It is shown that two-photon fluorescence images can be obtained almost throughout the entire grey-matter layers of the mouse neocortex by using optically amplified femtosecond pulses. The maximum imaging depth is not limited by the available excitation power but instead by the generation of out-of focus fluorescence.

In laser-scanning microscopy, acousto-optic (AO) deflection provides a means to quickly position a laser beam to random locations throughout the field-of-view. Compared to conventional laser-scanning using galvanometer-driven mirrors, this approach increases the frame rate and signal-to-noise ratio, and reduces time spent illuminating sites of no interest. However, random-access AO scanning has not yet been combined with multi-photon microscopy, primarily because the femtosecond laser pulses employed are subject to significant amounts of both spatial and temporal dispersion upon propagation through common AO materials. Left uncompensated, spatial dispersion reduces the microscope’s spatial resolution while temporal dispersion reduces the multi-photon excitation efficacy. In previous work, we have demonstrated, 1) the efficacy of a single diffraction grating scheme which reduces the spatial dispersion at least 3-fold throughout the field-of-view, and 2) the use of a novel stacked-prism pre-chirper for compensating the temporal dispersion of a pair of AODs using a shorter mechanical path length (2-4X) than standard prism-pair arrangements. In this work, we demonstrate for the first time the use of these compensation approaches with a custom-made large-area slow-shear TeO2 AOD specifically suited for the development of a high-resolution 2-D random-access AO scanning multi-photon laser-scanning microscope (AO-MPLSM).

Fluorescence is widely used as a spectroscopic tool or for biomedical imaging. To extend these measurements to small concentrations or to fluorophores with very low quantum yield we have developed nanostructured substrates made of silver nanoparticles covered with a spacerlayer of alumina. Factors of about 200 are obtained for fluorescence enhancement with two photon excitation. Lifetime measurements reveal additional information on the decay channels induced by the nanoparticle presence.

The one photon and two photon excitation spectral properties (absorption, emission spectra, singlet lifetime) of a very efficient two photon absorber, dimethyl-pepep, have been measured in solution. The one photon excitation peak lye near 525 nm and the emission falls at 600 nm, where autofluorescence of cells is weak. The value of the singlet-triplet conversion rate, obtained by two-photon excitation fluorescence correlation spectroscopy, has a quadratic dependence on the excitation power and is comparable to that shown by the dye rhodamine. Preliminary results on stained cells from yeast Saccaromices cerevisiae and Paramecium primaurelia show that the dye preferentially stains DNA in the cell. A direct comparison with a DNA stainer, Dapi, is also performed. Some measurements of the dye functionalized to react with lysine and n-terminal residues of protein are presented. Moreover, this dye can be employed in order to follow in detail some cellular processes such as nuclei division. In vitro fluorescence titration of dimethyl-pepep with calf thymus DNA allowed to estimate the values of the dye-DNA association constant versus ionic strength, and an affinity close to that of ethidium bromide is found.

We report on a novel laser source, emitting high energy (20 nanoJoule) femtosecond pulses, in a broad spectrum (250 nanometers). This source is easily tuned from 950 to 1200 nanometers, without any laser adjustment, and delivers sub-300 femtosecond pulses with a 10 nanometers spectral width.

We present a multiphoton microscope using a Cr⁴⁺:forsterite femtosecond laser with an emission wavelength of 1260 nm for the excitation of the multiphoton processes. This wavelength is well adapted to the "optical window" in biological tissues and permits to reach higher imaging depths than systems using more conventional titanium:sapphire laser sources. The paper describes the experimental set-up and reports on first results on human cornea and skin samples.

A preliminary investigation of tissue autofluorescence and uptake of the photosensitiser protoporphyrin IX (PpIX) has been investigated using multiphoton imaging of excised oesophageal tissue. The technique has indicated that changes in collagen structure may be a potential marker for high-grade dysplasia and adenocarcinoma. Changes in the localisation of PpIX with the development of malignancy in oesophageal tissue were also visualised.

The back-illuminated Electron Multiplying Charge Coupled Device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512 x 512 frame-transfer chip format at 10 MHz pixel readout speed, in optimising a demanding ultra low-light intracellular calcium flux microscopy set-up. The arrangement employed includes a spinning confocal Nipkow disk, which whilst facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this revolutionary technology over the previous standard in ultra low light CCD detection, the GenIII+ ICCD, and also portray the advanced temporal and spatial resolution capabilities of the EMCCD.

The understanding of tumour angiogenesis and response to vascular-targeted drugs are of increasing interest in cancer research. We present 3D images of the in vivo tumour vasculature captured utilising multi-photon microscopy together with the results of manual and semi-automated delineation of the vascular network using novel in-house-developed software and algorithms. The software presented is aimed at aiding in these investigations and other problems where linear or dendritic structures are to be delineated from 3D data sets. A new algorithm, CHARM, based on a compact Hough transform and the formation of a radial map, has been used to automatically locate vessel centres and measure diameters. The robustness of this algorithm to image smoothing and noise has been investigated. Statistical information characterising the network in terms of vascular parameters as well as more complex analyses, such as fractal dimension, are now possible and examples are presented.

We developed a confocal microscope for transmitted light to visualize fine details in phase objects like unstained biological specimens. The main difficulty of confocal microscopy in transmission is the alignment of illumination and detector pinholes. This alignment was achieved by using "electronic pinholes" on the detector side. As a first step, we were able to image cells in onion skin at greater depths and with higher resolution than by using conventional microscopy.

A technique is presented to utilize time-resolved fluorescence from acceptors in donor-acceptro Forster resonance energy transfer (FRET) fluorophore pairs to improve the efficiency and accuracy of FRET lifetime determinations. This technique was applied experimentally and the results presented. This technique could be useful in fluorescence-liftime imaging systems which can measure more than one wavelength channel simultaneously.

Confocal microscopy is one of the most widely used and non-invasive tool for the investigation of biological matter. It improves the performances of the optical microscope by reducing the excitation volume and enhancing the axial resolution due to the use of high numerical aperture lenses. We have adapted an inverted confocal optical microscope to the measurement of fluorescence emission dynamics (lifetime and fluorescence polarization anisotropy). The dynamic spectroscopy measurements are obtained with phase fluorometry and are based on a modulated linearly polarized laser beam which is fed to the epifluorescence port of the microscope. We report the test of the microscope by comparing the lifetime and fluorescence polarization anisotropy decay obtained in cuvettes in the standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor is introduced in the best fit functions used in the data analysis of the decays, the results obtained on microliters volumes is comparable to those obtained in cuvettes in the standard phase fluorometer spectrometer. An example of application is reported on short DNA fragments.

Photoisomerization properties of amphiphilic stilbazolium markers are used to provoke photo-induced flip-flop in model lipid bilayer membranes. The flip-flop mechanism and dynamics are determined using simultaneous two-photon excited fluorescence and second harmonic generation microscopy. In absence of illumination, trans- is the dominant conformation, however when an illumination pulse is applied to the membrane markers, photo-induced isomerization provokes a significant increase in the cis- population, whose flip-flop rate was determined to be at least a thousand times greater than that for the trans- marker. Following the illumination pulse, all markers rapidly relax to the trans-conformation.

We have used second harmonic generation (SHG) imaging to quantify a strong intrinsic SHG-signal from cellular and subcellular muscle fibre preparations. In isolated single muscle cells, the intrinsic SHG-signal periodically follows the striation pattern and strongly depends on the sarcomere length and the polarization of the illuminating laser beam. At the subcellular level, the SHG signal seems to be located mainly at the overlapping region of the (thin) actin and (thick) myosin filaments. Thus, SHG imaging resolves the arrangement of the contractile structures with high resolution non-invasively and without chromophores. It may also allow to study dynamic molecular interactions of the motor protein myosin with actin filaments during force production and muscle shortening.

The use of chiral harmonophores in second harmonic generation (SHG) microscopy of lipid bilayers should enable one to obtain a signal even when the distribution of the chromophores is centrosymmetric. In order to determine optimal chiral molecules, we performed polarization-resolved second harmonic reflection experiments. We found that chirality must arise from an excitonic coupling rather than from an asymmetric center. We selectively labeled giant unilamellar lipid vesicles and cell membranes with such a molecule, namely an acridine substituted Troger's base, as demonstrated by two-photon-excited fluorescence microscopy. We performed preliminary SHG microscopy experiments, but the poor efficiency of the current form of our molecule does not allow us to demonstrate chirality effects.

We use coherent anti-Stokes Raman scattering (CARS) for functional imaging in microscopy. In contrast to other methods, excitation and detection are performed in a wide-field (non-confocal) setup, similar to a combination of dark field and epi-fluorescence microscopy. Thus, imaging of the whole sample is performed at once, i.e. without scanning, which promises the possibility of fast microscopy with vibrational contrast. The use of a nanosecond laser system rather than typically used pico- or femtosecond systems facilitates a high spectroscopic resolution for various organic substances.

Spectrally encoded confocal microscopy (SECM) is a novel approach for high resolution, depth-sectioned imaging of tissue microstructure. By encoding one spatial dimension in wavelength, imaging probes can be simplified enabling endoscopic implementation. The novel use of a single-optical-axis element based on high index-of-refraction prisms and a transmission grating enables the design of narrow diameter SECM devices. Images were obtained from a 10.0 mm probe with 1.1 μm transverse resolution and a 650 μm FOV.

Due to the tendencies in biological research towards multiple stainings and increased use of newly developed dyes like fluorescent proteins, the application areas of multispectral confocal imaging increase rapidly. We present here the most recent advances in this technology particularily concerning but not limited to the excitation module, the detection module, and the beamsplitter optics of multispectral confocal microscopes.

An experimental setup for fluorescence lifetime imaging (FLIM) has been combined with total internal reflection fluorescence microscopy (TIRFM) in order to detect various membrane markers within living cells. The method is established using T47D human breast cancer cells transfected by a plasmid encoding for a membrane associated yellow fluorescent protein (EYFPmem). For further measurements the mitochondrial marker rhodamine 123 (R123) as well as the membrane marker laurdan are used. With increasing concentration R123 is accumulated outside the mitochondria, in particular within the plasma membrane, whereas mitochondrial fluorescence is quenched. Fluorescence lifetime of laurdan can be used to probe membrane dynamics, in particular the phase of membrane lipids. These lipids are in a rigid gel phase at temperatures around 24°C, whereas the gel phases and a liquid crystalline phase coexist at T ≥ 30°C. This phase pattern also depends on the age and the growth phase of the cells and may play a role in the uptake of pharmaceutical agents.

A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for online detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting on a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module (TCSPC) was used to determine the fluorescence lifetime from single spots and to record lifetime images (τ-mapping). To prove and calibrate the system, the time-resolved fluorescence characteristics of the mitochondrial marker Rhodamine 123 and 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acidhexylester)- induced protoporphyrine IX (PPIX) were investigated in solution and in cell culture. Different lifetimes could be found in different cell compartiments. During illumination, the lifetimes decreased significantly. From photobleaching experiments different metabolites of 5-ALA could be correlated with different fluorescence lifetimes. In conclusion FLIM, using ps diode lasers and TCSPC techniques is a valuable method to selectively identify and localize various metabolites of fluorescent probes during laser scanning microscopy.

Two photon microscopy is a powerful tool for cells or tissues imaging. However it presents the drawback of being a laser-scanning technique leading to long time acquisition for 3D images. To preserve biological samples from too long experiments and provide a more complete spectroscopic tool we developed a time-resolved multifocal multiphoton microscope. This setup allows us to speed up the acquisition and gives both intensity and lifetime images for all multifocal points.

We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localisation of protein-protein interactions in situ. Examples of new fluorescent protein variants with enhanced properties are given and the development of FRET biosensors for simultaneous measurement of multiple intra- and inter-molecular interactions is illustrated by experimental evidence of an energy transfer cascade via multiple acceptors. The juxtaposition of interacting population and FRET efficiency is elucidated, with a priori knowledge, by multi-exponential analysis.

We report the adhesion of human erythrocyte membranes mediated by monoclonal antibodies anti-glycophorin. The distribution of the linked antibodies on membrane was identified with selective fluorescence labels. To analyze the antibody distribution on interfacial region between two cells agglutinated and on its surface, three types of fluorescence marked strategy were evaluated. The 3D images were obtained in a CellScan and Confocal Laser Scanning Microscopy CLSM. We considered the FRET signal to characterize the agglutination of Red Blood Cells (RBC) by specific monoclonal antibodies (anti-glycophorin A or B). The fluorescence labeling demonstrated that distribution of antibody on erythrocyte membranes is not homogeneous. The fluorescence intensity on contact region in the agglutinated is bigger than the intensity on exterior surface. Tentatively, we interpreted these intensity differences in terms of the mobility of antibody linked to the glycocalix on cell surface. Such mobility has a large consequence in the morphology of cellular agglutinated.

We investigated normal and cancerous human colorectal tissues (fresh thick biopsy specimens) using Olympus Confocal laser scanning biological microscope (FV300). The different layers of autofluorescence images of the specimen were captured by 488 nm laser scanning and sectioning. Optical sectioning can be performed in the vertical plane. Laser scanning can be performed in the horizontal plane. By comparing the autofluorescence image of the normal colorectal tissue with cancerous tissue, the structures of the optical sectioning image layer were found to be significantly different. We have also obtained fibrous autofluorescence image inside tissue specimen. Our investigation may help provide some useful insight to other autofluorescence research studies like laser induced autofluorescence spectra of human colorectal tissue study as a diagnosis technique for clinical application.

The peculiarities of movement of red blood cells and blood plasma in very narrow capillaries are analyzed in this paper. Microvessels with diameter about the erythrocyte size are investigated in details. Spatial distributions of velocity of scatterers in such vessels are studied. Optical model of blood microflow in the smallest capillaries is developed. Process of diffraction of strongly focused laser beam from smallest blood vessel is considered. The shapes of spectra of scattering intensity fluctuations in speckle-microscope are studied. Relations between the spectral characteristics of dynamic speckles scattered from RBC, moving in blood plasma, and the hydrodynamic characteristics of microflow are found.

Using fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) we have explored the protein-protein interactions between fluorescent protein tagged fusion proteins of the activation pathways of PKC and NFkB. We observe FRET between CFP-IκB and YFP-p65 in unstimulated cells and when treated with TNFα. We also observed a reduction of the fluorescent lifetime of CFP-IκB in the absence of YFP-p65 when TNFα is present.

The synthesis and characterization of novel heteroaromatic-based two-photon absorption (TPA) dyes for bio-conjugation is described; the new isothiocyanate and maleimide derive from a class of novel efficient quadrupolar and octupolar/branched chromophores relying on the electronic effects of electron-poor and electron-rich simple heteroaromatic rings; the new systems exhibit very large TPA cross-sections, high chemical stability, and very low photobleaching.

Intracellular ion concentration images are essential for studying the biochemical processes inside a cell and among the ions Ca²⁺ and Na⁺ are at the top of listin terms of importance. The ions become "visible" when they bind to their specific fluorescent dyes. Each fluorochrome has its own distinct absorption and emission spectra and therefore observation of two ions in a conventional epifluorescence setup would entail switching the filter sets. A mechanical change of the filter sets can be cumbersome in imaging a dynamic phenomenon in an "in vivo" experiment. Also it can lead to registration errors when overlaying the images of different ions. Here we show how the use of an electrically tunable emission filter (Varispec, CRI, USA) can help solve the problem. We describe out setup based on an inverted microscope, Varispect and a cooled high resolution CCD camera. First we show the test results on fluorescent microspheres (Molecular Probes) and then the results obtained by using Sodium Green and Fluo-4 as indicators of Na and Ca ions respectively. We also show the results obtained with different excitation sources viz. mercury lamp, super luminescent LEDS and lasers.

Fluorescence Resonance Energy Transfer (FRET) spectroscopy is an ideal tool to assess protein-protein interactions in cells. FRET occurs if suitable fluorophores are less than 10nm apart and thus may be used to probe molecular scale interactions. To obviate the need for fixation and staining, the proteins of interest can be linked to Green Fluorescent Protein (GFP) variants, which can then be expressed in intact living cells. However, FRET is inherently difficult to validate and interpret in such environments, making suitable positive controls vital. In this study cyan and yellow variants of GFP (CFP and YFP) were linked by amino acid chains of known lengths (6, 14, 16, 18, and 22 amino acids). Ratiometric FRET measurements were obtained via the acceptor photobleaching technique. The data was analyzed by new FRET analysis software developed by this group, which will be made available to the public. The evaluation method is novel in that it assesses an entire field of view for FRET efficiencies, thus making high throughput data analysis possible. A perfect candidate for FRET studies is the cyclin-CDK switch, responsible for the regulation of mitosis. Preliminary results with CyclinB1-CFP and cdk1-YFP are presented.

The aim of this work is to establish a laboratory system, that embraces different areas such as optics, electronics, signal processing and software, to make possible the application of a linear sensor as detector in a scanning optical microscope (SOM) and to evaluate the application of different signal processing techniques on axial and lateral resolution. Thus a low-cost SOM laboratory prototype with reflection epi-illuminated configuration was assembled and a stage scanning type was selected to minimize the aberrations because low-cost optical components were employed. The line illumination was achieved using a low-cost anamorphic optical lens. In this paper a discription of the optical arrangement is presented. Also the acquisition system is reported regarding the circuitry developed with a microcontroller from PIC family to readout data from a linear sensor. A brief discription of the acquisition and visualization software running in microcontroller and personal computer (PC), respectively, is also included. The preliminary results presented in this paper were attained using plane mirror object mounted in a translation stage. A Matlab program was developed to implement different routines to estimate the axial resolution and evaluate its validity for the achievement of better depth discrimination.