Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

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Nonlinear magic: multiphoton microscopy in the biosciences

Femtosecond laser micromachining can be used either to remove materials or to change a material's properties, and can be applied to both absorptive and transparent substances. Over the past decade, this technique has been used in a broad range of applications, from waveguide fabrication to cell ablation. This review describes the physical mechanisms and the main experimental parameters involved in the femtosecond laser micromachining of transparent materials, and important emerging applications of the technology. Interactions between laser and matter are fascinating and have found a wide range of applications. This article gives an overview of the fundamental physical mechanisms in the processing of transparent materials using ultrafast lasers, as well as important emerging applications of the technology.

Femtosecond laser micromachining in transparent materials

Author(s): Vogel, Alfred; Venugopalan, Vasan | Abstract: The mechanisms of pulsed laser ablation of biological tissues were studied. The transiently empty space created between the fiber tip and the tissue surface improved the optical transmission to the target and thus increased the ablation efficiency. It was found that the structure and morphology also affect the energy transport among tissue constituents.

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Mechanisms of pulsed laser ablation of biological tissues.

When a molecule absorbs a photon of appropriate energy, a chain of photophysical events ensues, such as internal conversion or vibrational relaxation (loss of energy in the absence of light emission), fluorescence, intersystem crossing (from singlet state to a triplet state) and phosphorescence, as shown in the Jablonski diagram for organic molecules (Fig. 1). Each of the processes occurs with a certain probability, characterized by decay rate constants (k). It can be shown that the average length of time τ for the set of molecules to decay from one state to another is reciprocally proportional to the rate of decay: τ = 1/k. This average length of time is called the mean lifetime, or simply lifetime. It can also be shown that the lifetime of a photophysical process is the time required by a population of N electronically excited molecules to be reduced by a factor of e. Correspondingly, the fluorescence lifetime is the time required by a population of excited fluorophores to decrease exponentially to N/e via the loss of energy through fluorescence and other non-radiative processes. The lifetime of photophycal processes vary significantly from tens of femotoseconds for internal conversion1,2 to nanoseconds for fluorescence and microseconds or seconds for phosphorescence.1






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Figure 1


Jablonski diagram and a timescale of photophysical processes for organic molecules.

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Fluorescence Lifetime Measurements and Biological Imaging

Photodynamic therapy (PDT) employs a non-toxic dye, termed a photosensitizer (PS), and low intensity visible light which, in the presence of oxygen, combine to produce cytotoxic species. PDT has the advantage of dual selectivity, in that the PS can be targeted to its destination cell or tissue and, in addition, the illumination can be spatially directed to the lesion. PDT has previously been used to kill pathogenic microorganisms in vitro, but its use to treat infections in animal models or patients has not, as yet, been much developed. It is known that Gram-(−) bacteria are resistant to PDT with many commonly used PS that will readily lead to phototoxicity in Gram-(+) species, and that PS bearing a cationic charge or the use of agents that increase the permeability of the outer membrane will increase the efficacy of killing Gram-(−) organisms. All the available evidence suggests that multi-antibiotic resistant strains are as easily killed by PDT as naive strains, and that bacteria will not readily develop resistance to PDT. Treatment of localized infections with PDT requires selectivity of the PS for microbes over host cells, delivery of the PS into the infected area and the ability to effectively illuminate the lesion. Recently, there have been reports of PDT used to treat infections in selected animal models and some clinical trials: mainly for viral lesions, but also for acne, gastric infection by Helicobacter pylori and brain abcesses. Possible future clinical applications include infections in wounds and burns, rapidly spreading and intractable soft-tissue infections and abscesses, infections in body cavities such as the mouth, ear, nasal sinus, bladder and stomach, and surface infections of the cornea and skin.

Photodynamic therapy: a new antimicrobial approach to infectious disease?

Near-infrared laser pulses of a compact 80-MHz femtosecond laser source at 800 nm, a mean power of 15-100 mW, 170-fs pulse width, and millisecond beam dwell times at the target have been used for multiphoton-mediated nanoprocessing of human chromosomes. By focusing of the laser beam with high-numerical-aperture objectives of a scanning microscope to diffraction-limited spots and with light intensities of terawatts per cubic centimeter, precise submicrometer holes and cuts in human chromosomes have been processed by single-point exposure and line scans. A minimum FWHM cut size of ~100 nm during a partial dissection of chromosome 1, which is below the diffraction-limited spot size, and a minimum material removal of ~0.003mum (3) were determined by a scanning-force microscope. The plasma-induced ablated material corresponds to ~1/400 of the chromosome 1 volume and to ~65x10(3) base pairs of chromosomal DNA. A complete dissection could be performed with FWHM cut sizes below 200 nm. High-repetition-frequency femtosecond lasers at low mean power in combination with high-numerical-aperture focusing optics appear therefore as appropriate noncontact tools for nanoprocessing of bulk and (or) surfaces of transparent materials such as chromosomes. In particular, the noninvasive inactivation of certain genomic regions on single chromosomes within living cells becomes possible.

Nanodissection of human chromosomes with near-infrared femtosecond laser pulses.

We report on laser-assisted knocking out of genomic nanometer-sized regions within the nucleus of living cells The intranuclear nanosurgery was possible by application of highly intense near infrared femtosecond laser pulses The non-contact laser treatment was performed within a closed sterile cell chamber The destructive multiphoton effect was based on 10(12) W/cm2 light intensities and limited to a sub-femtoliter focal volume of a high numerical aperture objective We used the intracellular nanoscalpel for highly precise non-contact dissection of Hoechst-labelled chromosomes within a nucleus of a living Chinese hamster ovary cell Following laser treatment, the cell remained alive and did not show any signs of membrane perturbation The use of near infrared pulses provide the possibility of non-invasive intracellular nanoprocessing also within living tissue in depths of more than 100 microns

Intracellular nanosurgery with near infrared femtosecond laser pulses.

We studied spectral autofluorescence characteristics of dental caries. A wide range of carious lesions revealed characteristic emission of endogenous fluorophores with strong fluorescence bands in the red spectral region when excited with 407 nm line radiation of a krypton ion laser. Healthy hard dental tissue exhibited no emission bands in the red. The fluorescence spectra, fluorescence excitation spectra as well as the reflectance spectra of carious lesions were found to be typical for fluorescent porphyrins, mainly protoporphyrin IX. A possible source of these porphyrins within carious tissues is bacterial biosynthesis. Non-invasive sensitive in vivo caries detection by means of appropriate excitation sources and porphyrin fluorescence detectors should be possible.

Laser-induced autofluorescence spectroscopy of dental caries.

With the increase in the number of antibiotic resistant strains of microorganism, the search for alternative treatments of microbial infections becomes all the more important. We report a novel method for bacterial inactivation based on the optical excitation of the naturally occurring (endogenous) photosensitzing porphyrins by red light. In particular, the pathogenic Gram-positive porphyrin producing ATCC strains Propionibacterium acnes, Actinomyces odontolyticus and Porphyromonas gingivalis were investigated. Sensitive autofluorescence spectroscopy revealed that these bacteria naturally synthezise the fluorescent photosensitizer protoporphyrin IX. In addition, bacterial plaque samples of periodontitis patients were studied. Non-labeled fluorescent bacterial colonies were exposed to red light at 632.8 nm, 100 mW/cm2 light intensity and 360 J/cm2 energy density using a helium-neon laser. The survival rate after a single phototreatment with red light was found to be 0.58 +/- 0.09 in the case of Propionibacterium acnes, 0.30 +/- 0.04 in Actinomyces odontolyticus and 0.59 +/- 0.10 in Porphyrormonas gingivalis compared to non-exposed bacteria suspensions. No photoeffect was found for the bacterium Streptococcus mutans which exhibited no detectable porphyrin autofluorescence. Red-light exposed plaque samples of patients showed significant reduction of colony forming units by 50% as well as a pronounced photoeffect on the pigmented species Prevotella intermedia. Taken together, these results suggest the treatment with red light can be potentially employed as an therapeutic method to inactivate certain pathogenic strains of porphyrin producing bacteria without the use of external photosensitizers.

Red light kills bacteria via photodynamic action.

Laser-induced time-resolved autofluorescence from carious lesions of human teeth was studied by means of ultrashort pulsed laser systems, time-correlated single photon counting and time-gated imaging. Carious regions exhibited a slower fluorescence decay with a main 17 ns fluorescence lifetime than healthy hard dental tissue. The long-lived fluorophore present in carious lesions only emits in the red spectral region. Fluorescence decay time and spectral characteristics are typical of fluorescent metal-free porphyrin monomers. The spatial distribution of the long-lived endogenous porphyrin fluorophore within the tooth material was detected by time-gated nanosecond autofluorescence imaging. In particular, high contrast video images were obtained with an appropriate time delay of 15 ns to 25 ns between excitation and detection due to the suppression of short-lived autofluorescence of healthy tissue. First in vivo applications are reported indicating the potential of time-resolved fluorescence diagnostics for early caries- and dental plaque detection.

König K

Papers

Nanodissection of human chromosomes with near-infrared femtosecond laser pulses.

Intracellular nanosurgery with near infrared femtosecond laser pulses.

Laser-induced autofluorescence spectroscopy of dental caries.

Red light kills bacteria via photodynamic action.

Time-gated in vivo autofluorescence imaging of dental caries