We examine the effects of aberrations induced by a refractive index mismatch on the signal level and resolution of singlephoton (1–p) and two-photon (2–p), conventional and confocal scanning microscopes. In particular, we consider the aberrations introduced by an interface between oil/glass and water. Resolution is defined in terms of enclosed fluorescence, rather than full-width halfmaximum, revealing more useful information for heavily aberrated point spread functions (PSFs). It is shown that, at large focusing depths, the resolution of 2–p conventional and 1–p confocal microscopes are almost identical. The benefits of aberration correction are examined by removing Zernike aberration modes. With aberration correction, the best resolution is found for 1–p confocal and 2–p confocal modes. An approximation based upon geometrical optics is also introduced which shows that the axial resolution of heavily aberrated PSFs is roughly proportional to focusing depth.

In light microscopy the transverse nature of the electromagnetic field precludes a strongly focused longitudinal field component, thus confining polarization spectroscopy and imaging to two dimensions (x,y). Here we describe a simple confocal microscopy arrangement that optimizes for signal from molecules with transition dipoles oriented parallel to the optic axis. In the proposed arrangement, we not only generate a predominant longitudinally (z) polarized focal field, but also engineer the detection scheme in such a way that in a bulk of randomly oriented molecules, the microscope’s effective point-spread function is dominated by the contribution of those molecules that are oriented along the optic axis. Our arrangement not only implicitly allows for the determination of the orientation of transition dipoles of single molecules in three dimensions, but also highlights the contribution of z-oriented molecules in three-dimensional imaging.

Second harmonic generation (SHG) has been developed in our laboratories as a high-resolution nonlinear optical imaging microscopy for cellular membranes and intact tissues. SHG shares many of the advantageous features for microscopy of another more established nonlinear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce threedimensional images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It can be used to image membrane probes with high membrane specificity and displays extraordinary sensitivity in reporting membrane potential; it also has the ability to image highly ordered structural proteins without any exogenous labels.

Fluorescence resonance energy transfer (FRET) microscopy is a better method than the x-ray diffraction, nuclear magnetic resonance, or electron microscopy for studying the structure and localization of proteins under physiological conditions. In this paper, we describe four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBPa) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides twodimensional spatial distribution of steady-state protein–protein interactions. The two-photon imaging technique provides a better FRET signal (less bleedthrough and photobleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. The fluorescence lifetime methods allow us to monitor FRET signals at the moment of the protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution. This paper will provide a brief review of the above-mentioned FRET techniques.

During the last years, measurements considerably beyond the conventional ‘‘Abbe-Limit’’ of optical resolution in far field light microscopy were realized by several light microscopical approaches. Point spread function (PSF) engineering, spectral precision distance microscopy (SPDM), and related methods were used to demonstrate the feasibility of such measurements. SPDM allows the measurement of position and multiple distances between point-like fluorescent objects of different spectral signatures far below the optical resolution criterion as defined by the full width at half maximum of the PSF. Here, we report a software method to obtain online visualization of light distribution in the lateral and axial direction of any object detected in a spatially modulated illumination (SMI) microscope. This strongly facilitates routine application of SMI microscopy. The software was developed using Microsoft Visual C++ running on Windows NT. Furthermore, some aspects of the theoretical limits of the SPDM method were studied by virtual microscopy. For the case of SMI microscopy the precision of axial distance measurements was studied, taking into account photon statistics and image analysis procedures. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, precise distance measurements in the nanometer range can be determined, and that axial distances in the order of 40 nm are detectable with such precision.

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two-photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

The imaging of living cells and tissues using laser-scanning microscopy is offering dramatic insights into the spatial and temporal controls of biological processes. The availability of genetically encoded labels such as green fluorescent protein (GFP) offers unique opportunities by which to trace cell movements, cell signaling or gene expression dynamically in developing embryos. Two-photon laser scanning microscopy (TPLSM) is ideally suited to imaging cells in vivo due to its deeper tissue penetration and reduced phototoxicity; however, in TPLSM the excitation and emission spectra of GFP and its color variants [e.g., CyanFP (CFP); yellowFP (YFP)] are insufficiently distinct to be uniquely imaged by conventional means. To surmount such difficulties, we have combined the technologies of TPLSM and imaging spectroscopy to unambiguously identify CFP, GFP, YFP, and redFP (RFP) as well as conventional dyes, and have tested the approach in cell lines. In our approach, a liquid crystal tunable filter was used to collect the emission spectrum of each pixel within the TPLSM image. Based on the fluorescent emission spectra, supervised classification and linear unmixing analysis algorithms were used to identify the nature and relative amounts of the fluorescent proteins expressed in the cells. In a most extreme case, we have used the approach to separate GFP and fluorescein, separated by only 7 nm, and appear somewhat indistinguishable by conventional techniques. This approach offers the needed ability to concurrently image multiple colored, spectrally overlapping marker proteins within living cells.

In this study, we present a spectroscopic study of the entry pattern of a chemotherapeutic drug (AN-152) and its carrier hormone ([D-Lys6]LH-RH) into living cancer cells, with the help of our twophoton probes and a home-built localized microspectrofluorometer coupled with two photon laser scanning microscope (TPLSM). Due to the inherent localization ability of TPLSM, we were able to identify the drug and carrier location in different compartments of the cancer cells in vitro. The apparent doxorubicin-assisted nucleic accumulation of AN-152 suggests a possible nuclear action of the drug on cell proliferation.

Hypoxia caused by inadequate structure and function of the tumor vasculature has been found to negatively determine the prognosis of cancer patients. Hence, understanding the biological basis of tumor hypoxia is of significant clinical interest. To study solid tumor microenvironments in sufficient detail, large areas (several mm in diameter) need to be imaged at mm resolutions. We have used a novel confocal scanning laser MACROscope® (CSLM) capable of acquiring images over fields of view up to 2 cm32 cm. To demonstrate its performance, frozen sections from a cervical carcinoma xenograft were triple labeled for tissue hypoxia, blood vessels and hypoxiainducible transcription factor 1 alpha (HIF-1?), imaged using the CSLM and compared to images obtained using a standard epifluorescence microscope imaging system. The results indicate that the CSLM is a useful instrument for imaging tissue-based fluorescence at resolutions comparable to standard low-power microscope objectives.

Ablation characteristics of ultrashort laser pulses were investigated for pulse durations in the range of 130 fs–10 ps. Tissue samples used in the study were dental hard tissue (dentin) and water. We observed differences in ablation crater morphology for craters generated with pulse durations in the 130 fs–1 ps and the 5 ps–10 ps range. For the water experiment, the surface ablation and subsequent propagation of stress waves were monitored using Mach–Zehnder interferometry. For 130 fs–1 ps, energy is deposited on the surface while for longer pulses the beam penetrates into the sample. Both studies indicate that a transition occurs between 1 and 5 ps.

Ablation of human corneal tissue with 193 nm excimer laser energy generates fluorescence in the near ultraviolet and visible regions of the spectrum. The fluorescence spectra from five human corneas were collected during ablation in vitro. We find that the fluorescence spectrum changes continuously as the cornea is ablated from the epithelial surface towards the endothelium. We reduced the dimensionality of the large data set resulting from each cornea by a principal components analysis. The three most significant principal component eigenvectors suffice to describe the observed spectral evolution, and independent analysis of each tissue sample produces a similar set of eigenvectors. The evolution of the calculated eigenvector weighting factors during ablation then corresponds to the observed spectral evolution. In fact, this evolution is qualitatively consistent between corneas. We suggest that this spectral evolution offers promise as a real-time surgical feedback tool.

Background. The use of modern erbium: yttrium– aluminum–garnet (YAG) laser systems in opthalmic microsurgery requires a precise knowledge of the size and dynamics of the laser induced vapor bubbles. The aim of this work was to clarify the possibilities of controlling the vapor bubble shape and size by using an optimized fiber tip geometry for various ophthalmic applications with the erbium: YAG laser. Methods. The mid-infrared radiation of freerunning erbium: YAG laser was coupled optically into means of different low OH2 quartz fiber tips to investigate the vapor bubble formation in water by high-speed photography. The core diameter of four fiber tips ranged from 200 up to 940 µm. Fourteen fiber tips were polished at an angle graduated from 10° to 70° over the full core diameter (seven fiber tips) and over the half core diameter (seven fiber tips). Three fiber tips were produced to have a curvature at the distal end with curvature radii of 160, 230, and 420 µm. Results. The shape as well as the size of erbium: YAG laser induced vapor bubbles can be controlled systematically by using adequate fiber tip geometries. In detail, the used different angles and curvatures demonstrate that the propagation direction of the vapor bubbles can be estimated by optical modeling considering Snell’s law and the Fresnel laws at a quartzair boundary. Beside this, the size of a vapor bubble can be predetermined by choosing ideal fiber tip geometries to reduce or increase the radiant exposure at the distal end of the quartz fiber tip. Conclusions. The good possibility of controlling the shape and size of vapor bubbles offers a wider range of new applications, especially in ophthalmic microsurgery such as erbium YAG laser vitrectomy.

Pulses of high intensity laser light, when focused into transparent materials, may produce localized electron–ion plasmas through optical breakdown. By simultaneously incorporating the resulting volume of vaporized material within the focal volume of a high intensity ultrasound source, the photodisruption (1.05 µm wavelength) void served as a nucleation site for ultrasonic cavitation. Dilute suspensions of canine erythrocytes in phosphate buffered saline were exposed in a flow-through exposure chamber and the percentage of lysed cells was used as a measure of the biologically effective cavitation activity produced in the chamber. Brief (about 30 µs) acoustic emissions were detected from the photodisruption alone (indicating laser nucleation of bubbles), but the cell lysis produced was undetectable against the background. However, combined exposure greatly increased both the duration of the acoustic emissions (up to 1.5 ms) and the amount of cell lysis above an ultrasonic pressure amplitude threshold of about 4.3 MPa at 2.5 MHz. The amount of cell lysis (sometimes approaching 100%) increased with increasing ultrasonic intensity, laser pulse energy and laser PRF. Addition of 5% serum albumin enhanced the effect, apparently by stabilizing bubbles and nuclei. Photodisruptive laser nucleation of ultrasonic cavitation can provide controlled and synergistic enhancement of bioeffects.

We have determined the fluorescence characteristics of two long wavelength dyes, albumin blue 633 (AB633) and 670 (AB670), in plasma and blood to evaluate the possibility of making direct fluorescence sensing measurements in blood. Using binding and lifetime measurements we were also able to show that these dyes bind selectively to human serum albumin (HSA) in plasma and blood. By measuring changes in the mean lifetime of AB670 with changes in the HSA concentration, we showed that lifetime-based sensing can be used to monitor HSA concentrations using these albumin blue dyes. Anisotropy measurements for AB633 and AB670 in plasma and blood revealed high anisotropy values for these dyes in these media. Exploiting these high anisotropies, we were also able to determine HSA concentrations in plasma and blood mimics using changes in AB670 anisotropy with HSA concentration. These results show that, apart from being able to make fluorescence measurements directly in plasma and blood, it is possible to sense directly for specific plasma/blood components using fluorescent probes that bind preferentially to them.

Raman spectra of whole blood and oxy-hemoglobin (Hb) were measured under the same conditions with visible (514.5 nm) and near-infrared (NIR; 720 and 1064 nm) excitation, and the obtained spectra were compared in detail. The Raman spectrum of blood excited with visible light is dominated by very intense bands due to carotenoids, so that it was difficult to obtain information about Hb from the spectrum. The Raman spectra of whole blood and oxy-Hb excited with 720 nm light are very close to each other; both spectra are essentially Raman spectra of the heme chromophore that is preresonant with Q bands. Qualitative spectral analysis including band assignment and investigation of nature of resonance effect were carried out for the Raman spectra with 720 nm excitation. The spectra of whole blood and oxy-Hb excited with 1064 nm light contain contributions from nonresonance Raman spectra of the heme chromophore and Raman spectra of proteins. The 1064 nm excited spectra of blood and oxy-Hb are similar to each other but different in some features. For example, bands due to protein appear stronger in the spectrum of whole blood than in that of oxy-Hb which does not contain protein except globin part. The comparison between the 514.5, 720, and 1064 nm excited Raman spectra reveal that the excitation wavelength of 720 nm is more practical than that of visible light and 1064 nm in the Raman analysis of Hb, such as oxygenation, specially in situ measurement.

A synchrotron light source dedicated to medical applications has been designed at National Institute of Radiological Sciences. The storage ring, with circumference of 80 m, is designed for acceleration of 2.3 GeV and a stored current of 420 mA. It is equipped with two multipole wigglers to produce sufficient photon flux in a hard x-ray region required for medical applications. The purposes of the synchrotron light source are clinical performance of medical diagnoses clinically and research and development relating with medical applications. One of the most interesting applications for us is dualenergy x-ray computed tomography (CT). It gives the information about electron density of human tissue. The information plays an important role in advancing heavy-ion radiotherapy of cancers. Electron density can be derived from attenuation coefficients measured by different energy x rays. In this paper, a practical method of the dualenergy x-ray CT with synchrotron radiation is proposed with the theoretical consideration. The primitive experiment using monochromatic x rays emitted from radioisotopes proved the procedure of analysis mentioned here effective to derive electron densities from linear attenuation coefficients for two x rays of a different energy. The beamline dedicated to dual-energy x-ray CT is also proposed. It has a multipole wiggler as a light source and it mainly consists of a dual crystal monochromator and a rotating filter for attenuating photon flux of x rays and two-dimensional detector.