Multiphoton fluorescence lifetime imaging (FLIM) is gaining ground as a non-invasive and very sensitive method in life sciences, and even as a clinical tool. First clinical devices employing FLIM are on the market, e.g. MPTflex. A hot topic is using metabolic imaging to investigate melanoma lesions (Fig.1). This method utilizes imaging of the ratio of the amounts of the free and protein-bound forms of the intracellular autofluorescent metabolic co-enzyme nicotinamide adenine dinucleotide (NADH) [1,2,3,4]. In another study, we investigated safety aspects of nanoparticle based sun screens. Multiphoton FLIM enables tracing of nanoparticles after application on the skin . Furthermore, in case of penetration into the viable epidermis metabolic imaging can be employed to investigate toxicity on skin cells .
Traditionally, destructive sampling and analysis are used to determine the fate, kinetics and effects of exogenous materials in the body. Minimally invasive confocal and multiphoton microscopy (MPM) in 3D space over in time to deep tissue depths has enabled us to quantify endogenous fluorescent species in the body as well as exogenous fluorescent molecules, cells and nanoparticles that have been administered into the body and/or are applied to the skin, kidney and liver ex vivo and in vivo. Of particular importance has been the ability to get specificity in drug, metabolite and endogenous solute measurement in tissues in vivo by using specific spectral excitation and emission wavelengths, the use of fluorescence lifetime and the measurement of fluorescence anisotropy. We have applied MPM to characterise physiologically based pharmacokinetics of solutes, mesenchymal stem cells and nanoparticles in various organs. More recently, we have used MPM to examine stem cell and nanoparticle – tissue interactions directly in acute liver and kidney injury models, tumor models and inflammatory models. MPM has also been used to measure changes in the redox state of cells, as well the use of photochemical probes to measure adverse biochemical events such as the formation of reactive oxygen species. Sun-induced skin damage, with its sequelae of photoaging, actinic keratosis and various skin cancers is a particular issue for many of us in subtropical and temperate climates. Our group has therefore also used MPM to quantify the metabolic changes seen in melanoma lesions, the safety of nanoparticle sunscreens, whose use may prevent these lesions, and to aid in the mechanistic and regulatory evaluation of topical product efficacy, bioequivalence and safety. In conclusion, MPM fluorescence lifetime imaging microscopy (FLIM) is a promising technology to aid in product characterization and development as well as in the translational diagnosis of skin related pathologies in the clinic.
Ophthalmic imaging by fluorescence techniques is a tool which gets more and more established in eye disease diagnosis and research. All type of clinical imaging is usually restricted to the use of endogenous fluorophores present in the tissue. The excitation and emission spectra of these fluorophores are overlapping and poorly defined. Moreover, the apparent spectra are changed by variation in the relative concentration of fluorophores and by absorbers present in the tissue. Intensity images, even those with spectral resolution, therefore deliver very limited information on the state of the tissue.
A considerable improvement in the field of retinal imaging is obtained by using fluorescence lifetime imaging ophthalmoscopy (FLIO). The fluorescence lifetime measured by TCSPC is independent of the concentration, and enables the possibility to measure even the weak retinal autofluorescence. Moreover, it delivers direct information on the configuration of endogenous fluorophores, on binding to proteins or lipids, on the redox state, and on other metabolic parameters.
We will describe the technical problems of FLIO data and their solutions, demonstrate the performance of existing systems in ophthalmology and present some results.
Multiphoton fluorescence lifetime imaging (FLIM) is gaining ground as a non-invasive and very sensitive research tool, and even as a method in clinical applications. Skin science is the predestined field for the latter, since skin is optically accessible without surgery. A hot topic is using metabolic imaging to investigate melanoma lesions. This method utilizes imaging of the ratio of the amounts of the free and protein-bound forms of the intracellular autofluorescent metabolic co-enzyme nicotinamide adenine dinucleotide (NADH) [1,2,3,4]. Another important topic which is closely bound up with skin cancer risk is safety aspects of sun screens. Multiphoton FLIM enables tracing of nanoparticle after application on the skin. Furthermore, in case of penetration through the stratum corneum again metabolic imaging can be used to investigate toxicity on skin cells .
1. O. Warburg, On the origin of cancer cells. Science (1956) 123:309-14
2. L. Pires, M.S. Nogueira, S. Pratavieira, et al. Time-resolved fluorescence lifetime for cutaneous melanoma detection. Biomed Opt Express (2014) 5:3080-9.
3. S. Seidenari, F. Arginelli, M. Manfredini, Multiphoton Laser Microscopy with Fluorescence Lifetime Imaging and Skin Cancer. In: Skin Cancer (Baldi A, Pasquali P, Spugnini EP, eds): (2014) Springer New York, 279-90.
4. M.N. Pastore, H. Studier H, C.S. Bonder, and M.S. Roberts. Non‐invasive metabolic imaging of melanoma progression. Exp Dermatol. (2016) 26:607–614.
5. A. M. Holmes, J. Lim, H. Studier, and M.S. Roberts, Varying the morphology of silver nanoparticles results in differential toxicity against micro-organisms, HaCaT keratinocytes and affects skin deposition. Nanotoxicology (2016) 10:10, 1503-1514
Skin cancer is associated with abnormal cellular metabolism which if identified early introduces the possibility of intervention to prevent its progress to a deadly metastatic stage. This study combines multiphoton microscopy with fluorescence lifetime imaging (FLIM) using an orthotopic melanoma mouse model, to detect changes in redox states of single epidermal cells as a metabolic marker to monitor the progress of tumor growth. This method utilizes imaging of the ratio of the amounts of the free and protein-bound forms of the intracellular autofluorescent metabolic co-enzyme nicotinamide adenine dinucleotide (NADH). Here we investigate the impact of the primary tumor lesion on the epidermal layers at three different growth stages of melanoma lesion compared to normal skin as a control. We show a significant increase in the free-to-bound NADH ratio with the growth of the solid melanoma tumor, while concurrently the short and the long lifetime components of NADH remained constant. These results demonstrate the potential of FLIM for rapid, non-invasive and sensitive assessment of melanoma progression revealing its potential as a diagnostic tool for melanoma detection and as an aid for melanoma staging.
Metabolic imaging by NAD(P)H FLIM requires the decay functions in the individual pixels to be resolved into the decay components of bound and unbound NAD(P)H. Metabolic information is contained in the lifetime and relative amplitudes of the components. The separation of the decay components and the accuracy of the amplitudes and lifetimes improves substantially by using ultra-fast HPM-100-06 and HPM-100-07 hybrid detectors. The IRF width in combination with the Becker & Hickl SPC-150N and SPC-150NX TCSPC modules is less than 20 ps. An IRF this fast does not interfere with the fluorescence decay. The usual deconvolution process in the data analysis then virtually becomes a simple curve fitting, and the parameters of the NAD(P)H decay components are obtained at unprecedented accuracy.
The study of metabolic and oxygen states of cells in a tumor in vivo is crucial for understanding of the mechanisms responsible for the tumor development and provides background for the relevant tumor’s treatment. Here, we show that a specially designed implantable fiber-optical probe provides a promising tool for optical interrogation of metabolic and oxygen states of a tumor in vivo. In our experiments, the excitation light from a ps diode laser source is delivered to the sample through an exchangeable tip via a multimode fiber, and the emission light is transferred to the detector by another multimode fiber. Fluorescence lifetime of nicotinamid adenine dinucleotide (NAD(P)H) and phosphorescence lifetime of an oxygen sensor based on iridium (III) complex of enzothienylpyridine (BTPDM1) are explored both in model experiment in solutions, and in living mice. The luminescence spectroscopy data is substantiated with immunohistochemistry experiments. To the best of our knowledge, the measurements of both metabolic status and oxygenation of tumor in vivo by fluorescence/phosphorescence lifetime spectroscopy with a fiber-optic probe are done for the first time.
Liver disease is the fifth most common cause of death and unlike many other major causes of mortality, liver disease rates are increasing rather than decreasing. There is no ideal measurement of liver disease and although biopsies are the gold standard, this only allows for a spot examination and cannot follow dynamic processes of the liver. Intravital imaging has the potential to extract detailed information over a larger sampling area continuously. The aim of this project was to investigate whether multiphoton and fluorescence lifetime imaging microscopy could detect early liver damage and to assess whether it could detect changes in metabolism of fluorescein in normal and diseased livers. Four experimental groups were used in this study: 1) control; 2) ischemia reperfusion injury; 3) steatosis and 4) steatosis with ischemia reperfusion injury. Results showed that multiphoton microscopy could visualize morphological changes such as decreased fluorescence of endogenous fluorophores and the presence of lipid droplets, characteristic of steatosis. Fluorescence lifetime imaging microscopy showed increase in NADPH in steatosis with and without ischemia reperfusion injury and could detect changes in metabolism of fluorescein to fluorescein monoglurcuronide, which was impaired in steatosis with ischemia reperfusion injury. These results concluded that the combination of multiphoton microscopy and fluorescence lifetime imaging is a promising method of assessing early stage liver damage and that it can be used to study changes in drug metabolism in the liver as an indication of liver disease and has the potential to replace the traditional static liver biopsy currently used.
TCSPC (Time-Correlated Single-Photon Counting) FLIM data with megapixel resolution can be recorded by using bh TCSPC modules in combination with new 64 bit data acquisition software. The large memory space available in the 64 bit environment allows new FLIM procedures to be used. We demonstrate the performance for applications that require imaging of a large number of cells in a single field of view, for multi-wavelength FLIM, for spatial mosaic imaging, and for recording transient changes in the fluorescence decay after a stimulation of the sample. Image quality was further improved by integrating a parallel counter channel that bypasses the timing electronics of the TCSPC module. Photon numbers from this counter are not affected by dead-time effects. Lifetime images are built up by using intensity data from the parallel counter and fluorescence decay data from the TCSPC electronics.
We present in vivo measurements of the excitation-wavelength dependence of the autofluorescence of major endogenous fluorophores of human skin with a multiphoton tomograph. For the investigation high-resolution multiphoton images at different depths inside the skin were recorded and the main fluorophores identified. In particular, for the autofluorescence of the fluorophores keratin, NAD(P)H, elastin and for the second-harmonic-generation light induced by
collagen fibers clear trends are shown.
We report on multiphoton optical imaging with a laser scanning microscope (TauMapTM, Jenlab GmbH) in combination
with two different excitation fs-lasers: a 80 MHz Ti:sapphire oscillator generating spectrally tunable 100 fs pulses and a
1 GHz Ti:sapphire oscillator producing ultra broadband 6 fs pulses. While the ultra-broadband pulses enable
simultaneous excitation of several different types of fluorophores due their large spectral width, the 100 fs pulses are
spectrally more selective and require tuning the center wavelength to cover the same excitation range. The wavelength
selectivity was confirmed in measurements with microspheres with absorption maxima in the green and blue spectral
region. Furthermore, the potential of both lasers for imaging of human skin is evaluated.