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  • Author: Karsten König x
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Abstract

This review gives an overview of the history of fluorescence lifetime imaging (FLIM) in life sciences. FLIM microscopy based on an ultrafast laser scanning microscope and time-correlated single photon counting (TCSPC) was introduced in Jena/ Germany in 1988/89. FLIM images of porphyrin-labeled live cells and live mice were taken with an unique ZEISS confocal picosecond laser microscope. Five years later, the first in vivo FLIM on human volunteers started with time-gated cameras to detect dental caries based on one-photon wide-field pulsed laser excitation of autofluorescent bacteria. Another five years later, two-photon FLIM of autofluorescent skin was performed on a volunteer with a lab microscope in the frequency domain. The first clinical non-invasive optical, two-photon 3DFLIMbiopsieswere obtained fifteen years ago in patientswith dermatological disorders using a certified clinical multiphoton tomograph based on a tunable femtosecond titanium:sapphire laser and TCSPC. A current major FLIM application in cell biology is the study of protein-protein interactions in transfected cells by FLIM-FRETmicroscopy. Clinical FLIMapplications are still on a research level and include preliminary studies on (i) one-photon FLIM autofluorescence microscopy of patients with ocular diseases using picosecond laser diodes, (ii) time-gated imaging in brain surgery using a nanosecond nitrogen laser, and (iii) two-photon clinical FLIM tomography of patients with skin cancer and brain tumors with near-infrared femtosecond lasers and TCSPC.

Abstract

Laser tweezers or optical traps are established laser tools for optical noncontact manipulation of micron/submicron sized objects in liquids such as nonadherent biological cells in medium. Typical laser traps are based on optical gradient forces generated with high numerical aperture near-infrared (NIR) continuous wave (cw) lasermicroscopes. The laser-cell interaction is determined by a change of themomentum due to the beam direction being altered by refraction. In order to avoid laser absorption, NIR cw lasers such as the Nd:YAG laser at 1064 nm, the frequency doubled erbium:YAG fiber laser at 760 nm, the tunable cw Ti:sapphire ring laser, and laser diodes at wavelengths < 800 nmare employed. They are considered to be safe tweezer sources. However, two-photon absorption effects may occur due to the generation of high MW/cm2 laser intensities when using tightly focused cw laser beams at a power of 100mW or more. These nonlinear effects can be used for two-photon excited fluorescence spectroscopy of trapped objects. However, when using low-wavelength NIR (< 800 nm) traps, potential “UV-like” photodamaging effects have to be considered during cell manipulation. The use of the Nd:YAG laser at 1064nm is recommended when using laser tweezers for optical sperm transport for laser-assisted in vitro fertilization (IVF).

Abstract

The integration of an ultrafast laser into an optical microscope was a milestone as it has allowed for nonlinear imaging and precise nano- and microprocessing. Highly focused femtosecond laser pulses can be used to drill nanoholes, to perform nano- and microsurgery, and to generate sub-100nm structures even with lowenergy near-infrared (NIR) radiation. NIR nanoprocessing far below Abbe’s diffraction limit is based on multiphoton processes within the central part of the submicron focal spot, such as multiphoton ionization and plasma formation.With two-beam laser systems, structures with a feature size even in the sub-10nm range can be generated. Nanoprocessing can be performed at the surface as well as inside transparent materials, such as glass, polymers, biological cells and inside tissues, e.g. cornea. Transient TW/cm2 intensities are required. Interestingly, these enormous light intensities can be achieved with mean laser powers of a fewmilliwatt when using sub-20 fs laser pulses. Applications of fs NIR laser microscopes (nanoscopes) include 3D nanolithography, nanomachining such as nanowire production, optical cleaning, targeted transfection, optical reprogramming, and low power nanosurgery.

Abstract

Multiphoton tomography is a novel tissue imaging method with superior subcellular spatial resolution and picosecond temporal resolution. It provides noninvasively and label-free optical biopsies within seconds based on autofluorescence (AF) and second-harmonic generation (SHG). Besides 3D morphology, MPT enables optical metabolic imaging (OMI) by fluorescence lifetime imaging (FLIM) of the coenzymes NAD(P)H and flavins, the sensitive biosensors of cellular metabolism and oxidative stress. The add-on modules “CARS” and “microendoscope” provide further chemical information and deep tissue endomicroscopy. The multiphoton tomographs DermaInspect, MPTflex, andMPTflex-CARS have become CE certified medical devices. They are used in clinics around the world as well as in research centers of cosmetic and pharmaceutic companies. Major applications include early skin cancer detection, evaluation of skin modifications after long-term space flights, testing of anti-ageing substances, and intratissue nanoparticle tracking. Recently, the first intraoperative MPT-imaging during brain tumor surgery as well as MPT of human cornea have been reported. Besides clinical applications, the novel multiphoton tomographs are used as upright and inverted two-photon/SHG/FLIM microscopes to watch stem cells at work in transgenic mice, to perform in situ pharmacokinetics in the liver of small animals, to screen ex vivo human biopsies directly in the operation theater, and to image cell monolayers and 3D cell clusters.

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