An optical pulse modulation system for laser interference studies in the analytical ultracentrifuge

The mode of operation and construction of a high-speed laser light modulator is presented that is designed for use with the analytical ultracentrifuge. The system may be used with continuous wave lasers having optical powers not exceeding 100 W and has an optical bandwidth from 200 nm to 800 nm. The electronic modulation circuitry described is capable of producing optical pulses of 0.8 μsec duration at a firing frequency in excess of 100,000 pps and is designed to permit the laser source to operate in either a pulsed or nonpulsed mode. The unit is extremely versatile, and its characteristics permit full advantage to be taken of pulsed laser interferometry in the ultracentrifuge.     

Multiparameter wide‐field integrated optical imaging system‐based spatially modulated illumination and laser speckles in model of tissue injuries

In this report, an integrated optical platform based on spatial illumination together with laser speckle contrast technique was utilized to measure multiple parameters in live tissue including absorption, scattering, saturation, composition, metabolism, and blood flow. Measurements in three models of tissue injury including drug toxicity, artery occlusion, and acute hyperglycemia were used to test the efficacy of this system. With this hybrid apparatus, a series of structured light patterns at low and high spatial frequencies are projected onto the tissue surface and diffuse reflected light is captured by a CCD camera. A six position filter wheel, equipped with four bandpass filters centered at wavelengths of 650, 690, 800 and 880 nm is placed in front of the camera. Then, light patterns are blocked and a laser source at 650 nm illuminates the tissue while the diffusely reflected light is captured by the camera through the two remaining open holes in the wheel. In this manner, near‐infrared (NIR) and laser speckle images are captured and stored together in the computer for off‐line processing to reconstruct the tissue's properties. Spatial patterns are used to differentiate the effects of tissue scattering from those of absorption, allowing accurate quantification of tissue hemodynamics and morphology, while a coherent light source is used to study blood flow changes, a feature which cannot be measured with the NIR structured light. This combined configuration utilizes the strengths of each system in a complementary way, thus collecting a larger range of sample properties. In addition, once the flow and hemodynamics are measured, tissue oxygen metabolism can be calculated, a property which cannot be measured independently. Therefore, this merged platform can be considered a multiparameter wide‐field imaging and spectroscopy modality. Overall, experiments demonstrate the capability of this spatially coregistered imaging setup to provide complementary, useful information of various tissue metrics in a simple and noncontact manner, making it attractive for use in a variety of biomedical applications.     

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.     

A device for the rapid measurement of molecular model co-ordinates for X-ray crystallography

A device for the rapid and accurate measurement of model molecular co-ordinates, to be used in conjunction with a Richards optical comparator, is described. The device may be operated in either a manual or automatic mode. The manual mode allows an operator to find the co-ordinates of a desired atom by optical superposition of the transmitted image of a small marker light upon the reflected image of the atom to be measured. The automatic mode allows the operator to position the marker light automatically by entering preselected co-ordinates from an electronic console. This mode of operation facilitates the rapid construction and comparison of structures the atomic co-ordinates of which are already known. The device utilizes pulsed stepping motors to position the marker light and incorporates modularized solid-state circuitry throughout. Several applications of the device are described.     

The importance of optical optimization in whole slide imaging (WSI) and digital pathology imaging

In the last 10 years, whole slide imaging (WSI) has seen impressive progress not only in image quality and scanning speed but also in the variety of systems available to pathologists. However, we have noticed that most systems have relatively simple optics axes and rely on software to optimize image quality and colour balance. While much can be done in software, this study examines the importance of optics, in particular optical filters, in WSI.Optical resolution is a function of the wavelength of light used and the numerical aperture of the lens system (Resolution = (f) wavelength/2 NA). When illumining light is not conditioned correctly with filters, there is a tendency for the wavelength to shift to longer values (more red) because of the characteristics of the lamps in common use. Most microscopes (but remarkably few WSI devices) correct for this with ND filter for brightness and Blue filter (depends on the light source) for colour correction.Using H&E slides research microscopes (Axiophot, Carl Zeiss MicroImaging, Inc. NY. Eclipse 50i., Nikon Inc. NY) at 20x, an attached digital camera (SPOT RT741 Slider Color, Diagnosis Instruments., MI USA), and a filter set, we examined the effect of filters and software enhancement on digital image quality. The focus value (as evaluated by focus evaluation software developed in house and SPOT imaging Software v4.6) was used as a proxy for image quality. Resolution of tissue features was best with the use of both the Blue and ND filters (in addition to software enhancement). Images without filters but with software enhancement while superficially good, lacked some details of specimen morphology and were unclear compared with the images with filters.The results indicate that the appropriate use of optical filters could measurably improve the appearance and resolution of WSI images.     

光动力治疗中光的有效吸收光剂量及其确定

光动力治疗中真正有效的光剂量是达到病变组织并且被组织中的光敏剂所吸收的那部分剂量,即有效吸收光剂量。明确组织中的有效吸收光剂量可以指导临床治疗,从而避免治疗剂量不足(治疗不彻底)或剂量过量(造成正常组织的热损伤)。而确定PDT中光的有效吸收剂量时,需测定组织中考察点的光辐射能流率。在目前计算和模拟组织中光辐射能流率的方法中,都需要首先确定所研究组织的光学特性参数。本文概述了常用的测定组织中光辐射能流率及组织光学特性参数的测量方法。     

大叶黄杨叶片表皮组织的聚光效应及其对叶片内部光分布的影响

利用自制的光纤微探测器研究了大叶黄杨 (EuonymusjaponicusT .)叶片内部的光分布。叶片表皮组织具有聚光效应 ,用金相砂纸磨去叶片的上表皮组织可以去除这种效应。用 6 6 0nm的红光照射叶片时 (从上表皮方向 ) ,叶片内部光量迅速下降 ,在不到 10 0 μm的路径上光量下降到初始值的 2 0 %。叶片内部光分布微分曲线说明 :叶片表皮组织的存在有利于叶片内部各组织之间光吸收的均匀化。分析叶片内部红光分布曲线 (照射上表皮方向以及照射下表皮方向 ) ,海绵组织对红光 (6 6 0nm)吸收较少 ,这可能是海绵组织的一种生理生态意义上适应性的反映     

The utility of antisera to canine growth hormone and canine prolactin for immunocytochemical staining of the dog pituitary gland

Summary Fluorescent microspectrophotometry using dichroic mirror vertical epi-illumination of tissue sections stained with the PAS reaction (periodic acid and pararosaniline Schiff reagent) provides a measure of the relative concentration of 1:2 glycols within and between tissue sections. In PAS reacted sections of agarose gel, pararosaniline Schiff fluorescence increases linearly as the concentration of agarose increases (r=0.97, p<0.05). The concentration of glycogen within liver as measured by a phenol-based tissue assay is linearly correlated with pararosaniline Schiff fluorescence of formalin fixed liver sections (r=0.87, p<0.05). These relationships are unaffected by alcina blue or hematoxylin. Heretofore the amount of color reaction as measured by densitometry at the pararosaniline absorption peak was claimed to be an unreliable indicator of the amount of reactive glycol present in tissue. Our observations indicate that when the concentration of Schiff reagent exceeds an empiric limit relative to available polysaccharides, the Schiff reagent-tissue complex reflects light at the excitation wavelength instead of fluorescing the emission spectra. This can be circumvented by using dilute pararosaniline-Schiff reagent, shortening the staining period, and lowering the temperature of the staining medium.While routine PAS staining reactions are followed by washing in running water to develop the red color seen with broad spectrum illumination, water development is unnecessary for the dye-tissue complex to fluoresce. The fluorescent emission peak and the maximum excitation peak of both developed and undeveloped pararosaniline-Schiff-reagent-tissue complexes are 645–50 nm and 540–45 nm, respectively. These spectral characteristics are not changed by binding to oxidation products of different glycoproteins or polysaccharides. Intense exposure to room light, but not 100 repetitive short (0.13 s) exposures, causes partial photodecomposition.Quantitative assessment of cytofluorescence requires definition of the optical system used to measure emission. In the microspectrophotometer employed in this study, dichroic mirrors reflect light with variable efficiency depending on wavelength from the light source to the stage, and variably block light reflected or emitted from the specimen, serving as crude barrier filters. These dichroic mirror characteristics are influenced by the exact nature of the optical coating on the surface of each individual mirror. Since the optical coating of similar mirrors may vary, the properties of individual mirrors must be considered in the interpretation of spectral data and in determining the proper optical conditions for quantification of cytofluorescence.This investigation was supported by National Institutes of Health Research Service Award IF32 NS 517701 PTHA from the National Institute of Neurological and Communicative Disorders and Stroke and the National Cancer Institute Grant R01 CA 17341     

Assessment of methanogenie activity in anaerobic digestion: Apparatus and method

An apparatus is described for the rapid measurement and recording of methanogenic activity in anaerobic fermentations, and its application is demonstrated in the evaluation of the anaerobic contact process, using pear waste. The method is based on recording the rate of manometer liquid displacement in a Warburgtype vessel by means of optical sensors, appropriate electronic circuitry, and an event marking recorder or time-interval printer. Optimum conditions for measuring methanogenic activity included a pH of 6.7–6.9, a final phosphate buffer concentration of 0.07–015M, and formic and acetic acid contents of over 500 and 200 mg/liter, respectively. In comparisons of fermenter liquid and settled effluent, methanogenic activity can be assumed to be proportional to the number of methane formers present. The apparatus should be generally useful in recording rates of gas production or consumption.     

Site of Perception of the Far-Red Inhibition of Flowering in Pharbitis nil Choisy

The site of perception of the inhibition of flowering by far-redlight (729 nm) was investigated in the short-day plant Pharbitisnil, using a specially designed optical fibre apparatus withinterference filters. The cotyledon was found to be the primarysite of perception and no inhibition was obtained when the apexalone was irradiated. However, when the apex and cotyledon weresimultaneously irradiated there was a reduction in the numberof terminal flower buds compared with the cotyledon alone. Forwhole plants, far-red light centred at 729 nm (10 nm bandwidth)was considerably more inhibitory than at 719 nm. ¹Present address: Department of Biology, Guru Nanak Dev University,Amritsar—143005, Panjab, India. (Received April 28, 1986; Accepted June 23, 1986)     

New method for spectrofluorometer monochromator wavelength calibration

A method is presented for wavelength calibration of spectrofluorometer monochromators. It is based on the distortion that the characteristic absorption bands of glass filters (holmium or didymium oxide), commonly used for calibration of spectrophotometers, introduce in the emitted fluorescence of fluorophores like indole, diphenyl hexatriene, xylene or rhodamine 6G. Those filters or a well characterized absorber with sharp bands like benzene vapor can be used for the same purpose. The wavelength calibration accuracy obtained with this method is better than 0.1 nm, and requires no modification in the geometry of the spectrofluorometer sample compartment.     

A reaction cell for simultaneous measurements of fluorescence and absorption in a hemoglobin solution at various oxygen pressures

An apparatus was constructed to carry out measurements of fluorescence, optical absorption and oxygen partial pressure in a hemoglobin or other solution simultaneously, and its performance was examined. This apparatus has a rhombiform optical cell in place of the usual square optical cell used in commercially available spectrofluorometers. Fluorescence emitted at the region near the cell surface in the solution could be detected satisfactorily and easily even if the solution had strong light absorption bands at both the excitation and the emission wavelengths in the presence of high concentrations of a chromophore. This apparatus was particularly effective for studies on the interactions of a fluorescent allosteric effector with hemoglobin at various degrees of deoxygenation. Consequently, it was proved experimentally that the fluorescence of β-naphthyl triphosphate bound to hemoglobin is completely quenched. Moreover, simultaneous and continuous measurements of the oxygen-binding equilibrium of hemoglobin and the allosteric effector-binding to hemoglobin as a function of oxygen partial pressure could be satisfactorily carried out, and it is confirmed that β-naphthyl triphosphate binds not only to deoxyhemoglobin but also to fully oxygenated hemoglobin and lowers strongly the oxygen affinity of hemoglobin as an allosteric effector.     

位相分辨偏振灵敏光学相干层析术:组织双折射特性的成像与定量分析

我们研制了一种基于光纤的位相分辨偏振灵敏光学相干层析成像系统。该系统中的偏振状态控制设量在参考臂而非光源臂上,因而使得光抵达样品的传输效率大大提高。鉴于光源的部分偏振性,入射于样品上的光含有任意偏振状态的分量,通过对参考光偏振状态的调制,就可相干地提取对应于入射光四种正交偏振状态并经样品后向散射的光信号。基于斯托克斯矢量夹角在无损光纤系统传输的变换不变性,我们能利用测量臂中光信号的斯托克斯参数来确定双折射样品深度分辨的位相延迟信息。利用所研制的偏振灵敏光学相干层析成像系统,不仅确认了韧带和软骨的双折射性质,而且定量分析了不同条件下韧带的双折射变化．研究结果表明：韧带松弛可使其双折射特性明显减弱,而韧带经拉伸后,其双折射特性的变化却不明显。     

Quantitative non-radioactive in situ hybridization. Model studies and studies on pituitary proopiomelanocortin cells after adrenalectomy

Non-radioactive in situ hybridization using biotinylated oligodeoxynucleotides and a detection protocol involving monoclonal antibiotin antibodies and the alkaline phosphatase-anti-alkaline phosphatase system was employed for quantitation by image analysis. Calibrations of the image analysis system with neutral density filters revealed that the grey levels recorded were strongly linearly correlated to the absorbance (r2 = 0.97) in the range studied in tissue specimens (0-0.8 optical density or absorbance units). Several methodological parameters, including light source stability, section thickness, probe concentration and development time were initially optimized. Model systems revealed that the grey level measured varied linearly with the logarithm of the target concentration. Moreover, histophysiological studies on adrenalectomized and sham-operated rats documented that previous biochemical data on an 8- to 10-fold increase in anterior lobe proopiomelanocortin (POMC) mRNA levels 8 days after adrenalectomy are accounted for both by an increased ACTH cell concentration and content of POMC mRNA, as well as by increases in ACTH cell sizes and cell numbers. Also in agreement with biochemical data, image analysis did not reveal significant differences between OD's of melanotrophs in adrenalectomized and sham-operated animals. To our knowledge, these data are the first to document that non-radioactive in situ hybridization can be employed for relative quantitation. A particular advantage of this approach is the good morphological definition which permits parallel analyses of densitometric values, cell sizes and cell areas/cell numbers.     

Light Scattering Properties Vary across Different Regions of the Adult Mouse Brain

Recently developed optogenetic tools provide powerful approaches to optically excite or inhibit neural activity. In a typical in-vivo experiment, light is delivered to deep nuclei via an implanted optical fiber. Light intensity attenuates with increasing distance from the fiber tip, determining the volume of tissue in which optogenetic proteins can successfully be activated. However, whether and how this volume of effective light intensity varies as a function of brain region or wavelength has not been systematically studied. The goal of this study was to measure and compare how light scatters in different areas of the mouse brain. We delivered different wavelengths of light via optical fibers to acute slices of mouse brainstem, midbrain and forebrain tissue. We measured light intensity as a function of distance from the fiber tip, and used the data to model the spread of light in specific regions of the mouse brain. We found substantial differences in effective attenuation coefficients among different brain areas, which lead to substantial differences in light intensity demands for optogenetic experiments. The use of light of different wavelengths additionally changes how light illuminates a given brain area. We created a brain atlas of effective attenuation coefficients of the adult mouse brain, and integrated our data into an application that can be used to estimate light scattering as well as required light intensity for optogenetic manipulation within a given volume of tissue.     

Measuring Hearing Organ Vibration Patterns with Confocal Microscopy and Optical Flow

A new method for visualizing vibrating structures is described. The system provides a means to capture very fast repeating events by relatively minor modifications to a standard confocal microscope. An acousto-optic modulator was inserted in the beam path, generating brief pulses of laser light. Images were formed by summing consecutive frames until every pixel of the resulting image had been exposed to a laser pulse. Images were analyzed using a new method for optical flow computation; it was validated through introducing artificial displacements in confocal images. Displacements in the range of 0.8 to 4 pixels were measured with 5% error or better. The lower limit for reliable motion detection was 20% of the pixel size. These methods were used for investigating the motion pattern of the vibrating hearing organ. In contrast to standard theory, we show that the organ of Corti possesses several degrees of freedom during sound-evoked vibration. Outer hair cells showed motion indicative of deformation. After acoustic overstimulation, supporting cells contracted. This slowly developing structural change was visualized during simultaneous intense sound stimulation and its speed measured with the optical flow technique.     

On-line optical fiber detection in a preparative free-flow electrofocusing apparatus

Many analytical isoelectric focusing (IEF) instruments are equipped with on-line detection, e.g., conductivity or UV-vis absorbance. Most of their preparative counterparts have not integrated such detection systems and thus require labor-intensive off-line analysis to quantify separation results. This paper describes the incorporation of an optical fiber based, on-line detection system that allows one to follow the evolution of the protein bands in a preparative IEF apparatus. An array of four optical fibers was designed to deliver light to the annulus of a free-flow electrophoresis apparatus, to detect the transmitted light passing through the separation media and to determine the protein concentration at vertical positions along the annulus of a vortex-stabilized focusing chamber using a 1024 bit CCD line camera. The final concentration of the major myoglobin band was 21.0 mg/mL at electric field strengths as high as 333 V/cm. Spectrophotometric analysis indicated a final concentration of 18.9 mg/mL, 10% less than that reported by the optical fibers.     

Measurement of light gradients and spectral regime in plant tissue with a fiber optic probe

A method is described in which light gradients and spectral regime can be measured within plant tissue using fiber optics. A fiber optic probe was made by modifying a single optical fiber (200 μm diameter) so that it had a light harvesting end that was a truncated tip 20–70 μm in diameter. The probe was a directional sensor with a half-band acceptance angle of 17–20°. Light measurements were made as the fiber optic probe was driven through plant tissue by a motorized micromanipulator, and the light that entered the fiber tip was piped to a spectroradiometer. By irradiating green leaf tissue of the succulent Crassula falcata L. with collimated light and inserting the probe from different directions, it was possible to measure light quality and quantity at different depths. Collimated light was scattered completely by the initial 1.0 mm of leaf tissue, which also greatly attenuated all light except the green and far-red. Light scatter contributed significantly to light quantity and had a pronounced spectral structure. Immediately beneath the irradiated surface the amount of light at 550 nm was 1.2 times that of the incident light. The light gradient declined rapidly to 0.5 times incident light at 1.4 mm depth. In contrast, the amount of light at 750 nm increased during the initial 0.5 mm to 2.9 times incident light and then declined linearly to 0.5 times incident light at the dark side of the leaf (4.5 mm). The implications of the magnitude of the contribution of light scatter to the light gradient is also discussed.     

Detection of polymeric forms of dihydroquercetin by optical absorption and light scattering

Two methods for the detection of long polymers in dihydroquercetin (DHQ) preparations has been developed. The first method is based on UV spectrophotometry. It was shown that the quantity of long polymers in aqueous solutions can be estimated by the ratio of the absorption bands at 328 and 290 nm, since the 328-nm band was attributed to the monomeric form of DHQ, whereas the 290-nm band was attributed to both the monomeric and polymeric forms. The second method is based on the high-sensitive measurement of light-scattering intensity in aqueous solutions of diluted DHQ preparations using a spectrofluorometer with crossed monochromators. It has been shown that the filtration of DHQ solutions through Millipore filters with a pore diameter of 0.05–0.45 microns makes it possible to nearly completely eliminate long polymers and their aggregates. Long polymers at high concentrations can aggregate. The longest polymers and their aggregates may be 0.1 mm in length, which leads to fluctuations in the light-scattering intensity on the second and minute time scale.     

Protein rotational motion in solution measured by polarized fluorescence depletion.

A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator generates a several-microsecond pulse of linearly polarized light which produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. When the sample is then probed with light polarized parallel to the excitation pulse, fluorescence recovers over 0-1,000 microseconds as the sum of two exponentials. One exponential corresponds to triplet decay and the other to the rotational relaxation. An exciting pulse perpendicular to the probe beam is then applied. Fluorescence recovery following this pulse is the difference of the same two exponentials. Equations for fluorescence recovery kinetics to be expected in various experimentally significant cases are derived. Least-squares analysis using these equations then permits the triplet lifetime and rotational correlation time to be determined directly from PFD data. Instrumentation for PFD measurements is discussed that permits photobleaching recovery measurements of lateral diffusion coefficients using the same microscope system. With this apparatus, both rotational and translational diffusion coefficients (Dr, Dt) were measured for EITC-labeled bovine serum albumin in glycerol solutions. Values obtained for Dr and Dt are discussed in light of both the PFD models and the experimental system.