Dual-mode reflectance and fluorescence near-video-rate confocal microscope for architectural, morphological and molecular imaging of tissue

We have developed a near‐video‐rate dual‐mode reflectance and fluorescence confocal microscope for the purpose of imaging ex vivo human specimens and in vivo animal models. The dual‐mode confocal microscope (DCM) has light sources at 488, 664 and 784 nm, a frame rate of 15 frames per second, a maximum field of view of 300 × 250 μm and a resolution limit of 0.31 μm laterally and 1.37 μm axially. The DCM can image tissue architecture and cellular morphology, as well as molecular properties of tissue, using reflective and fluorescent molecular‐specific optical contrast agents. Images acquired with the DCM demonstrate that the system has the sub‐cellular resolution needed to visualize the morphological and molecular changes associated with cancer progression and has the capability to image animal models of disease in vivo. In the hamster cheek pouch model of oral carcinogenesis, the DCM was used to image the epithelium and stroma of the cheek pouch; blood flow was visible and areas of dysplasia could be distinguished from normal epithelium using 6% acetic acid contrast. In human oral cavity tissue slices, DCM reflectance images showed an increase in the nuclear‐to‐cytoplasmic ratio and density of nuclei in neoplastic tissues as compared to normal tissue. After labelling tissue slices with fluorescent contrast agents targeting the epidermal growth factor receptor, an increase in epidermal growth factor receptor expression was detected in cancerous tissue as compared to normal tissue. The combination of reflectance and fluorescence imaging in a single system allowed imaging of two different parameters involved in neoplastic progression, providing information about both the morphological and molecular expression changes that occur with cancer progression. The dual‐mode imaging capabilities of the DCM allow investigation of both morphological changes as well as molecular changes that occur in disease processes. Analyzing both factors simultaneously may be advantageous when trying to detect and diagnose disease. The DCM's high resolution and near‐video‐rate image acquisition and the growing inventory of molecular‐specific contrast agents and disease‐specific molecular markers holds significant promise for in vivo studies of disease processes such as carcinogenesis.     

Quantitative measurement of the resolution and sensitivity of confocal microscopes using line-scanning fluorescence correlation spectroscopy

Spatial resolution and the sensitivity to detect a fluorophore are the two most important optical parameters that characterize a confocal microscope. However, these are rather difficult to estimate quantitatively. We show that fluorescence correlation spectroscopy (FCS) provides an easy and reliable measure of these quantities. We modify existing schemes for performing FCS on a commercial confocal microscope to carry out these measurements, and provide an analysis routine that can yield the relevant quantities. Our method does not require any modification of the confocal microscope, yet it yields a robust measure of the resolution and sensitivity of the instrument.     

Double-pulse fluorescence lifetime imaging in confocal microscopy

A theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides high time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non-steady-state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime determination.     

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.     

Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology

We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.     

Three-dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.     

Quantitative comparison between full-spectrum and filter-based imaging in hyperspectral fluorescence microscopy

We implement a filterless illumination scheme on a hyperspectral fluorescence microscope to achieve full-range spectral imaging. The microscope employs polarisation filtering, spatial filtering and spectral unmixing filtering to replace the role of traditional filters. Quantitative comparisons between full-spectrum and filter-based microscopy are provided in the context of signal dynamic range and accuracy of measured fluorophores' emission spectra. To show potential applications, a five-colour cell immunofluorescence imaging experiment is theoretically simulated. Simulation results indicate that the use of proposed full-spectrum imaging technique may result in three times improvement in signal dynamic range compared to that can be achieved in the filter-based imaging.     

Direct observation of the asphaltene structure in paving-grade bitumen using confocal laser-scanning microscopy

The structure of the asphaltene phase in the bitumen is believed to have a significant effect on its rheological properties. It has traditionally been difficult to observe the asphaltene phase in unaltered samples of bitumen. The maltenes are thought to form a continuous phase in which the asphaltenes are ‘dispersed’. In this study, confocal laser‐scanning microscopy (CLSM) operating in fluorescence mode was used to examine the structure of paving‐grade Safaniya and San Joaquin bitumen. The asphaltene fraction fluoresces in the 515–545 nm wavelength range when irradiated with light with a wavelength of 488 nm. The major advantages of CLSM are that the bitumen sample requires little pretreatment or preparation that may affect the original dispersion of asphaltenes and the bitumen is observed at ambient temperature and pressure. This reduces the possibility of producing images that are not representative of the original material. CLSM was able to show the distribution of maltene and asphaltene components in bitumen. The asphaltene aggregates in the bitumen were observed to be 2–7 µm in size and formed a dispersed ‘sol’ structure in the continuous maltene matrix rather than a network ‘gel’ structure. Surprisingly, the structure and fluorescence of the asphaltene phase does not appear to alter radically upon oxidative ageing. The structure of the asphaltene phase of an AR4000 San Joaquin bitumen was found to be more homogeneous than that of Safaniya bitumen, illustrating the range of structures that can be observed in bitumens by this method.     

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.     

Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B-labelled albumin in arterial tissue

Measurements of the transport of circulating sulphorhodamine B-labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ , are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine-like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15 nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovsky's fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative-free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde-based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde-based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50 °C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.     

Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy

Monomolecular films of polymerized dimethyl-bis[pentacosadiinoic-oxyethyl] ammonium bromide (EDIPAB) provide one- and two-photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3-D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi-confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB-layers can be used as substrates for the cultivation of cells.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

Viability of endolithic micro-organisms in rocks from the McMurdo Dry Valleys of Antarctica established by confocal and fluorescence microscopy

The rocks of the McMurdo Dry Valleys desert in Antarctica harbour endolithic communities of micro‐organisms such as lichens, fungi, cyanobacteria and bacteria. Establishing the physiological status and viability of these microbial colonies in their natural microhabitat has far‐reaching implications for understanding the microbial ecology of the harsh environment of this polar desert. Here we describe the use of confocal microscopy and a specific fluorescent probe (FUN‐1) to evaluate the metabolic activity of fungal cells. Application of confocal microscopy also served to identify living and dead bacteria or cyanobacteria using the fluorescent assay reagents Live/Dead SYTO 9 and propidium iodide or SYTOX Green, respectively. In addition, through the use of epifluorescence microscopy, live/dead bacteria and cyanobacteria could be detected by estimating fluorescence from their cell components provoked by simultaneously staining with nucleic acids stains such as DAPI and SYTOX Green.     

Novel methods for the quantification of changes in actin organization in chondrocytes using fluorescent imaging and linear profiling

We present three novel reproducible methodologies for the quantification of changes in actin organization from microscope images. Striation and integrative analysis were devised for the investigation of trans-cellular filaments and F-actin localization, respectively, in response to physiological or mechanical actin-modulatory conditions. Additionally, the Parker-Qusous (PQ) formula was developed as a measure of total quantity of F-actin, independent of cell volume changes, whereby fluorescence intensity was divided by the cube root of cell volume, squared. Values obtained were quantified in Mauricean Units (Mu; pixel/μm(3)). Upon isolation, there was a 49% decrease in total F-actin fluorescence from 1.91 ± 0.16 pixel/μm(3) (Mu) to 0.95 ± 0.55 Mu, whereas upon culture, an apparent increase in total fluorescence was deemed insignificant due to an increase in average cell volume, with a rise, however, in striation units (StU) from 1 ± 1 to 5 ± 1 StU/cell, and a decrease in percentage cortical fluorescence to 30.45% ± 1.52% (P = 7.8 × 10(-5)). Freshly isolated chondrocytes exhibited a decrease in total F-actin fluorescence to 0.61 ± 0.05 Mu and 0.32 ± 0.02 Mu, 10 min posthypertonic and hypotonic challenges, respectively. Regulatory volume decrease was inhibited in the presence of REV5901 with maintenance of actin levels at 1.15 Mu. Following mechanical impact in situ, there was a reduction in total F-actin fluorescence to 0.95 ± 0.08 Mu and 0.74 ± 0.06 Mu under isotonic and hypotonic conditions, respectively, but not under hypertonic conditions. We report simple methodologies for quantification of changes in actin organization, which will further our understanding of the role of actin in various cellular stress responses. These techniques can be applied to better quantify changes in localization of various proteins using fluorescent labeling.     

Optimizing imaging parameters for the separation of multiple labels in a fluorescence image

A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image. Equations are derived that allow the calculation of the signal‐to‐noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels. We then ask how such imaging parameters have to be chosen for optimal fluorophore separation. We optimize the signal‐to‐noise ratio or optimize a newly defined ‘figure of merit’, which is a measure of efficiency in the use of emitted photons. The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information. A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected. The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching. Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.     

Quantitative imaging of green fluorescent protein in cultured cells: Comparison of microscopic techniques,use in fusion proteins and detection limits

To determine the application limits of green fluorescent protein (GFP) as a reporter gene or protein tag, we expressed GFP by itself and with fusion protein partners, and used three different imaging methods to identify GFP fluorescence. In conventional epifluorescence photomicroscopy, GFP expressed in cells could be distinguished as a bright green signal over a yellow-green autofluorescence background. In quantitative fluorescence microscopy, however, the GFP signal is contaminated by cellular autofluorescence. Improved separation of GFP signal from HeLa cell autofluorescence was achieved by the combination of confocal scanning laser microscopy using 488-nm excitation, a rapid cut-on dichroic mirror and a narrow-bandpass emission filter. Two-photon excitation of GFP fluorescence at the equivalent of ? 390 nm provided better absorption than did 488-nm excitation. This resulted in increased signal/background but also generated a different autofluorescence pattern and appeared to increase GFP photobleaching. Fluorescence spectra similar to those of GFP alone were observed when GFP was expressed as a fusion protein either with glutathione-S-transferase (GST) or with glucokinase. Furthermore, purified GST?GFP fusion protein displayed an extinction coefficient and quantum yield consistent with values previously reported for GFP alone. In HeLa cells, the cytoplasmic GFP concentration must be greater than ? 1 μM to allow quantifiable discrimination over autofluorescence. However, lower expression levels may be detectable if GFP is targeted to discrete subcellular compartments, such as the plasma membrane, organelles or nucleus.     

Extension of imaging depth in two-photon fluorescence microscopy using a long-wavelength high-pulse-energy femtosecond laser source

We experimentally demonstrate, for the first time to the best of our knowledge, two-photon fluorescence imaging with a femtosecond optical parametric amplifier. In particular, we systematically compare the imaging depths of two-photon fluorescence microscopes based on three different excitation sources, including a femtosecond oscillator, a femtosecond regenerative amplifier and the optical parametric amplifier. The results show that the optical parametric amplifier can greatly extend the penetration depth by approximately 227% as compared with that obtained with the femtosecond oscillator due to effective suppression of scattering at longer wavelength and enhanced excitation efficiency enabled by higher pulse energy.     

Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1

Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.     

Time-resolved fluorescence microscopy using an improved europium chelate BHHST for the in situ detection of Cryptosporidium and Giardia

Fluorescent immunoconjugates prepared with the europium chelate BHHCT (4,4'-bis(1',1',1',2',2',3',3'-heptafluoro-4',6'-hexanedion-6'-yl)-chlorosulfo-o-terphenyl) have previously been reported as suitable labels for time-resolved fluorescence applications. BHHCT is limited by a tendency to destabilize immunoglobulins when covalently bound to the protein at moderate to high fluorophore to protein ratios (F/P). We report a new derivative of BHHCT prepared by appending a short hydrophylic tether to the chlorosulfonate activating group on BHHCT. The new derivative, BHHST (4,4'-bis-(1',1',1',2',2',3',3'-heptafluoro-4',6'-hexanedion-6'-yl)sulfonylamino-propyl-ester-N-succinimide-ester-o-terphenyl), was activated to bind at the tether terminus with a succinimide leaving group that displayed less aggressive coupling activity and improved storage stability. BHHST has been used to prepare a stable and useful immunoconjugate with the anti-Cryptosporidium monoclonal antibody CRY104. The BHHST immunoconjugate provides more than a 10-fold enhancement in the signal to noise ratio (SNR) of labeled oocyst fluorescence over background when observed using TRFM techniques. An immunoconjugate was also prepared with BHHST and (goat) anti-mouse that effectively labeled Giardia cysts in situ. Detection of cysts with the TRFM was achieved with an 11-fold increase in SNR when a gate-delay of 60 micros was employed. The storage half-life of both immunoconjugates is extended more than 20-fold when compared to immunoconjugates prepared with BHHCT.