Introduction
Fluorescence microscopy
Fluorescence microscopy (FM) is a technique traditionally used for determining biological structures [33]; its basic concept is summarised in Figure 1a. The biological specimen under examination is labelled with one or more fluorescent probes before being placed in the microscope. A single photon from the light source (usually a Hg lamp) has sufficient energy to excite an electron in the fluorescence moiety of the specimen-bound probe, taking it from an un-excited 'ground' state to an excited state. The excited electron subsequently decays back to its ground state and in so doing loses energy in the form of an emitted photon that has a longer wavelength than the photon from the light source. This phenomenon is known as the Stokes shift. The objective of FM is to detect only probe-emitted photons, leaving wavelengths originating from the light source invisible. An excitation filter is placed in the light's pathway to ensure the specimen is illuminated only by light with a wavelength-band that will optimally excite the specific specimen-bound probe. The microscope also contains an emission filter and a dichroic mirror that reflects the excitation light away from the filter but allows emitted photons of a specific wavelength to pass through to the detector. Specific filter settings are used when specimens have been stained with multiple probes, for instance for co-localisation of molecules of interest. To establish the precise location of a bound probe in a specimen, fluorescent images can be recorded in combination with (contrast) bright field images (Figure 2). To image live cells an inverted fluorescence microscope is used with the sample placed in a cell chamber, and cells are adhered to a glass rather than a plastic support because of the superior optical properties of glass and its lack of autofluorescence.









Confocal Laser Scanning Microscopy
Images obtained by FM can only provide two-dimensional (2-D) information in the plane of focus. However, depth - the third dimension - is also important in biological imaging [38]. As a consequence, confocal laser scanning microscopy (CLSM) became popular and increasingly important in the mid-1980s. As the name implies, the microscope uses a laser beam to supply the energy for excitation. It is emitted as a monochromatic light bundle, so that an Argon laser, for instance, emits photons with a wavelength of 488 nm. Excitation light provided by a laser is uniformly focused within a small spot on the biological sample, and a photo multiplier tube then detects the fluorescent light emitted from the sample. The CLSM also contains a mirror system to make the laser beam scan across the plane of focus of the specimen (i.e. in the x and y directions), and a pinhole aperture between the sample and the detector (Figure 3a) prevents the detection of out of focus fluorescence (Figure 3c). With the aid of computerised data storage and processing, a 2-D emission scan - also called an x-y scan - is obtained. By taking a consecutive series of x-y scans at different depths in the specimen, 3-D images can also be made. To achieve this the microscope stage is repeatedly raised or lowered by small distances (= 0.25-0.50 µm) and each time a new x-y scan is made. Eventually the complete series of images can be converted into a 3-D image by special imaging software (Movie 1; obtained by multi-photon excitation microscopy [MPEM]). Thus, CLSM provides superior resolution, due to a reduction in background information, as well as the possibility of 3-D images. Modern CLSM systems use more than one laser(line) to enable excitation of more than one fluorescent probe [49] and the technique is now widely used for 3-D localisation of multiple fluorescent reporter probes, for instance in co-localisation studies when two indicator probes emit a fluorescent signal within the same 3-D image pixel. In conventional FM, multiple probes can also be detected but only in two dimensions, thus probes that are located at different depths in a cell will appear to be at the same depth. Clearly, 3-D structure analysis is more informative for most cells in their native state.











Multi-photon excitation microscopy
Despite the considerable advantages that CLSM offers compared to conventional FM, the technique does still have its limitations (e.g. limited z-axis resolution, more pronounced photobleaching and background signal). Most of these, however, can be overcome by using multi-photon excitation microscopy (MPEM), and consequently it has become the preferred method for live cell and tissue imaging. To achieve excitation of an electron in the fluorescence moiety of the probe bound to the molecule of interest, two or more photons co-operate and must arrive within a short period of one another (10-16 seconds) to effect excitation - individually these photons have insufficient energy to achieve excitation. Following its excitation, the electron relaxes to a level from which it decays back to the ground state, just as it does in one-photon fluorescence, and in the process emits the same wavelength spectrum (Figure 1b). The energy of individual photons used in two-photon excitation is about half that of the photons used for single-photon excitation - or marginally higher due to energy loss in the two-photon event. An infra red (IR) excitation laser line (tuneable between wavelengths of 700 and 1060 nm) is used, which is remarkable because the emitted light has a shorter wavelength. To achieve excitation of a single-photon ultra violet (UV) excitation probe, which normally occurs at 358 nm, either two photons of 700 nm or three photons of approximately 1050 nm should be sufficient (Figure 4; [18, 46]).

Multi-photon excitation has numerous specific advantages in biomedical microscopy [44], most of which are described below.



No out of focus fluorescence:
Two-photon events do not occur outside the focal point of the laser so that light emission from the probe occurs only within the focal plane (Figure 3b and c). In CLSM, out of focus fluorescence does take place but an aperture placed in the pathway of emitted light prevents it being detected. The size of this aperture determines the z-axis resolution in CLSM - a small aperture gives rise to maximum z-resolution with a minimum of in focus emitted photons. In contrast, MPEM does not require such a device in the emission light-path and all fluorescence light, even after being scattered in the biological specimen, can be detected directly in the descanned mode. This feature gives MPEM much greater detection sensitivity than CLSM can offer (Figure 5; [6]).




Minimal photon damage effects
Exposure of biological material to excitation light is potentially damaging to the sample. Photons can modify molecules, an effect known as photo-damage, and produce by-products that may be toxic to the living specimen (photo-toxicity) [14]. Damage to the fluorescent probe is also possible and induces fading of the signal, known as photobleaching. There are two ways in which these effects are minimised in the MPEM system: First, the light source for MPEM is IR which means the excitation photons have low energy. In contrast, single-photon excitation works at short wavelengths (approximately half the wavelength of two-photon excitation), which give off more energy. Indeed, UV light is notorious for causing molecular damage [18]. Photo damage can occur during MPEM, at the focal point where two-photon excitation events occur, but the most modern microscopes use a femtosecond-pulsed excitation laser (with a frequency of 80 MHz) [2] which minimises exposure time (Figure 6).





Monitoring physiological processes in living cells
MPEM allows longer time-lapse experiments to be performed [18] (e.g. the investigation of physiological and metabolic processes [32, 40]) by minimising photo damage, photobleaching and photo toxicity. It can be used, for instance, to observe proliferating or differentiating cells [9] in embryos (Movie 1) or tissues; even after 24 hr in a MPEM, hamster embryos labelled with DAPI and mitotracker red do not have serious developmental problems, in contrast to similar experiments using CLSM when development of embryos was completely arrested (Figure 7; [39]).



Hamster embryos were stained with mitotracker and imaged at the 2-cell stage (Start) for either 8 or 24 hr, at intervals of 2.5 or 15 min. All stained embryos were imaged at 24 hr, which marked the end of the experiment (End). Embryos imaged at 514 nm (i.e. by single-photon excitation in a conventional CLSM) did not develop further, i.e. subjection to CLSM arrested embryo development. In contrast, embryos imaged at 1047 nm (i.e. by MPEM) developed into blastocysts. As a control, unstained embryos were subjected to the same radiation conditions (either 514 nm or 1047 nm) to ensure the effect was not due to phototoxicity by the mitotracker probe. Only control embryos imaged at an excitation wavelength of 1047 nm developed into blastocysts. The bar graph reveals that illumination of embryos at 1047 nm every 2.5 min for 24 hr did not impede blastocyst development in the presence of mitotracker. In contrast, illumination at 514 nm, 532 nm or 568 nm at 15 min intervals for a total of 8 hr and in the absence of mitotracker completely arrested development of 2-cell embryos. The viability of embryos was tested by transferring them back into the donor, and only those illuminated at 1047 nm developed into healthy offspring (the hamster depicted was from a multi_photon imaged embryo). [39]

Maximal penetration depth in biological samples
The multi-photon excitation principle allows a sample penetration depth of up to 800 µm [6, 26]. Three features are responsible for this enhanced penetration: (i) IR light penetrates the samples better than light of shorter wavelength, which scatters more easily; (ii) two-photon excitation only takes place in the focal spot and prevents out of focus bleaching of the fluorescent probe (fading of a probe reduces the depth of penetration); (iii) light emission can be detected directly in the descanned mode, giving rise to greater sensitivity compared with CLSM in which light must travel through a pinhole aperture before it reaches the fluorescence detector. The extra sensitivity allows fluorescence detection deeper in the specimen (Figure 8) and is therefore useful for analysis of oocytes [13], tissue sections thicker than can currently be used in CLSM [21, 22], optically dense materials [36] (e.g. bone or cartilage tissue [47]), intact small animals [drosophila, zebra fish and C. elegans; 23, 24], developing embryos [Movie 1, obtained by MPEM; 39, 41]) and of fresh biopsy material [19]. The latter, in particular, could be of interest to veterinary clinicians wishing to identify pathological changes in patients.


Greater spectral excitation range
Modern MPEM uses wide-spectrum pulsed excitation lasers with an excitation photon spectrum ranging from 700 to 1100 nm. Fluorescent probe excitation occurs in the spectral range from UV to IR and is superior to excitation of single-photon probes used in CLSM, for which the most commonly available laser lines are limited to 458 nm, 488 nm, 568 nm or 628 nm. This feature makes MPEM a more flexible technique. It can be used to detect unusual fluorescent probes, single-photon UV excitable probes (often used for DNA and RNA imaging [15]), as well as for ratio Ca2+ imaging [20, 42]. The technique is preferred to UV single-photon fluorescence microscopy because it is less detrimental to biological samples [34].

Live cell or tissue imaging
An inverted multi-photon excitation microscope can be equipped with a temperature and gas controlled cell incubator and with temperature-adjustable objectives to make it ideal for live imaging from a subcellular to whole organism level. Although similar experiments can be performed using a well-equipped CLSM, MPEM has superior qualities such as a longer illumination wavelength, femtosecond-pulsed laser illumination and the ability to remove out of focus fluorescence. These factors help reduce photo- damage, toxicity and bleaching of the probe and enable direct detection of descanned fluorescence, which requires lower excitation energy than CLSM. Fluorescence can also be detected to a greater depth in a sample, thereby allowing examination of biopsy specimens, embryos or small animals [25, 30, 31, 43]. For the first time it is possible to perform in situ, time-resolved biochemical research with maximum spatial resolution in 3-D (Movies 2 and 3).










Detection of molecular interactions
Molecular dynamics can be visualised using a high-powered laser pulse to create a photobleached spot (e.g. in a fluorescent-labelled membrane). Recovery of fluorescence occurs when fluorescent molecules diffuse into the dark membrane spot. Using this 'fluorescence recovery after photobleaching' (FRAP) technique, the velocity of lateral diffusion and the recovery rate can be measured (Figure 9). FRAP is important in the study of membrane dynamics (e.g. in raft formation), and the precise z-positioning of MPEM ensures that the bleached spot is limited to the membrane of interest (e.g. the plasma membrane), rather than the underlying membrane systems. There is also minimal photo-damage [4, 27].




Another way to observe molecular dynamics is to detect interactions between two fluorescent probes that together form a fluorescent resonance energy transfer (FRET) couple (Figure 10) [28]. FRET occurs only when the molecules detected by the two probes are very close to one another in the sample, i.e. when the fluorescence emitted from the two probes originates from virtually the same location in the specimen (in reality this only occurs when the two molecules interact with each other, i.e. are bound). MPEM excites the donor probe with two IR wavelength photons and the excited electron from the donor probe falls back to the ground state after emitting a photon with a probe-specific wavelength. This occurs without interaction with the acceptor probe. The acceptor probe is selected for non-excitability during two-photon excitation. However, in the event of FRET interaction between the donor and acceptor does occur. The excited (donor) electron relaxes and returns to ground energy level, at the same time releasing energy that is transferred to the acceptor probe, which subsequently uses it to excite an electron in its own fluorescent moiety. In general, the acceptor probe is excited by a photon and yields a similar amount of energy to that released by the donor probe. After relaxation, the excited (acceptor) electron also returns to ground level and a fluorescence emission that is specific to the acceptor probe is detected. Because the acceptor probe has a different colour, image analysis can reveal where in the sample the two fluorescent probes are bound to each other. The distance required for FRET to occur is so small that the phenomenon demonstrates not only co-localisation but also effective binding of the two molecules concerned. Thus, molecular interactions can be monitored in situ in specific structures from the sub-cellular to whole organism level. MPEM is very useful for FRET imaging because it eliminates out of focus fluorescence, has optimal z-resolution, causes minimal photo-damage and has low fluorescence detection sensitivity [48]. FRET allows intermolecular binding/dissociation as well as intramolecular binding (e.g. Ca2+ chelation [42]) or cleavage to be temporally visualised in living samples [37].






Fluorescent probes have characteristic fluorescent lifetimes (usually 2 to 5 nanoseconds). During this period excitation and relaxation of an electron in the fluorescent moiety of the molecule takes place with the emission of a low energy photon (Figure 10, part C). In the case of FRET, the lifetime of the donor probe is shortened due to the transfer of energy from the excited electron of the donor probe to an electron in the acceptor probe during the process of relaxation (Figure 10). A new technique, known as fluorescence lifetime imaging microscopy (FLIM), has evolved specifically to measure this feature, which occurs upon FRET (Figure 11; [1]). FLIM provides more quantitative information about FRET phenomena than spectral analyses of emitted light, because not all of the energy transferred from the donor probe will result in the emission of photons from the acceptor probe. The lifetime of fluorescence emission can be visualised within 3-D pixel resolution of the microscope [11]. The technique can also be employed to observe very rapid molecular changes in living specimens (nanosecond resolution).



Summary of applications of MPEM
The following 3-D biomedical imaging applications can be executed by MPEM:
1. 3-D biomedical imaging of specimens with low optical penetration or with large penetration distances: For example, thick (up to 1 mm) slices of brain [7] - to examine small synaptic vesicle trafficking [9] and exocytosis events [25] - and other tissues [21, 22]; small animals, (e.g. zebra fish, using GFP-tagged proteins [10], Drosophila [35], or C. elegans [23]); embryos [39]; and tissue biopsies [19] (e.g. cartilage [47] and bone tissues).

2. Long-term observation experiments with minimal molecular damage: Imaging of Ca2+ dynamics and signalling (e.g. in pre-synaptic nerve terminal turnover [12, 16], in brain tissue [20], or during the process of fertilisation [13]); imaging of the functional status of cell organelles (e.g. mitochondria in apoptotic processes [8]) and embryos; the developmental organisation of multi-cellular structures in embryos [39]; the cellular internalisation of fluorescent liposomes in drug targeting systems and drug delivery within cells [45], which can be performed on cell and tissue samples in vitro and in vivo (e.g. delivery to antigen-presenting cells at the site of inflammation [11], or to tumour cells [4]). It is also possible to follow the effects of cytoplasmic and nuclear delivery of DNA constructs using transfection systems in real-time studies [3]. Cellular processes such as intracellular transport of prohormones, release of neurotransmitters and the activation of oocytes can all be favourably imaged by MPEM.

3. High z-resolution of molecular processes with minimal out of focus fluorescence and photobleaching: Multiple photon excitation only takes place in the diffraction-limited volume of the focal point, thus reducing out of focus emission. Therefore, MPEM is superior for z-positioned local uncaging of caged components (e.g. Ca2+ and neurotransmitters [29, 5]), photobleaching for FRAP experiments [27], single-molecule spectral imaging FRET [28], and for FLIM [11], in order to show protein-protein interactions involved in the formation of signalling complexes, conformational changes or modifications such as peroxidation phosphorylation in bioactive molecules.


Conclusions
Recent advances in optical microscopy have incorporated spectroscopy to monitor the biochemical status of structures inside living cells and tissues. Molecular interactions, dynamics and processing can be observed and measured with great accuracy in 3-D, and fluorescence can be applied to living cells with exceptional sensitivity. In addition to conventional techniques used to measure fluorescence intensity, other spectroscopic protocols can be applied to image the proximity, interaction and dynamics of molecules and to detect environmental changes. A huge demand for MPEM (Figure 12) is anticipated because of its superior ability to image living cells or tissue. Minimal photo-damage allows the dynamics of biological systems to be imaged on a time scale ranging from seconds to hours [14], and tuneable, pulsed (femto-second) laser enables the selective excitation of fluorophores in the spectral range from UV to IR light. The use of a pulsed laser in MPEM makes fluorescence real-time imaging straightforward, and FRET and spectral imaging can also be implemented. By using IR photons that have lower scatter and absorption characteristics, the introduction of MPEM has also significantly improved the penetration depth of imaging techniques and thus allows fluorescence imaging of optically dense samples, such as bone and cartilage tissue. Traditional single-photon excitation technology allows imaging in layers no deeper than 25 to 50 ?m below the sample surface, whereas penetration depths of 800 ?m have been achieved using MPEM techniques. Multi-photon absorption occurs primarily at the focal point in the specimen and forms the physical basis for optical sectioning. Photobleaching, a problem in traditional confocal microscopy, is minimised in multi-photon microscopy. Moreover, uncaging or photobleaching processes can be performed in the diffraction-limited volume of the focused laser beam point. Finally, high-throughput MPEM screening [17], referred to as cellomics, is set to expand the research fields of functional proteomics and genomics, because of its ability to observe complex molecular processes in living cells, tissues and organisms.





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