An optical microsensor to measure fluorescent light intensity in biofilms

We have developed an optical microsensor to quantify fluorescent light intensity distribution in biofilms. The optical system consisted of a beam splitter, light couplers, filters and a spectrophotometer able to accept the fiberoptic cable to measure fluorescent light intensity. The emitted light, fluorescence from the biofilm, was collected at the tip of the optical microsensor and was transferred to a spectrophotometer via a fiberoptic cable. The total fluorescent light intensity was evaluated from the emission spectrum by numerical integration. The newly developed fiberoptic microsensor was tested using a Staphylococcus aureus strain producing yellow fluorescent protein (YFP) grown as biofilm. We used a 405-nm violet laser diode for excitation, and measured the emission intensity between 480 nm and 540 nm. The optical microsensor that quantifies fluorescent light intensity is a promising tool in biofilm research which often requires detection and quantification of fluorescent light intensity distribution generated by various fluorescent proteins.     

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.     

Construction of implantable optical fibers for long-term optogenetic manipulation of neural circuits

In vivo optogenetic strategies have redefined our ability to assay how neural circuits govern behavior. Although acutely implanted optical fibers have previously been used in such studies, long-term control over neuronal activity has been largely unachievable. Here we describe a method to construct implantable optical fibers to readily manipulate neural circuit elements with minimal tissue damage or change in light output over time (weeks to months). Implanted optical fibers readily interface with in vivo electrophysiological arrays or electrochemical detection electrodes. The procedure described here, from implant construction to the start of behavioral experimentation, can be completed in approximately 2-6 weeks. Successful use of implantable optical fibers will allow for long-term control of mammalian neural circuits in vivo, which is integral to the study of the neurobiology of behavior.     

生物组织的折射和折射率

光在生物组织中的传播与组织的光学性质有关。光通过组织时,光强和光的偏振状态会发生变化。而折射率是组织光学用来评价组织改变光线行进方向的基本参量。本文以菲涅耳公式为理论依据,用空气一组织界面的反射率、生物组织薄膜的反射率和生物组织反射光的倔振分量,推算生物组织的折射率。     

A method for studying histochemical reactions in intact human dentine with a polarizing incident light microscope

Localization and distribution of non-specific esterases has been studied in intact human dentine, by reflected light microscopy. The method of specimen preparation described here permits the visualization of optical sections in depth within the specimen at high optical resolution. Non-specific esterase was found deposited as discrete bands across the tubules. or as droplets, or as a diffuse microsomal variety in the dentinal tubules and in the interglobular spaces. It was possible to distinguish the droplet variety from the microsomal variety, of esterase within the same tubule, by means of a novel optical method using antiflex and differential interference contrast systems of reflected light microscopy. It was found that the coefficient of reflection of dentine diminished gradually from the enamel to the pre-dentine and was inversely related to the scattering of light in dentine. This scattering plays an important role in the formation of the image with reflected light microscopy. The reflected light microscope offers an economically attractive alternative or a supplementary mode of microscopy to the confocal scanning microscopes for studying intact dentine at varying depths.     

鼻咽组织荧光寿命影响因素研究

本文在研究了离体人体鼻咽正常组织和癌变组织的荧光寿命的基础上,实验研究了生理盐水的浓度、组织光学特性参数、激发光源的偏振性对癌变和正常鼻咽组织的荧光寿命的影响。实验结果表明：组织的光学特性参数对组织的荧光寿命有不同程度的影响;而不同浓度的生理盐水和光源的偏振性对组织的荧光寿命没有显著的影响。荧光寿命与该发射荧光的强度没有关系,只决定于局部环境,受微环境的物理化学性质因素的影响,因此荧光寿命作为人体组织癌变的检测方法,有着很好的应用前景。     

A novel near-infrared indocyanine dye-polyethylenimine conjugate allows DNA delivery imaging in vivo

Near-infrared (NIR) fluorescence light has been applied to monitor several biological events in vivo since it penetrates tissues more efficiently than visible light. Dyes exhibiting NIR fluorescence and having large Stokes shift are key elements for this promising optical imaging technology. Here, we report the synthesis of a novel conjugate between a near-infrared indocyanine dye and an organic polyamine polymer (polyethylenimine, PEI) (IR820-PEI) with high chemical stability and good optical properties. IR820-PEI absorbs at 665 nm, emits at 780 nm, and displays a large Stokes shift (115 nm). Moreover, the reported conjugate is able to bind DNA, and the delivery process can be monitored in vivo with noninvasive optical imaging techniques. These characteristics make IR820-PEI one of the most effective and versatile indocyanine dye polymeric-conjugate reported so far.     

Pitfalls in the study of steady state kinetics of enzymes: spurious inhibition patterns due to stray light errors

When reaction velocity measurements of enzyme reactions are carried out with single beam, single monochromator spectrophotometers, stray light in the spectrophotometer can produce systematic errors in the apparent velocities when highly absorbing solutions (optical density >2.0) are used. These errors can give rise to spurious “inhibition” patterns of the steady state kinetics. Because of a suspected error of this kind, this laboratory has recently reinvestigated the kinetics of glucose 6-phosphate dehydrogenase from Escherichia coli and found that the reported noncompetitive inhibition of the enzyme by DPNH is explained more readily by an unnoticed effect of stray light on the apparent reaction velocity than by a true enzyme inhibition. Methods for estimating and correcting such errors in spectrophotometers are presented in detail.     

Numerical study on the thawing process of biological tissue induced by laser irradiation

Most of the laser applications in medicine and biology involve thermal effects. The laser-tissue thermal interaction has therefore received more and more attentions in recent years. However, previous works were mainly focused on the case of laser heating on normal tissues (37 degrees C or above). To date, little is known on the mechanisms of laser heating on the frozen biological tissues. Several latest experimental investigations have demonstrated that lasers have great potentials in tissue cryopreservation. But the lack of theoretical interpretation limits its further application in this area. The present paper proposes a numerical model for the thawing of biological tissues caused by laser irradiation. The Monte Carlo approach and the effective heat capacity method are, respectively, employed to simulate the light propagation and solid-liquid phase change heat transfer. The proposed model has four important features: (1) the tissue is considered as a nonideal material, in which phase transition occurs over a wide temperature range; (2) the solid phase, transition phase, and the liquid phase have different thermophysical properties; (3) the variations in optical properties due to phase-change are also taken into consideration; and (4) the light distribution is changing continually with the advancement of the thawing fronts. To this end, 15 thawing-front geometric configurations are presented for the Monte Carlo simulation. The least-squares parabola fitting technique is applied to approximate the shape of the thawing front. And then, a detailed algorithm of calculating the photon reflection/refraction behaviors at the thawing front is described. Finally, we develop a coupled light/heat transport solution procedure for the laser-induced thawing of frozen tissues. The proposed model is compared with three test problems and good agreement is obtained. The calculated results show that the light reflectance/transmittance at the tissue surface are continually changing with the progression of the thawing fronts and that lasers provide a new heating method superior to conventional heating through surface conduction because it can achieve a uniform volumetric heating. Parametric studies are performed to test the influences of the optical properties of tissue on the thawing process. The proposed model is rather general in nature and therefore can be applied to other nonbiological problems as long as the materials are absorbing and scattering media.     

Improved autoradiographic procedure for cytogenetics: inverted coverslips and double illumination

The method described here has the advantage of presenting a clear image of both chromosomes and silver grains. Chromosomes are stained through the emulsion with Hoechst 33258. As two different sources of light are employed-epi-ultraviolet illumination and transmitted visible light-a separate photographic record of each optical plane can be obtained of chromosomes, silver grains, and chromosomes and grains together.     

Monitoring cytochrome redox changes in the mitochondria of intact cells using multi-wavelength visible light spectroscopy

We have developed an optical system based on visible light spectroscopy for the continuous study of changes in the redox states of mitochondrial cytochromes in intact mammalian cells. Cells are suspended in a closed incubation chamber in which oxygen and nitric oxide (NO) concentrations can be monitored during respiration. Simultaneously the cells are illuminated with a broad-band tungsten-halogen light source. Emergent light in the visible region (from 490-650 nm) is detected using a spectrophotometer and charge-coupled device camera system. Intensity spectra are then converted into changes in optical attenuation from a 'steady-state' baseline. The oxidised-minus-reduced absorption spectra of the mitochondrial cytochromes are fitted to the attenuation spectra using a multi-wavelength least-squares algorithm. Thus, the system can measure changes in the redox states of the cytochromes during cellular respiration. Here we describe this novel methodology and demonstrate its validity by monitoring the action of known respiratory chain inhibitors, including the endogenous signalling molecule NO, on cytochrome redox states in human leukocytes.     

Optical coherence microscopy. A technology for rapid, in vivo, non-destructive visualization of plants and plant cells

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 microm in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 x 5 x 10 microm) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a three-dimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.     

A 50-cm-path-length spectrophotometer cuvette for enzyme kinetic studies

A 10-cm cuvette that can be used in a commercial spectrophotometer and that uses internal reflections to produce a light path up to 50 cm is described. The apparent light path can be varied from 30 to 50 cm by adjusting the proportion of the entering light undergoing two, four, or more reflections. The cuvette allows use of substrate concentrations as low as 60 nm when reduced pyridine nucleotide concentrations are monitored at 340 nm in enzyme kinetic studies.     

A high performance micro-dual-wavelength-spectrophotometer (MDWS)

The dual wavelength spectrophotometer (DWS) has proven to be the most sensitive device to monitor minute optical absorbance changes, which are inaccessible to conventional single or double beam spectrophotometers. The typical set ups, e.g. extensively used for Ca2+ or phytochrome measurements, are huge, expensive and cumbrous. Therefore, a novel high performance micro-dual-wavelength spectrophotometer (MDWS) was developed. It is miniaturized and no moving parts such as vibrational mirrors or rotating filter wheels involved. Its specifications are superior compared to the conventional set up being capable of detecting minute optical changes (reflection, absorbance, transmittance) at particular wavelengths.     

Rapid melting apparatus for studying thermal denaturation of polynucleotides.

A spectrophotometer attachment is described which allows complete thermal denaturation of heating/cooling cycles (0 to 90 to 0°C) of polynucleotides to be obtained in less than 5 min. Solutions with a wide range of optical densities (0.15 to 500) can be studied and several examples are given to demonstrate the performance and application of the device.     

The reaction of protein tryptophanyl residues with reduced nicotinamide adenine dinucleotide. Structure of a model adduct.

This paper describes in detail a simple, light producing and handling system which differs greatly from the commercial unit presently used by most investigators, and which overcomes a number of its disadvantages. The apparatus is designed so that it can easily be substituted for the light producing, dispersing, and collimating section of the commercial photoelectric scanners, with no further changes in the commercial scanners except the substitution of optical flats for the collimating and condensing lenses in the bottom and top, respectively, of the rotor chamber. The most outstanding feature of this new system is that the use of a faster monochromator ($\text{f}\text{75.3}$) and a cylindrical lens theoretically increases light intensity by up to 100 times that of the commercial scanner, under otherwise identical conditions. Other advantages include the following: (1) For collecting and collimating light, two pairs of mirrors give a precell optical system focused in the radial direction at all wavelengths of light; (2) these components and the lamp are located on a table outside the centrifuge, so virtually any size of lamp can be used; (3) the entire precell optical system is a self-contained unit which, within reasonable limits, may be moved to any desired location without markedly affecting the quality of the collimated light; and (4) the optical path length has been shortened, providing less dissipation of light energy. The principles behind the selection and design of the key components are discussed. New alignment procedures and apparatus developed to aid in fast, easy, accurate alignment are described and discussed. Several components for use with a system using a computer-controlled stepping motor scanner for collection of data are also described. This system is simple enough and is documented in sufficient detail so that other interested workers, even those with little or no optics experience, can duplicate and use the system.     

Optical multichannel analyzer as a scanner for the ultracentrifuge. I. Single beam operation

The use of a commercial optical multichannel analyzer as a scanner for the ultracentrifuge is described. A uv-sensitive silicon vidicon tube serves as the light detector. The 5 by 12.5 mm surface of the vidicon is divided into 500 channels, scanned about 30 times/sec. An optical system was constructed which provided a reduced image of cell and counter-balance with the radius in the direction of channel number. The video signal from the vidicon is converted into digital data for each channel, the number of counts being proportional to the light intensity for that radial position. Storage registers are used to accumulate the counts for each channel, for any desired number of scans up to 9,999. For these studies, blue light from an H85C3 ac mercury lamp was used to illuminate the cell. Rotor and lamp pulses, visible on the real-time cathode-ray-tube monitor, were examined theoretically, and shown to have a negligible effect on the accumulated image. Only single-beam operation of the optical multichannel analyzer is described. The conversion of intensity data to absorbance as a function of radial position is described. The accuracy of the optical multichannel analyzer was verified by comparison of absorbances with values determined with a spectrophotometer.As a final test of the optical multichannel analyzer, the molecular weight of myoglobin was studied by sedimentation equilibrium. The reduced scatter in the experimental points permitted a more detailed analysis than usually performed. The final conclusion is that, even with single-beam operation at this early stage of development, the optical multichannel analyzer performs better than the double-beam, commercial scanner. Features already built into the optical multichannel analyzer lend themselves to automatic operation, either with a minicomputer or a specially constructed unit. Possible means of accomplishing double-beam and multiple cell operation are discussed.     

Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers

We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm². The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 × 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman’s layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics.Dedicated on the occasion of the 66th birthday of Professor Dr. Karl-Juergen Halbhuber     

Starch determination in plant tissues using a computerized image analysis system

Starch is the main reserve compound in woody plant species. Changes in starch content are clear indicators of a variety of plant developmental processes. Thus, carbohydrate extraction and other analytical methods have been widely used to measure changes in starch content. However, the use of these methods can be limited by the fact that starch is often compartmentalized in very small portions of tissue. While changes in these small structures can be histochemically characterized and localized under the microscope, they cannot be quantified. As an alternative, an image analysis system attached to a microscope has been developed to detect quantitative variations in starch in particular tissues or cells. The system has been successfully used to study the differences in starch content of sections from pistillar structures in apricot ( Prunus armeniaca L.). The procedure is based on the measurement of the optical density of black and white images obtained from the microscope. Two staining methods, I₂KI (potassium iodide-iodine) and PAS (periodic acid Schiff's reagent), and two embedding techniques, paraffin and JB4 plastic resin, were compared. The best results were obtained using I₂KI-stained sections of paraffin-embedded material. Since the procedures used are non-destructive for the tissues studied, additional information can be obtained, on the same section, by the subsequent use of additional stains. The method described here can be used to detect quantitative variations in starch content under the microscope in different plant tissues and thus to follow changes in starch reserves in small structures.     

Characterization of Light Lesion Paradigms and Optical Coherence Tomography as Tools to Study Adult Retina Regeneration in Zebrafish

Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.