Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new,efficient upconverting fluorophore

Three-dimensional confocal imaging of polymer samples was achieved by the use of two-photon excited fluorescence in both positive and negative contrast modes. The fluorophore was a new and highly efficient two-photon induced upconverter, resulting in improved signal strength at low pumping power. Because of the relatively long wavelength of the excitation source (798 nm from a mode-locked Ti:Sap-phire laser), this technique shows a larger penetration depth into the samples than provided by conventional single-photon fluorescence confocal microscopy. Single-photon and two-photon images of the same area of each sample show significant differences. The results suggest the possibility of using two-photon confocal microscopy, in conjunction with highly efficient fluorophores, as a tool to study the surface, interface, and fracture in material science applications.     

4Pi-confocal images with axial superresolution

We present two-photon excitation 4Pi-confocal images of clustered fluorescence beads demonstrating three-dimensional far-field light microscopy with unprecedented resolution. For an excitation wavelength of 760 nm, the lateral and axial resolution amounts to 200 and 145 nm, respectively. The four-fold improved axial resolution is achieved by engineering the point-spread function through a suitable combination of aperture enlargement, two-photon excitation, confocalization and three-point deconvolution. In contrast to their confocal counterparts, 4Pi-confocal images do not exhibit the typical axial elongation. The axial resolution in the 4Pi-confocal images corresponds to about one-fifth of the wavelength and surpasses the lateral resolution by 25%.     

Imaging of optically thick specimen using two-photon excitation microscopy.

The in-depth imaging properties of two-photon excitation microscopy were investigated and compared with those of confocal microscopy. Confocal imaging enabled the recording of images from dental biofilm down to a depth of 40 microm, while two-photon excitation images could be recorded at depths greater than 100 microm. Two-photon excitation point spread functions (PSFs) were recorded at depths ranging from 0 to 90 microm depth using 220-nm diameter fluorescent beads immersed in water. PSFs were measured using both a high numerical aperture oil immersion objective and a water immersion objective. The experiments carried out using the oil immersion objective showed a rapid degradation of both the axial and lateral resolution due to spherical aberrations. In addition, the detected fluorescence intensity rapidly decreased as a function of depth. The experiments carried out using the water immersion objective showed no significant degradation of both the axial and lateral resolution and the fluorescence intensity.     

Isotropic high-resolution three-dimensional confocal micro-rotation imaging for non-adherent living cells

Recently, micro-rotation confocal microscopy has enabled the acquisition of a sequence of micro-rotated images of nonadherent living cells obtained during a partially controlled rotation movement of the cell through the focal plane. Although we are now able to estimate the three-dimensional position of every optical section with respect to the cell frame, the reconstruction of the cell from the positioned micro-rotated images remains a last task that this paper addresses. This is not strictly an interpolation problem since a micro-rotated image is a convoluted two-dimensional map of a three-dimensional reality. It is rather a 'reconstruction from projection' problem where the term projection is associated to the PSF of the deconvolution process. Micro-rotation microscopy has a specific difficulty. It does not yield a complete coverage of the volume. In this paper, experiments illustrate the ability of the classical EM algorithm to deconvolve efficiently cell volume despite of the incomplete coverage. This cell reconstruction method is compared to a kernel-based method of interpolation, which does not take account explicitly the point-spread-function (PSF). It is also compared to the standard volume obtained from a conventional z-stack. Our results suggest that deconvolution of micro-rotation image series opens some exciting new avenues for further analysis, ultimately laying the way towards establishing an enhanced resolution 3D light microscopy.     

4Pi-confocal microscopy of live cells

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3-fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live-cell microscopy.     

Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation

We compare conventional infrared laser based three-photon excitation with a visible laser based two-photon excitation scheme for imaging the ultraviolet fluorophore serotonin in solution and in live cells. To obtain a signal level of 1000 photons per second per mM serotonin solution, we need a back aperture power of 5 mW at 550 nm (for two-photon excitation) and 33 mW at 740 nm (for three-photon excitation). The detectivity of serotonin (defined as the concentration of serotonin that yields a signal equivalent to three times the standard deviation of the signal obtained from the buffer alone) is 12 microM for two-photon, and 220 microM for three-photon excitation. Surprisingly, for live cell imaging of vesicular serotonin in serotonergic cells, three-photon excitation appears to provide better image contrast than two-photon excitation. The origin of this is traced to the concentration-dependent shift of the serotonin emission spectrum.     

Three-dimensional microscopy migrates to the web with "PowerUp Your Microscope"

"PowerUp Your Microscope" is a software package designed and realized for the optimization of 3D optical microscopy image quality using the Internet and inverse problems computational approaches. The package is mainly devoted to 3D microscopy users, being operative for wide-field, confocal, and multiphoton microscopy. It provides the microscopy community with an extremely easy and comparatively powerful access to advanced image restoration methods. The core of the computational section is the optical system modeling and inverse deconvolution implementation, which is strongly linked to Web-based software and technology. This project constitutes a real and effective migration to the Web, extending computational approaches to image restoration to the whole microscopy user community, regardless of their background.     

Imaging of mouse experimental melanoma in vivo and ex vivo by combination of confocal and nonlinear microscopy

We investigated possibilities of the combination of the one- and two-photon excitation microscopy for examination of the experimental melanoma tissue in vivo, in mice under general anesthesia, and ex vivo on freshly harvested specimens. Our aim was to obtain sufficiently informative images of unstained tumor tissues and their modifications after hyperthermia treatment. The mouse experimental melanoma structure was studied and compared with normal tissue from the same animal by using confocal and nonlinear microscopy techniques based on (i) one-photon excitation (1PE) fluorescence, (ii) 1PE reflectance, (iii) second harmonic generation imaging, and (iv) two-photon excitation autofluorescence. We checked different spectral conditions and other settings of image acquisition, as well as combinations of the above imaging modalities, to fully exploit the potential of these techniques in the evaluation of treated and untreated cancer tissue morphology. Our approach enabled to reveal the collagen fiber network in relation with the other tissues, and to identify invasive tumor cells. It also proved to be useful for the examination of interrelationships between functional and morphological aspects based on optical properties of the tissues, especially in studies of changes between the tumor and control tissue, as well as changes induced by physical treatments, e.g., delivery of microwave hyperthermia treatment. These differences were also evaluated quantitatively, when we found out that the maximum Euler–Poincaré characteristic reflects well the melanoma morphological structure. The results showed that the proposed investigative approach could be suitable also for a direct evaluation of tissue modifications induced by clinical interventions. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Effects of aberrations and specimen structure in conventional, confocal and two-photon fluorescence microscopy

Specimen-induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as point-like, linear, planar and volume structures, when imaged by conventional, confocal and two-photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two-photon microscopes over conventional instruments when aberrations are present.     

Refractive-index-induced aberrations in two-photon confocal fluorescence microscopy

The effect of refractive index mismatch on the image quality in two-photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high-aperture objectives when the image plane is moved deeper into the sample. When exciting at 740 nm and recording the fluorescence around 460 nm in a glycerol-mounted sample using a lens of a numerical aperture of 1·4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 μm. In an aqueous sample, the same decrease is observed at a depth of 3 μm. By reducing the numerical aperture to 1·0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermat's principle. Advice is given concerning how the effects can be taken into account in practice.     

Biological imaging using fast laser scanning heterodyne differential phase confocal microscopes

Differential phase microscopy has proved invaluable in the study of live, unstained, thin biological samples because of its ability to image changes in refractive index and topography. Similarly, because of its optical sectioning capability, confocal microscopy is now a well-established technique in the study of relatively thick live biological samples. This paper describes the development and application of two differential phase heterodyne confocal microscopes, and compares their performance. The use of these systems for imaging in-vitro cell and tissue cultures is considered and compared with confocal reflection microscopy. It is demonstrated that the differential phase capability reveals subcellular structural information not readily seen in the confocal reflection images. This technique opens up the possibility of imaging thick unstained live tissues, avoiding cell damage and artefacts associated with staining procedures. Furthermore, the differential phase images can be used to provide a visual frame within which stained features can be located.     

Wavelength considerations in confocal microscopy of botanical specimens

We have used a multiple-laser confocal microscope with lines at 325, 442, 488, 514 and 633 nm to investigate optical sectioning of botanical specimens over a wide range of wavelengths. The 442-nm line allowed efficient excitation of Chromomycin A3, with minimal background autofluorescence, to visualize GC-rich heterochromatin as an aid to chromosome identification. Sequential excitation with 442- and 488-nm light enabled ratio imaging of cytosolic pH using BCECF. The red HeNe laser penetrated deep into intact plant tissues, being less prone to scattering than shorter blue lines, and was also used to image fluorescent samples in reflection, prior to fluorescence measurements, to reduce photobleaching. Chromatic corrections are more important in confocal microscope optics than in conventional microscopy. Measured focus differences between blue, green and red wavelengths, for commonly used objectives, were up to half the optical section thickness for both our multi-laser system and a multi-line single-laser instrument. This limited high-resolution sectioning at visible wavelengths caused a loss in signal. For ultraviolet excitation the focus shift was much larger and had to be corrected by pre-focusing the illumination. With this system we have imaged DAPI-stained nuclei, callose in pollen tubes using Aniline Blue and the calcium probe Indo-1.     

Two-photon microscopy: imaging in scattering samples and three-dimensionally resolved flash photolysis.

Two-photon molecular excitation microscopy has several advantages over conventional confocal fluorescence microscopy, including the ability to section deeper into scattering samples and to allow spatially resolved flash photolysis. We describe and examine the benefit of incorporating non-descanned fluorescence detection in our microscope system. In a scattering sample where almost no signal could be obtained at a depth of 50 microm with confocal detection, non-descanned detection resulted in an improvement of signal strength by more than an order of magnitude at depths >40 microm. The spatio-temporal properties of stationary spot two-photon excited flash photolysis (TPEFP) in drops of test solutions and cardiac myocytes were also examined. At input powers that produce >10% of the maximum rate of DM-nitrophen photolysis, serious photodestruction of the reporter fluorochrome (Fluo-3) at the photolysis spot occurred. At power levels of approximately 4 mW for periods <50 ms, we were able to produce small repeatable calcium release events using DM-nitrophen in cardiac myocytes, which were similar to naturally occurring calcium sparks. The properties of these artificial calcium sparks were very similar to signals obtained from drops of test solutions, suggesting that the apparent rate of calcium diffusion in myocytes is similar to the rate of diffusion of Fluo-3 in solution. Using TPEFP, we also examined the ability of a combination of EGTA and a low-affinity calcium indicator to track the time course of calcium release. Although the addition of EGTA improved the temporal fidelity of the rise of the calcium signal, it did not significantly reduce the spread of the fluorescence signal from the photolysis spot.     

3D restoration with multiple images acquired by a modified conventional microscope

A problem in high magnification microscopy is the blurring in the imaging of an object. In this article, we demonstrate a restoration technique that simultaneously makes use of the confocal image and the wide-field image. These images can be acquired by a modified conventional microscope. In front of the light-source, there is an array of pinholes. There are no pinholes at the detection plane. Instead, one or more pixels from the CCD camera are used, where the pinholes would have been. Using all pixels gives the wide-field image, but using a selected subset can give a confocal image. The array is used to speed up the process of acquiring the image. Note that the speed of acquisition is proportional to the number of pinholes. We show that the restoration from the two images can lead to a better result than using only one of the images. If this is the case, we show that a distance of 5 times the diameter of the pinholes can give the same results as a distance of 20 times after deconvolution. This offers an increase in acquisition time of a factor 16.     

In-vivo observation of cells with a combined high-resolution multiphoton-acoustic scanning microscope

We present a combined multiphoton-acoustic microscope giving collocated access to the local morphological as well as mechanical properties of living cells. Both methods relay on intrinsic contrast mechanisms and dispense with the need of staining. In the acoustic part of the microscope, a gigahertz ultrasound wave is generated by an acoustic lens and the reflected sound energy is detected by the identical lens in a confocal setup. The achieved lateral resolution is in the range of 1 mum. Contrast in the images arises mainly from the local absorption of sound in the cells related to viscose damping. Additionally, acoustic microscopy can access the sound speed as well as the acoustic impedance of the cell membrane and the cell shape, as it is an intrinsic volume scanning technique. The multiphoton image formation bases on the detection of autofluorescence due to endogenous fluorophores. The nonlinearity of two-photon absorption provides submicron lateral and axial resolution without the need of confocal optical detection. In addition, in the near-IR cell damages are drastically reduced in comparison with direct excitation in the visible or UV. The presented setup was aligned with a dedicated procedure to ensure identical image areas. Combined multiphoton/acoustic images of living myoblast cells are discussed focusing on the reliability of the method.     

Spatial control of pa-GFP photoactivation in living cells

Photoactivatable green fluorescent protein (paGFP) exhibits peculiar photo-physical properties making it an invaluable tool for protein/cell tracking in living cells/organisms. paGFP is normally excited in the violet range (405 nm), with an emission peak centred at 520 nm. Absorption cross-section at 488 nm is low in the not-activated form. However, when irradiated with high-energy fluxes at 405 nm, the protein shows a dramatic change in its absorption spectra becoming efficiently excitable at 488 nm. Confocal microscopes allow to control activation in the focal plane. Unfortunately, irradiation extends to the entire illumination volume, making impracticable to limit the process in the 3D (three-dimensional) space. In order to confine the process, we used two advanced intrinsically 3D confined optical methods, namely: total internal reflection fluorescence (TIRF) and two-photon excitation fluorescence (2PE) microscopy. TIRF allows for spatially selected excitation of fluorescent molecules within a thin region at interfaces, i.e. cellular membranes. Optimization of the TIRF optical set-up allowed us to demonstrate photoactivation of paGFP fused to different membrane localizing proteins. Exploitation of the penetration depth showed that activation is efficiently 3D confined even if limited at the interface. 2PE microscopy overcomes both the extended excitation volume of the confocal case and the TIRF constraint of operating at interfaces, providing optical confinement at any focal plane in the specimen within subfemtoliter volumes. The presented results emphasize how photoactivation by non-linear excitation can provide a tool to increase contrast in widefield and confocal cellular imaging.     

Virtual biopsy of the joint tissues using near-infrared, reflectance confocal microscopy. A pilot study

Standard noninvasive imaging techniques applied to joints provide gross morphological features, insufficient for assessing histological detail. On the other hand, biopsying is invasive, time consuming, and may involve unwanted processing artifacts. Near-infrared reflectance confocal microscopy is a technique that allows serial, high-resolution optical sectioning through intact tissues without employing exogenous fluorescent stains. The aim of this work was to evaluate the potential utility of near-infrared reflectance confocal microscopy for providing immediate histological information on meniscus, articular cartilage, epiphyseal plate, bone, muscle, and tendon. Images from near-infrared reflectance confocal microscopy were compared with mirror routine histology sections. Characteristic architectural features were readily visualized in the three dimensions of space. Additionally, the use of experimental contrast agents highlighted the localization of nuclei. Limitations include penetration depth and minor optical artifacts. In conclusion, near-infrared reflectance confocal microscopy is a useful technique for immediate, nondestructive, serial "virtual" sectioning through intact tissues, being thus a potential adjunct to current imaging techniques in orthopedics.     

Evaluating performance in three-dimensional fluorescence microscopy

In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3-D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is 'better', in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal-to-noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide-field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in-focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out-of-focus background to in-focus signal, and defines an upper limit to the useful range of the three most common modes of imaging.     

Confocal microscopy and image processing in the study of plant nuclear structure

We describe measurements of the point spread function (PSF) for a confocal microscope and compare them with the PSF for a conventional (wide-field) fluorescence microscope. In situ hybridization with probes to telomere and ribosomal rDNA sequences, combined with three-dimensional (3-D) microscopy, has been used to study interphase nuclei in root tissue of Pisum sativum and Vicia faba. Nearly all the telomeres in both species are located at the nuclear envelope, and are highly clustered in the Vicia tissues, suggesting specific binding interactions. rDNA labelling in P. sativum shows four brightly staining knobs, corresponding to condensed regions of the rDNA genes from the two pairs of nucleolar organizer genes in this species, arranged approximately tetrahedrally around each nucleolus. Deconvolution using the measured PSFs can be used to improve these images, revealing a fibrous substructure in the perinucleolar knobs, and a large amount of interconnecting internal structure, which we suggest represents rDNA both in the fibrillar centres and also more diffuse, widely dispersed rDNA. Finally we show that accurate conventional data coupled with deconvolution can produce 3-D reconstructions comparable to those obtainable with confocal microscopy, but that the clearest images are obtained by applying deconvolution to the confocal data.