Karl-Juergen Halbhuber

Two‐photon microscopy of deep intravital tissues and its merits in clinical research

Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two‐photon excitation microscopy including 2‐photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2‐photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in‐tandem combination of 2‐photon fluorescence and second harmonic generated signal microscopy as two‐modality microscopy allows for in situ co‐localization imaging of various microstructural components in the whole‐mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2‐photon‐controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two‐modal 2‐photon microscopy/tomography, acting as an efficient and sensitive non‐injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non‐linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.

High-resolution two-photon excitation microscopy of ocular tissues in porcine eye

Two‐photon excitation laser scanning microscopy (TPM), based on nonlinear optical (NLO) response under high irradiance, is currently being extensively employed for diagnostic purposes in biomedical fields and becomes more and more an interesting imaging technique in the intact bulk tissue examination. In this study, this nonlinear‐excitation imaging technique including two‐photon‐mediated autofluorescence (2PF) and second harmonic generation (SHG) was employed to investigate the microstructures in the whole‐mount scleral, retinal, and corneal tissues of porcine eyes with intracellular spatial resolution and high signal‐to‐noise ratio.

Photodynamic-induced inactivation of Propionibacterium acnes

We report on photodynamically induced inactivation of the skin bacterium Propionibacterium acnes (P. acnes) using endogenous as well as exogenous photosensitizers and red light sources. P. acnes is involved in the pathogenesis of the skin disease acne vulgaris. The skin bacterium is able to synthesize the metal-free fluorescent porphyrins protoporphyrin IX (PP) and coproporphyrin (CP) as shown by in situ spectrally-resolved detection of natural autofluorescence of human skin and bacteria colonies. These naturally occurring intracellular porphyrins act as efficient endogenous photosensitizers. Inactivation of P. acnes suspensions was achieved by irradiation with He-Ne laser light in the red spectral region (632.8 nm). We monitored the photodynamically-induced death of single bacteria using a fluorescent viability kit in combination with confocal laser scanning microscopy. In addition, the photo-induced inactivation was calculated by CFU (colony forming units) determination. We found 633 nm-induced inactivation (60 mW, 0.12 cm2 exposure area, 1 hour irradiation) of 72% in the case of non-incubated bacteria based on the destructive effect of singlet oxygen produced by red light excited endogenous porphyrins and subsequent energy transfer to molecular oxygen. In order to achieve a nearly complete inactivation within one exposure procedure, the exogenous photosensitizer Methylene Blue (Mb) was added. Far red exposure of Mb-labeled bacteria using a krypton ion laser at 647 nm and 676 nm resulted in 99% inactivation.

Optical tomography of human skin with subcellular spatial and picosecond time resolution using intense near infrared femtosecond laser pulses

We describe the novel high resolution imaging tool DermaInspect 100 for non-invasive diagnosis of dermatological disorders based on multiphoton autofluorescence imaging (MAI)and second harmonic generation. Femtosecond laser pulses in the spectral range of 750 nm to 850 nm have been used to image in vitro and in vivo human skin with subcellular spatial and picosecond temporal resolution. The non-linear induced autofluorescence originates mainly from naturally endogenous fluorophores/protein structures like NAD(P)H, flavins, keratin, collagen, elastin, porphyrins and melanin. Second harmonic generation was observed in the stratum corneum and in the dermis. The system with a wavelength-tunable compact 80 MHz Ti:sapphire laser, a scan module with galvo scan mirrors, piezoelectric objective positioner, fast photon detector and time-resolved single photon counting unit was used to perform optical sectioning and 3D autofluorescence lifetime imaging (t-mapping). In addition, a modified femtosecond laser scanning microscope was involved in autofluorescence measurements. Tissues of patients with psoriasis, nevi, dermatitis, basalioma and melanoma have been investigated. Individual cells and skin structures could be clearly visualized. Intracellular components and connective tissue structures could be further characterized by tuning the excitation wavelength in the range of 750 nm to 850 nm and by calculation of mean fluorescence lifetimes per pixel and of particular regions of interest. The novel non-invasive imaging system provides 4D (x,y,z,t) optical biopsies with subcellular resolution and offers the possibility to introduce a further optical diagnostic method in dermatology.

N,N-Dialkylaminostyryl dyes: specific and highly fluorescent substrates of peroxidase and their application in histochemistry.

Fluorescent labeling of immuno-bound or endogenous peroxidase (PO) activity has been achieved to date by means of phenol derivatives with a low substitution degree. Here it is demonstrated that N,N-dialkylamino-styryl dyes can also act as fluorescent substrates of PO. They undergo enzymatically cross-linking reactions to surrounding cell constituents in an analogous manner thus permitting highly fluorescent and permanent labeling. This approach is narrowly related to the catalyzed reporter deposition (CARD) technique based on tyramine conjugates and the recently described catalytic cross-linking approach of hydroxystyryl derivatives. The substitution patterns for optimal cross-linking capability and the spectral properties of obtained specific reaction products were studied using an iterative semi-empirical approach. The best staining performance is achieved with N,N-dimethylaminoaryl derivatives. Their N,N-dialkyl homologues as well as the primary aryl amine pendants failed as PO substrates. Due to their basic character, novel substrates occasionally tend to unspecific interactions (staining nuclei, mast cells, or keratin). Centering this side specificity and repressing the staining capability of PO was achieved by chemical modification of the respective dye leading to new specific probes for keratin and cytoplasmatic RNA. In conclusion, catalytic cross-linking of heterocyclic 4-N,N-dimethylamino-styryl dyes represents a promising approach for the permanent fluorescent staining of PO in fixed cells and tissues, complementing the CARD technique. In contrast to CARD-related approaches, new substrates are characterized by a broad excitation and emission range of fluorescence and the outstanding spatial resolution of specific fluorescence signaling known so far from their 4-hydroxystyryl analogues. They currently represent the smallest fluorescent substrates of PO. Histochemical and immuno-histochemical applications share several outstanding features: High detection sensitivity, spatial resolution of fluorescence signaling, and photo stability. 4-N,N-dimethylamino-styryl substrates are compatible with their phenol and phenol–ester analogues. Their combination facilitates the trichromatic immuno-histochemical demonstration of three different targets simultaneously at one excitation wavelength in a conventional epi-fluorescence microscope.

Cell damage in UVA and CW/Femtosecond NIR microscopes

Cell damage in UV and NIR laser microscopes by highly focused micromanipulation and fluorescence excitation microbeams has been studied. Damage in erythrocytes, spermatozoa and Chinese hamster ovary cells was detected by monitoring morphology changes, autofluorescence detection, cloning assay, and viability screening. It was found that 364 nm/365 nm UVA radiation induced irreversible cell damage at radiant exposures as low as <10 J/cm2. NIR CW microradiation used in laser tweezers was also able to damage cells via a two-photon excitation process, in particular, when using <800 nm trapping beams. Non- destructive two-photon excitation in femtosecond NIR microscopes is possible within a narrow intensity window. The lower limit is determined by two-photon absorption coefficients and detector efficiency, the higher by intracellular optical breakdown in the extranuclear region. Above certain wavelength-dependent intensity thresholds in femtosecond microscopy, cells were completely destroyed by fragmentation concomitant with plasma generation. The influence of excitation and micromanipulation microbeams should be considered when studying physiology and metabolism of vital cells.

In-vivo Corneal Nonlinear Optical Tomography based on Second Harmonic and Multiphoton Autofluorescence Imaging induced by Near-Infrared Femtosecond Lasers with Rabbits

The intratissue multiphoton autofluorescence imaging (MAI) and the second harmonic generation (SHG) based on nonlinear process of femtosecond nanojoule laser pulses at wave length of 750-850 nm emitted from solid-state Titanium: Sapphire Chameleon have been used as a highly precise non-destructive tool to realize the in-vivo differentiation of corneal layers with the assistance of intratissue optical tomography and to visualize the keratocyte structures and collagen lamellas with submicron resolution. Multiphoton nonlinear imaging occurs only with high light intensity on an order of MG-GW/cm2 and photon flux density of more than 1024 photons cm-2s-1 in a 0.1femtoliter intrastromal focus volume obtained by diffraction-limited focussing with high-numerical objectives. This technique, acting as a novel diagnostic tool, proved to be essential for femtosecond (fs) nanojoule (nJ) cornea surgery to determine the interest of region preoperation, to visualize and verify the outcomes immediately after the laser surgery and has potential to become a powerful tool in advancing understanding of corneal biomechnics and cellular reactions after laser induced lesion.

Cloning assay thresholds on cells exposed to ultrafast laser pulses

The influence of the peak power, laser wavelength and the pulse duration of near infrared (NIR) ultrashort laser pulses on the reproduction behavior of Chinese hamster ovary (CHO) cells has been studied. In particular we determined the cloning efficiency of single cell pairs after exposure to ultrashort laser pulses with an intensity in the range of GW/cm2 and TW/cm2. A total of more than 3500 non- labeled cells were exposed to a highly focused scanning beam of a multiphoton laser microscope with 60 microsecond pixel dwell time per scan. The beam was provided by a tunable argon ion laser pumped mode-locked 76 MHz Titanium:Sapphire laser as well as by a compact solid-state laser based system (Vitesse) at a fixed wavelength of 800 nm. Pulse duration (tau) was varied in the range of 100 fs to 4 ps by out-of-cavity pulse- stretching units consisting of SF14 prisms and blazed gratings. Within an optical (laser power) window CHO cells could be scanned for hours without severe impact on reproduction behavior, morphology and vitality. Ultrastructural studies reveal that mitochondria are the major targets of intense destructive laser pulses. Above certain laser power P thresholds, CHO cells started to delay or failed to undergo cell division and, in part, to develop uncontrolled cell growth (giant cell formation). The damage followed a P2/(tau) relation which is typical for a two-photon excitation process. Therefore, cell damage was found to be more pronounced at shorter pulses. Due to the same P2/(tau) relation for the efficiency of fluorescence excitation, two- photon microscopy of living cells does not require extremely short femtosecond laser pulses nor pulse compression units. Picosecond as well as femtosecond layers can be used as efficient light sources in safe two photon fluorescence microscopy. Only in three photon fluorescence microscopy, femtosecond laser pulses are advantageous over picosecond pulses.

In vivo animal follow-up studies on intrastromal surgery with near-infrared nanojoule femtosecond laser pulses

We report on the histological results of in-vivo animal follow-up studies on refractive femtosecond laser surgery. Non-invasive flap-free intrastromal ablation as well as flap generation has been performed with MHz nanojoule near infrared femtosecond laser pulses. In particular, the dynamics of corneal wound healing have been studied. Wound-healing effects could be detected up to 90 days post-operation in the case of lasermediated flap generation. The flap-free intrastromal cavity was identified until the 28th day post-treatment. Interestingly, eosinophil granulocytes were observed. The follow-up studies confirmed that the near infrared femtosecond laser at near-nanojoule pulse energy is a highly precise and an attractive tool for intraocular refractive surgery, especially for flap-free intrastromal surgery.

Multiphoton imaging of corneal tissue with near-infrared femtosecond laser pulses: corneal optical tomography and its use in refractive surgery

The two-photon-mediated autofluorescence and second harmonic generation (SHG) are acting as a novel diagnostic tool to perform tissue optical tomography with submicron resolution. The three-dimensional corneal ultrastructure of whole depth can be probed without any staining or mechanical slicing. Compared with photodisruptive surgical effects occurring at TW/cm2 light intensity, multiphoton imaging can be induced at MW-GW/cm2 photon intensity. The multiphoton microscopy based on nonlinear absorption of femtosecond laser pulses at the wavelength of 715-930nm emitted from solid-state Ti: sapphire system is being used as a precise non-invasive monitoring tool to determine the interest of region, to visualize and to verify the outcomes in the invivo intrastromal laser nanosurgery. More interesting, the activated keratocytes have been also observed in-vivo 24 hours after the laser nanosurgery with this system. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the corneal tissue and for assessing the intrastromal nanosurgery. With the help of the multiphoton-mediated imaging, the next generation of laser refractive surgery approaches based on the nonamplified femtosecond lasers with higher precision and less complications are being evaluated systematically.