Time-resolved fluorescence microscopes

As described in the previous section, the femtosecond fluorescence up-conversion microscope enabled us to visualize microscopic samples based on position-depen-dent ultrafast fluorescence dynamics. However, in the imaging measurements using the fluorescence up-conversion microscope, XY scanning was necessary as when using FLIM systems. To achieve non-scanning measurements of time-resolved fluorescence images, we developed another time-resolved fluorescence microscope. 

The time-resolved techniques that are usually used for FLIM are based on electronic-basis detection methods such as the time-correlated single photon counting or streak camera. Therefore, the time resolution of the FLIM system has been limited by several tens of picoseconds. However, fluorescence microscopy has the potential to provide much more information if we can observe the fluorescence dynamics in a microscopic region with higher time resolution. Given this background, we developed two types of ultrafast time-resolved fluorescence microscopes, i.e., the femtosecond fluorescence up-conversion microscope and the... 

Paraffin-embedded prostatic tissue sections (5 xm thick) are deparaffinized and rehydrated (Scorilas et al., 2000). A 5% universal tissue conditioner (Biomeda) is applied for 10 min at room temperature to block any nonspecific binding. The sections are incubated with biotinylated monoclonal mouse antihuman PSA antibody ( 2mg/l) (Diagnostic Systems Laboratories [coded 8301]) for 1 hr at 37°C. The sections are stained with (SA)Z-(biotin)x-PVA-(BCPDA)y-Eu3+ for 25 min at 37°C. After each step, the sections are rinsed briefly with 0.5 ml/1 Tween 20 solution. The slides are dried with a stream of cold air, and the resultant fluorescence is observed with a time-resolved fluorescence microscope. 

Hennink EJ, de Haas R, Verwoerd NP et al (1996) Evaluation of a time-resolved fluorescence microscope using a phosphorescent Pt-Porphine model system. Cytometry 24 312-320... 

Connally R, Veal D, Piper J (2004) Flash lamp excited time-resolved fluorescence microscope suppresses autofluorescence in water concentrates to deliver 11-fold intm ase signal to noise ratio. J Biomed Opt 9 725-734... 

FinaUy, it must be underhned that a photosensitizer can undergo an extensive relocahzation within the ceU owing to the disruption or modihcation of the initial binding sites, as documented by time-resolved fluorescence microscopic studies. This can signihcantly ampHfy the overaU photoinduced damage. 

Itaya et al,(99) have described a TIR system for obtaining time-resolved fluorescence decay curves induced by laser flash illumination of polymer films in a microscope configuration. Presumably, use of this configuration can be extended to studies on biological cells. 

Time-resolved fluorescence from sub-picosecond to the nanosecond time-scale of dye molecules like coumarins has been widely used to study solvation dynamics in liquids [1], As the dye is photoexcited, its dipole moment abruptly changes. Then by monitoring the time-dependent fluorescence energy one can have access to the solvent dynamical response to the charge reorganization in the dye. The microscopic interpretation of these experiments has greatly benefited from Molecular Dynamics (MD) studies [2], Recently, few experimental [3-5] and theoretical [6,7] works have been performed on solvation dynamics in liquid mixtures. A number of new phenomena can arise in mixtures which are not present in pure solvents, like association, mutual diffusion and preferential solvation [6]. We present here a... 

We measured the time-resolved fluorescence from an organic microcrystal, perylene, by using the newly developed fluorescence microscope. Perylene has two types of crystal structure, a-and P- perylene. In a-pcrylene, four molecules exist in a unit cell, whereas two molecules in P-perylene. We chose one a-perylene crystal under the microscope by checking the fluorescence spectrum. At room temperature, a-perylene exhibits very broad fluorescence in the visible region with the intensity maximum around 600 nm. The fluorescence located at 480 nm has been assigned to the emission of the free exciton. The free exciton is relaxed to... 

Time-resolved fluorescence spectroscopy of polar fluorescent probes that have a dipole moment that depends upon electronic state has recently been used extensively to study microscopic solvation dynamics of a broad range of solvents. Section II of this paper deals with the subject in detail. The basic concept is outlined in Figure 1, which shows the dependence of the nonequilibrium free energies (Fg and Fe) of solvated ground state and electronically excited probes, respecitvely, as a function of a generalized solvent coordinate. Optical excitation (vertical) of an equilibrated ground state probe produces a nonequilibrium configuration of the solvent about the excited state of the probe. Subsequent relaxation is accompanied by a time-dependent fluorescence spectral shift toward lower frequencies, which can be monitored and analyzed to quantify the dynamics of solvation via the empirical solvation dynamics function C(t), which is defined by Eq. (1). 

We developed two kinds of multidimensional fluorescence spectroscopic systems the time-gated excitation-emission matrix spectroscopic system and the time- and spectrally resolved fluorescence microscopic system. The former acquires the fluorescence intensities as a function of excitation wavelength (Ex), emission wavelength (Em), and delay time (x) after impulsive photoexcitation, while the latter acquires the fluorescence intensities as a function of Em, x, and spatial localization (%-, y-positions). In both methods, efficient acquisition of a whole data set is achieved based on line illumination by the laser beam and detection of the fluorescence image by a 2D image sensor, that is, a charge-coupled device (CCD) camera. 

In this chapter we have mentioned only a few of the more important future developments which can be foreseen in colloid science. Many of these will depend on the availability of modern instrumentation and of powerful computer facilities. In addition to the techniques dealt with in this chapter, mention should also be made of the contributions from greatly improved electron microscopic techniques, ultracentrifuges, and X-ray equipment. Other techniques that will become of increasing significance include dielectric measurements, electrical birefringence, and time-resolved fluorescence. 

Holzwarth [14] describes techniques for measuring and analyzing time-resolved fluorescence he discusses applications to dynamics of proteins, nucleic acids, and membranes. Time-resolved two-dimensional microscopic images show promise of revealing detailed information about the location and motions of fluorophores in cells. 

As a result of enormous development in the technology and production of pulse lasers, laser diodes, detector systems, and powerful computers in recent decades, steady-state and time-resolved fluorometers now belong to the standard equipment of biochemical and macromolecular laboratories. For example, there are apparatuses combined with microscopes that are suitable for time-resolved fluorescence measurements of individual organelles in living cells. However, the widespread use of fluorescence techniques generates certain danger, which is connected with their routine use. We would like to point out that the fluorescence spectroscopy is an indirect technique and that the interpretation of results needs great care and precaution. It almost always requires additional information on the system. 

The combination of time-resolved laser spectroscopic techniques with a laser scanning microscope opens new possibilities for the investigation of dynamical processes with high spatial resolution. This is demonstrated in [15.143b] by time-resolved fluorescence measurements of carcinoma cells compared with normal cells. 

Damage to the respiratory chain is correlated with a decrease in ATP production and a lack of certain enzymes and cytochromes. These defects can be detected by measuring the autofluorescence of flavin molecules in intact and respiratory deficient yeast strains with advanced microscopic techniques [15.114]. The combination of time-resolved laser spectroscopic techniques with a laser scanning microscope opens new possibilities for the investigation of dynamical processes with high spatial resolution. This is demonstrated in [15.114b] by time-resolved fluorescence measurements of carcinoma cells compared with normal cells. 

Polymers are not homogeneous in a microscopic scale and a number of perturbed states for a dye molecule are expected. As a matter of fact, non-exponential decay of luminescence in polymer systems is a common phenomenon. For some reaction processes (e.g, excimer and exciplex formation), one tries to fit the decay curve to sums of two or three exponential terms, since this kind of functional form is predicted by kinetic models. Here one has to worry about the uniqueness of the fit and the reliability of the parameters. Other processes can not be analyzed in this way. Examples include transient effects in diffusion-controlled processes, energy transfer in rigid matrices, and processes which occur in a distribution of different environments, each with its own characteristic rate. This third example is quite common when solvent relaxation about polar excited states occurs on the same time scale as emission from those states. Careful measurement of time-resolved fluorescence spectra is an approach to this problem. These problems and many others are treated in detail in recent books (9,11), including various aspects of data analysis. 

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