Commercial Fluorescence Lifetime Spectrometers have long suffered from the lack of a simple, compact and relatively
inexpensive broad spectral band light source that can be flexibly employed for both quasi-steady state and time resolved
measurements (using Time Correlated Single Photon Counting [TCSPC]). This paper reports the integration of an
optically pumped photonic crystal fibre, supercontinuum source1 (Fianium model SC400PP) as a light source in
Fluorescence Lifetime Spectrometers (Edinburgh Instruments FLS920 and Lifespec II), with single photon counting
detectors (micro-channel plate photomultiplier and a near-infrared photomultiplier) covering the UV to NIR range. An
innovative method of spectral selection of the supercontinuum source involving wedge interference filters is also
discussed.
KEYWORDS: Luminescence, Single photon, Fluorescence resonance energy transfer, Data acquisition, Fluorescence anisotropy, Diffusion, Confocal microscopy, Anisotropy, Molecules, Time metrology
A new plate reader (Nanotaurus) has been developed by Edinburgh Instruments that has the principle design features of a confocal microscope and utilises the technique of Time Correlated Single Photon Counting for data acquisition. The advantages of Fluorescence Lifetime Measurements in the nanosecond time scale and analysis methods to recover lifetime parameters are discussed based on experimental data. First working assays using changes of lifetime parameters are presented that clearly demonstrate the advantages of the new instrument for biochemical assays and show strong promise for cell-based assays, by utilising the independence of lifetime parameters from sample volume and concentration.
The method of Time Correlated Single Photon Counting requires high repetitive light sources (>100kHz) with pulse widths of ideally less than approximately 20ps. While these light sources have been available for some time now in the form of Ti:Sapphire lasers, picosecond pulsed diode lasers (<90ps) and light emitting diodes (<700ps), they all have the drawback of either having no spectral tunability, or tunability over a very narrow spectral range (10nm-100nm).
While this is often sufficient for specific laboratory setups for measurements of fluorescence lifetimes, commercial Fluorescence Lifetime Spectrometers have suffered for a long time from the lack of the availability of simple, compact and relatively inexpensive broad spectral band light sources that can be employed for Time Correlated Single Photon Counting.
A new light source as an integral part of a commercial fluorescence lifetime spectrometer will be discussed that allows tunability over a wide spectral band of more than 500nm.
Alexander Savitsky, K. Lopatin, Oleg Lobanov, N. Golubeva, Marina Poroshina, Elena Chernyaeva, L. Solovieva, Eugeny Lukyanets, Maria Galpern, B. Roder, Dirk Nather
Photodynamic therapy (PDT) of malignant tumors is based on the property of some photosensitizers to be accumulated by tumour tissues'. On subsequent illumination with light of the wavelength absorbed by these photosensitizers tumour cells can be destroyed. Hematoporphyrin derivative and similar agents presently have some negative features. First, these agents do not possess optimal absorption spectrum. In particular the absorbance in the red region is not very high. Second, the composition of these agents varies for different preparations and with the storage time. Third, they do not possess a sufficient selectivity for malignant cells. Furthermore, they are also accumulated in skin causing its damage upon exposure to light. Thus at present the developing of the effective PDT photosensitizers is of great interest. Recently some of this interest has been focussed on the phthalocyanines (PC). These dyes have sufficient molar extinction coefficient (<10 M1cm1) in red region of optical spectrum (650 700 nm). They are non-toxic. Some of them are effective photosensitizers and produce singlet oxygen. They are relatively easy synthesized and resistant to chemical degradation. PC can have different groups added to the external macrocycle perifery. These groups mainly determine chemical properties and solubility. They offer possibility to bind the dyes to any other molecules, for example, proteins. The properties of naphthalocyanines (NC) are rather similar to the ones of PC, but the most efficient absorption band is in region of 750 - 800 nm. An important problem is to increase the ratio between the level of photosensitizer in malignant and normal tissues. One of the possible solutions is the use of photoimmunotoxins, i.e. conjugates of photosensitizers with monoclonal antibodies (mAb) against cancer antigens or tumor marker. Experiments with immunoglobulin-hematoporphyrin2 and dimetoxyhematoporphyrin3 as well as chiorin e6 conjugates4 showed that they retained both photochemical properties of free dyes and immunological ones of antibodies.
The luminescence emission of singlet molecular oxygen (1O2) generated by bacteriopheophytin a, a near infrared emitting photosensitizer, was measured using a new high sensitivity spectrometer system for time and spectral resolved near infrared detection. The instrument uses a low energy pulsed nitrogen laser (40 (mu) J per pulse) to optically excite the photosensitizer and is capable of a time resolution of 40 ns per data point and an instrument response function of 350 ns FWHM (full width at half maximum). The use of a low energy (and relatively low cost) source provides sufficient system sensitivity to measure time resolved spectra in the near infrared with high spectral and temporal resolution. The simultaneous detection, with high accuracy and repeatability, of both the temporal and spectral dependence of the photo-processes of 1O2 generation, especially with near infrared emitting photosensitizers, may further stimulate the current intensive investigations concerning the activity of 1O2 to bio-molecules.
Tetrapyrroles obtain very good photophysical properties making them efficient photosensitizers in photodynamic therapy. Most of them have high triplet quantum yields, long triplet lifetimes and triplet state energies suitable for singlet oxygen generation. These photophysical parameters differ depending on the chemical structure and interactions with the environment. Today a large variety of tetrapyrroles are synthetized or chemically separated from natural substances for investigation of their photophysical properties with the aim of using them in photodynamic therapy. In this paper, some general aspects of energy conversion and energy transfer of tetrapyrroles in relation to their photophysical and biophysical properties relevant for photodynamic activity are discussed. Essential optical methods for the investigation of these parameters, as well as the determination of segmental mobilities of sensitizer-carrier complexes, are discussed using the example of phthalocyanines.
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