidth for real-time readout: 2 MByte lambda-stacks composed of 16 spectral channels, 256 x 256 pixel image with 12-bit dynamic range can be transferred at 30 frames per second. The modular design of the mC-PhCC can be readily extended to accommodate PMTs of more anodes. Data acquisition from a 64-anode PMT has been verified. As a demonstration of system performance, spectrally resolved images of fluorescent latex spheres and ex-vivo human skin are reported. The multicolor multiphoton microscope is suitable for highly sensitive, real-time, spectrally-resolved three-dimensional imaging in biomedical applications.

Data Display↗

PubMed2005

Multi-photon excitation of intrinsic protein fluorescence and its application to pharmaceutical drug screening.

The majority of proteins contain intrinsic fluorophores as natural sensors of molecular structures, dynamics, and interactions. The intrinsic protein fluorescence signal allows for the label-free and, hence, undisturbed and rapid study of protein-ligand interactions. Ultraviolet-based drug screening is hampered by the background, photobleaching, light scattering, inner filter effects, and interfering assay compounds. Such problems can be overcome by means of molecular three-photon excitation (3PE) with infrared femtosecond light pulses since longer excitation wavelengths result in less Raleigh scattering, and the subfemtoliter (confocal-like) 3PE volume minimizes out-of-focus photobleaching, background generation, and inner filter effects. We demonstrate the general feasibility of 3PE for protein spectroscopy and illustrate the technique's excellent potential for high-throughput screening. By using the intrinsic fluorescence intensity of a protein-substrate, we were able to discriminate between ligands of different affinities in binding assays.

Computer-Aided Design↗

PubMedDate not supplied

Mitigating thermal mechanical damage potential during two-photon dermal imaging.

Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 microm. 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.

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