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.
Alteration of cellular energy metabolism is a principal feature of tumor and stem cells. Here we analyze the metabolic interactions between cancer cells and fibroblasts in a co-culture model and the metabolic heterogeneity of tumors and metabolic changes in mesenchymal stem cells during adipogenic differentiation based on the fluorescence of the metabolic cofactors NADH, NADPH, and FAD. We registered a metabolic switch from oxidative phosphorylation to glycolysis with slight acidification of the cytosol in cancer cells in a co-culture model. In the tumor tissue we detected metabolic heterogeneity with more glycolytic metabolism of cancer cells in the stroma-rich zones. The shift of cellular energy metabolism from glycolysis to oxidative phosphorylation and the activation of lipogenesiswere observed in adipocytes. Data aboutmetabolic alterations in cancer and stemcells are important formonitoring the progression of cancers, the development of anticancer drugs and stemcell therapy.
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.
We describe multiphoton imaging with sample-temperature control to monitor animal cells and cells of intact plants during freezing, thawing and heating processes based on autofluorescence intensity and lifetime. The sample temperature can be set with a heating and freezing stage to any value in the range between liquid nitrogen temperature (−196 °C; 77 K) and +600 °C (873 K) and changed with adjustable heating/freezing rates between 0.01 K/min and 150 K/min. Multiphoton imaging is realized with near-infrared femtosecond-laser excitation with different setups employing different laser sources. To illustrate the capabilities, imaging of animal cell samples with and without a cryoprotectant during freezing at cooling rates is presented. Lowering the temperature led to a significant increase of the intracellular fluorescence intensity and modifications. Fluorescence lifetime imaging indicated an increase of the mean lifetime with decreasing temperature. Furthermore, to illustrate imaging of plant samples, Arabidopsis thaliana leaves were employed. The measurements revealed thermally-induced changes of fluorescence lifetime and intensity as well as morphological alterations in the distribution of chloroplasts. The measurements illustrate the general usefulness of multiphoton imaging to investigate freezing and thawing effects on animal and plant cells even at temperatures commonly used for cryopreservation.
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.
Nonlinear contrast methods such as two-photon excited autofluorescence (in combination with fluorescence lifetime imaging) and second-harmonic generation have been combined with a further multiphoton contrast mechanism called coherent anti-Stokes Raman scattering. We describe the principle and the instrumentation for its implementation in tomographs for dermatological applications and present multimodal optical skin biopsies.
The development of clinical multiphoton technologies has led to new, labelfree approaches for non-invasive, in vivo imaging of human skin. Recent studies have shown that multiphoton imaging can be used to assess a wide range of biological processes, including cancer, cellular metabolism, and the effects of skin treatments. The imaging devices MPTflex and its earlier version, DermaInspect, have been employed in a broad range of clinical applications spanning from characterizing and understanding keratinocyte metabolism to malignant melanoma detection and diagnosis, pigment biology, cosmetic treatments, and skin aging. The promising results indicate that in the near future, real-time non-invasive nonlinear “optical biopsies” can be performed at the bedside.
Dysfunctions and dystrophies severely affect the cornea’s function. In point of fact, cornea diseases are the second major cause of blindness worldwide. Corneal diagnosis in clinical practice heavily relies on imaging techniques such as slit lamp microscopy, confocal microscopy, or optical coherence tomography. However, these fail to provide information on the cell’s metabolic state or the structural organization of the corneal stroma. With two-photon microscopy and fluorescence lifetime imaging this information can be obtained. Therefore, corneal pathology diagnosis may be improved. The feasibility of corneal characterization by two-photon imaging has been demonstrated in ex vivo samples and in vivo animal models. In this chapter, we report on the use of two multiphoton microscopy instruments for imaging the human cornea: a 5D multiphoton laser scanning microscope and the multiphoton tomograph MPTflex. Human corneas unsuitable for transplantation but otherwise normal and pathological samples obtained after surgery were imaged and characterized based on their autofluorescence and second-harmonic generation signals. Two possible clinical applications of two-photon microscopy are discussed: (i) the assessment of tissue viability before corneal transplantation and (ii) the differential diagnosis of corneal pathologies, further demonstrating the advantages of this imaging modality for corneal diagnosis.
Recent advancements in optical microscopy are challenging our understanding of the brain. In this chapter, we show the potential of optical microscopy techniques to tackle different aspects of brain structure and function, from wholebrain neuroanatomy to neural network plasticity and functionality. We will first address novel implementations of light microscopy for cellular resolution imaging of neuronal anatomy spanning the whole brain. Afterwards, we will illustrate real-time brain rewiring of single synaptic contacts visualized through two-photon microscopy in vivo. Then, the functionality of microcircuits is investigated with nonlinear microscopy combined with fluorescent indicators of neuronal activity. Nevertheless, a single technique is not enough for targeting the articulate organization of the brain; a wider view is more efficiently gained by combining complementary approaches. In the last section of this chapter, we show examples of this multiscale approach by discussing correlative imaging obtained by combining different microscopy techniques. At the end, we discuss the perspective of a wider methodological framework fusing multiple levels of brain investigation possibly leading to an omni-comprehensive view of brain machinery.
A key challenge in pharmacology and toxicology is understanding the exposure, fate and effects of drugs and nanomaterials on the body.Nanoparticles arewidely used in topically applied consumer products. For example, titanium dioxide and zinc oxide are used in commercial sunscreen formulations to afford the skin protection from harmful ultraviolet radiation. There are still ongoing safety concerns regarding the long-term toxicity and fate of nanomaterials within complex biological systems with a key question being whether topically applied nanoparticles can penetrate human skin and reach the viable epidermis to cause localized and potentially systemic toxicity under ‘in-use’ conditions. Recent advances in imaging technology have provided us with the tools to map the distribution of native, unlabeled, nanomaterials after application to biological tissue. Another key organ for in situ monitoring is the liver as it is the major site for drug metabolism and excretion in the body. Clearance of drugs from the body can be greatly altered in liver disease, resulting in toxicity and altered effects of the drugs. It is therefore crucial to understand liver functional changes in diseased livers. Recent advances in imaging technology have provided us with insight into mapping the distribution of native, unlabeled, nanomaterials after application to a range of organs within the body. This chapter deals with the use of multiphoton fluorescence microscopy in combination with fluorescence lifetime imaging techniques to assess the safety concerns regarding nanomaterials discussing the key challenges faced.