Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter ¹. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury. Open in a separate windowClick here to view.^{(76M, flv)}     

Irradiations of rabbit myofibrils with an ultraviolet microbeam. I. Effects of ultraviolet light on the myofibril components necessary for contraction

Glycerinated rabbit psoas myofibrils, F-actin, and myofibril ghosts were irradiated with ultraviolet light (UV) to investigate how UV blocks myofibril contraction. Myofibril contraction is most sensitive to 270- and 290-nm wavelength light. We irradiated I and A bands separately with 270- and 290-nm wavelength light using a UV microbeam and constructed dose-response curves for blocking sarcomere contraction. For both wavelengths, irradiations of A bands required less energy per area to block contraction than did irradiations of I bands, suggesting that the primary effects of both 270- and 290-nm wavelength light in stopping myofibril contraction are on myosin. We investigated whether the primary effect of UV in blocking I-band contraction is the depolymerization of actin by comparing the relative sensitivities of I-band contraction, F-actin depolymerization, and thin filament depolymerization to 270- and 290-nm light. We also compared the dose of UV required to depolymerize F-actin in solution with the dose needed to block I-band contraction and the dose required to alter thin filament structure in myofibril ghosts. The results confirm that UV blocks I-band contraction by depolymerizing actin. We discuss how the results might be relevant to the hypothesis that an actomyosin-based system is involved in chromosome movement.     

A circular dichroism microspectrophotometer

A microscope capable of measuring the CD of intact single eukaryotic cells, DNA microcrystals, and other microscopic structures has been constructed and tested. It can measure the CD spectra in the 200- and 800-nm wavelength range and consists of a modification to a standard Cary 60 CD machine in combination with a Zeiss uv microspectrometer. Preliminary CD spectra of red blood cells and lymphocytes are presented.     

Rhodamine-phalloidin and anti-tubulin antibody staining of spindle fibres that were irradiated with an ultraviolet microbeam

Summary We irradiated chromosomal spindle fibres in crane-fly spermatocytes with an ultraviolet microbeam of 270 nm wavelength light with total energies near those that cause actin filaments in myofibrils to depolymerize; after irradiation we stained the cells with rhodamine-labelled phalloidin and with anti-tubulin antibodies. In some cells, the irradiation reduced both phalloidin and tubulin staining of the chromosomal spindle fibres; in other cells, the irradiations reduced phalloidin staining but not tubulin staining; in yet other cells, the irradiations reduced tubulin staining but not phalloidin staining. In all irradiated cells in which phalloidin staining was reduced in the irradiated areas phalloidin staining also was reduced poleward from the irradiated areas. These results show that phalloidin staining of chromosomal spindle fibres is not dependent on the presence of kinetochore microtubules, and, therefore, that actin filaments are present in the spindle fibres in vivo. We suggest that actin filaments present in spindle fibres in vivo may be involved in causing chromosome movements during anaphase.     

High intensity solid-state UV source for time-gated luminescence microscopy.

BACKGROUND: The unique discriminative ability of immunofluorescent probes can be severely compromised when probe emission competes against naturally occurring, intrinsically fluorescent substances (autofluorophores). Luminescence microscopes that operate in the time-domain can selectively resolve probes with long fluorescence lifetimes (tau > 100 micros) against short-lived fluorescence to deliver greatly improved signal-to-noise ratio (SNR). A novel time-gated luminescence microscope design is reported that employs an ultraviolet (UV) light emitting diode (LED) to excite fluorescence from a europium chelate immunoconjugate with a long fluorescence lifetime. METHODS: A commercial Zeiss epifluorescence microscope was adapted for TGL operation by fitting with a time-gated image-intensified CCD camera and a high-power (100 mW) UV LED. Capture of the luminescence was delayed for a precise interval following excitation so that autofluorescence was suppressed. Giardia cysts were labeled in situ with antibody conjugated to a europium chelate (BHHST) with a fluorescence lifetime >500 micros. RESULTS: BHHST-labeled Giardia cysts emit at 617 nm when excited in the UV and were difficult to locate within the matrix of fluorescent algae using conventional fluorescence microscopy, and the SNR of probe to autofluorescent background was 0.51:1. However in time-gated luminescence mode with a gate-delay of 5 mus, the SNR was improved to 12.8:1, a 25-fold improvement. CONCLUSION: In comparison to xenon flashlamps, UV LEDs are inexpensive, easily powered, and extinguish quickly. Furthermore, the spiked emission of the LED enabled removal of spectral filters from the microscope to significantly improve efficiency of fluorescence excitation and capture.     

Involvement of phytochrome and a blue light photoreceptor in UV-B induced flavonoid synthesis in parsley (Petroselinum hortense Hoffm.) cell suspension cultures

Flavonoid synthesis in cell suspension cultures of parsley (Petroselinum hortense Hoffm.) occurs only after irradiation with ultraviolet light (UV), mainly from the UV-B (280–320 nm) spectral range. However, it is also controlled by phytochrome. A P_fr/P_tot ratio of approximately 20% is sufficient for a maximum phytochrome response as induced by pulse irradiation. Continuous red and far red light, as well as blue light, given after UV, are more effective than pulse irradiations. The response to blue light is considerably greater than that to red and far red light. Continuous red and blue light treatments can be substituted for by multiple pulses and can thus probably be ascribed to a multible induction effect. Continuous irradiations with red, far red and blue light also increase the UV-induced flavonoid synthesis if given before UV. The data indicate that besides phytochrome a separate blue light photoreceptor is involved in the regulation of the UV-induced flavonoid synthesis. This blue light receptor seems to require the presence of P_fr in order to be fully effective.Abbreviations HIR high irradiance response - P_fr far red absorhing form of phytochrome - P_tet total phytochrome - UV ultraviolet light     

Quinacrine fluorescence and Q-banding patterns of human chromosomes. I. Effects of varying factors.

Various factors involved in the production of "Q-bands" have been studied. It was found that a Zeiss standard WL fluorescent microscope required a shorter exposure time for photography as compared to a Zeiss photomicroscope. The minimal exposure time was obtained when the standard WL microscope was equipped with a UV light source containing a DC powered mercury burner and a concave mirror. Further, the pH and type of water used in the staining, washing and mounting of the slide were also important factors in producing clear and well differentiated "Q-bands". It also appears that the factors involved in the production of "Q-bands" effect the enhancement or quenching of fluorescence by poly d(A-T)-poly d(A-T) and salmon sperm DNA or poly dG-poly dC respectively. This preliminary report also suggests that DNA or polynucleotides with a specific base sequence may play an important role in Q-banding patterns on chromosomes.     

Application of laser optical tweezers in immunology and molecular genetics.

Optical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T cells into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis). In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the chinese hamster karyotype (CHV 79) can be easily micro-dissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.     

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated ^1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated ^4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot ^8-10. We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.     

Rapid continuous monitoring of enzyme activity in tissue sections: experience with the M85A and Zeiss UMSP-30 systems

We have previously described methods for the continuous monitoring of formazan deposition in tissue sections with a system based on the Vickers M85A microdensitometer. However, this instrument only allows the monitoring of a single field in each section. We have now developed a new system based on the Zeiss UMSP-30 microspectrophotometer. This machine is entirely computer controlled and by virtue of its fast-scanning stage allows the rapid (less than 0.5 s) sequential monitoring of multiple fields (up to 35) in each section. Thus a number of cell types may be studied simultaneously and work which used to take a full working day with the M85A system now can be performed in 45 min. As with the M85A the Zeiss system has full capability for data analysis (i.e. calculation of initial velocity rates, etc.). We have found that continuous monitoring of tissue sections by microdensitometry is a precise, sensitive and biochemically valid method of studying enzyme activity within the cellular matrix.     

Objective detection of apoptosis in rat renal tissue sections using light microscopy and free image analysis software with subsequent machine learning: Detection of apoptosis in renal tissue

Objective

The current study proposes an automated machine learning approach for the quantification of cells in cell death pathways according to DNA fragmentation.

INFLUENCE OF SUCCESSIVE AND COMBINED ULTRAVIOLET A AND B IRRADIATIONS ON MATRIX METALLOELASTASES PRODUCED BY HUMAN DERMAL FIBROBLASTS IN CULTURE

To study the cumulative influence of UV irradiations on skin matrix alterations, human skin fibroblasts were irradiated successively three-fold, at 24h intervals, with UVA (3×5J/cm²), UVB (3×8mJ/cm²), UVA plus UVB (3×5J/cm²and 3×8mJ/cm²) and the levels of 92kDa gelatinase (pro-MMP₉), 72kDa gelatinase (pro-MMP₂) and plasma-membrane elastase type protease were determined, following subsequent 24-h culture in 10% serum-containing medium. UV irradiations had only minor influence (1.4-fold increase for UVB) on secreted levels of pro-MMP₂and decreased the amount of plasma membrane elastase produced by cells. It did however, for UVA and UVB alone, induce a significant increase of 66kDa activated MMP₂production: 2.5- and 1.7-fold respectively. Such enhancement was not observed when combined irradiations were administered. UV exposure possessed a much higher influence on pro-MMP₉secretion by dermal fibroblast enhancing enzyme levels by 2.5-, 6.5- and 5-fold for UVA, UVB and UVA+UVB, respectively.     

Ultrastructural localization of endogenous calcium in the teleost retina

Summary The ultrastructural localization of endogenous calcium in the retina of adult cichlid fishOreochromis mossambicus (Teleostei) was studied using the cytochemical osmiate-bichromate method of Probst (1986). The specificity of this method for calcium localization was proven by means of EGTA treatment of ultrathin sections and electronspectroscopic-imaging technique (ESI) with an energy-filtering transmission electron microscope (CEM 902, Zeiss). Large amounts of electron-dense calcium containing deposits were found in the outer segments of rods, in the synaptic vesicles of receptor terminals and bipolar cells, in the perinuclear space of photoreceptors and in the endoplasmic reticulum of different cell types, especially in the inner segment and fibres of photoreceptor cells. In the inner plexiform layer calcium was detected in the extracellular space with greater accumulations in the synaptic cleft. Principal differences in the localization of calcium between rods and cones and between several types of synapses and vesicles are shown. The possible role of calcium in the subcellular structures of retinal cells is discussed.     

Electrical properties of a light-addressable microelectrode chip with high electrode density for extracellular stimulation and recording of excitable cells

A light-addressable microelectrode chip with 3600 TiN electrodes was fabricated. Amorphous silicon (a-Si:H) serves as a photo conductor. The electrodes on the chip are addressed by a laser spot and electrical properties of the system are determined. DC measurements show a dark to bright dynamic of 10(6)-10(7). The AC impedance dynamic @ 1 kHz/100 mV and thus the signal-to-noise-ratio is determined to 60. This value is quite sufficient for electrophysiological measurements. For the first time, recordings from cardiac myocytes are reported using the principle of light-addressing. Measurements were done with a standard laser scan microscope (Zeiss LSM 410).     

Automated image acquisition and processing using a new generation of 4K x 4K CCD cameras for cryo electron microscopic studies of macromolecular assemblies

We have previously reported the development of AutoEM, a software package for semi-automated acquisition of data from a transmission electron microscope. In continuing efforts to improve the speed of structure determination of macromolecular assemblies by electron microscopy, we report here on the performance of a new generation of 4 K CCD cameras for use in cryo electron microscopic applications. We demonstrate that at 120 kV, and at a nominal magnification of 67000 x, power spectra and signal-to-noise ratios for the new 4 K CCD camera are comparable to values obtained for film images scanned using a Zeiss scanner to resolutions as high as approximately 1/6.5A(-1). The specimen area imaged for each exposure on the 4 K CCD is about one-third of the area that can be recorded with a similar exposure on film. The CCD camera also serves the purpose of recording images at low magnification from the center of the hole to measure the thickness of vitrified ice in the hole. The performance of the camera is satisfactory under the low-dose conditions used in cryo electron microscopy, as demonstrated here by the determination of a three-dimensional map at 15 A for the catalytic core of the 1.8 MDa Bacillus stearothermophilus icosahedral pyruvate dehydrogenase complex, and its comparison with the previously reported atomic model for this complex obtained by X-ray crystallography.     

Peptide stimulation of phagocytosis in Amoeba proteus

Summary Normal articular cartilages from the weightbearing areas of the femoral condyles of the knee joints of 11 patients (3–20 years old) and of 35 Schwarzkopf sheep (3 months to 2 years old) were studied using the electron microscope. The study has shown that the matrix of normal articular cartilage is not only composed of collagen fibrils and proteoglycans, but also contains two types of elastic system fibres. Small elastic fibres can be identified in the superficial and lower radiate zones of cartilage of man and sheep. Similar to elastic fibres in other tissues, they consist of a central amorphous core and are surrounded by aggregates of 10 nm microfibrils. Another type of elastic system fibres, oxytalan fibres, are found in the intermediate and upper radiate zones of the articular cartilage.     

Preparation and imaging of nuclear spreads from cells of the zebrafish embryo

We describe a method for preparing nuclear spreads from cells of live, unfixed zebrafish embryos at the late-gastrula (∼8000 cell) stage of development. The method consists of a sequence of four steps: (1) a slow, gentle lysis, in low to moderate salt concentration, of cells and then nuclei, to release DNA-containing fibres; (2) spreading of the released fibres by a transverse fluid flow; (3) electrostatic, and possibly also covalent, attachment of the spread fibers to poly(l-lysine)-coated glass microscope slides; and (4) continued incubation to produce periodic cleavage of the DNA within the fibres, apparently through activation of endogenous nucleases. The nuclear spreads are imaged with epifluorescence, at a spatial resolution approaching the Rayleigh limit (∼230 nm for blue light). The epifluorescent signal is provided from Hoechst 33258 bound specifically to the DNA, from a dye-coupled antibody conjugate bound specifically to histone H1 in the fibres, or from a DNA nick end-labelling assay. The spontaneous cleavage of DNA-containing fibres in step (4) of the above procedure can be blocked by the chelating agents EGTA and EDTA, by the caspase-2,3,7 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde, and by the caspase-1,4,5 inhibitors N-acetyl-Tyr-Val-Ala-Asp-aldehyde and N-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone. These data suggest that the spontaneous cleavage of fibres is catalysed by nucleases that become activated through a caspase-mediated mechanism. The involvement of caspase-dependent nucleases would suggest that an apoptosis pathway is activated in the spreads during their prolonged incubation. If bona fide apoptosis is induced in living zebrafish embryos by treatment with camptothecin (a topoisomerase I poison), and then nuclear spreads are prepared, we observe a similar fragmentation of the spread fibres. However, in this case the fragmentation is more rapid and complete. We hypothesize that, during the early phase of apoptosis, one or more endogenous nucleases are activated by a caspase-mediated mechanism. The nuclease(s) then specifically recognize and cleave a susceptible, periodically repeating feature of interphase chromatin. Received: 19 September 1997; in revised form: 14 November 1997 / Accepted: 15 November 1997     

Neuroprotective effects of a new skin care formulation following ultraviolet exposure

Background: Chronic ultraviolet (UV) exposure is a major environmental factor involved in extrinsic skin ageing (photo‐ageing). Skin nerve fibres are significantly reduced in number following UV irradiation and new skincare compounds with neuroprotective effects are thus highly warranted. Objectives: We developed a new skincare formulation from a plant extract and evaluated its neuroprotective effects of ex vivo UV irradiation. Materials and methods: The new skincare emulsion was formulated from Echinacea purpurea extract and was enriched with antioxidants (patent no. PROV020110087075). Skin samples were obtained from 20 healthy patients enrolled for plastic surgery and were immediately treated with placebo (SPF 15) or test emulsions. Skin samples were exposed to UVA and UVB for 60 min. Nerve fibres were identified by immunofluorescence using a monoclonal antibody, anti‐human CD56. Cell damage was quantified by image analysis. Results: UVA and UVB significantly reduced (40–60%) densities of nerve endings in control samples treated with placebo (P < 0.001). Samples treated with test emulsion completely blocked UV‐related effects on skin nerve endings. These neuroprotective effects were similarly observed regardless of age or tissue analysed (breast versus abdomen). Conclusions: Our new skincare formulation obtained from E. purpurea provides important neuroprotective effects of UV irradiation and could be used together with SPFs to prevent chronic deleterious effects of solar exposure.     

Enhanced survival by photoreactivation and liquid holding following UV damage of TN-368 insect cells

These studies demonstrate that the TN-368 lepidopteran insect cell line, which is extremely resistant to the lethal effects of ionizing radiation, is also quite resistant to 254-nm ultraviolet light. While resistance to ionizing radiation in TN-368 cells has been associated with superior DNA repair processes, previous findings have indicated no correlation between survival ability and amount of unscheduled DNA synthesis in response to ultraviolet light. The present studies were undertaken to define the TN-368 ultraviolet light survival response, the ability of the cells to repair UV-induced damage by photoreactivation, the capacity of the cells to undergo UV repair during liquid holding in the dark, and the relationship between photoreactivation and liquid-holding recovery. Survival was assayed by colony formation. 254-nm irradiations were performed using germicidal lamps and photoreactivation was accomplished using black lights. Photoreactivable sectors of UV damage at 50 and 10% survival are 0.65 and 0.68, respectively. Survival responses, both with and without photoreactivation, have a small initial shoulder followed by an exponential region, and finally the curves continue to decrease but with decreasing slope. F0, Fq, and extrapolation number for the exponential portion of the curves are 77.5 J/m2, 16.8 J/m2, and 1.7 for non-photoreactivated cells and 234 J/m2, 56.1 J/m2, and 1.7 for those exposed to photoreactivating light. In the primarily exponential survival region, the fluences required to produce equivalent levels of survival in photoreactivated cells range from approximately 10.8 to 23.3 times as great as cells receiving UV light alone. The maximum survival enhancement of cells maintained under liquid-holding conditions over cells plated immediately following 100-400 J/m2 irradiations appears to be about 2-fold and occurs at 3-6 h of holding. Photoreactivation alone has a greater enhancement of survival than when photoreactivation follows liquid holding, but when liquid holding follows photoreactivation, the enhancement surpasses that of photoreactivation alone.     

Analysis of the EBT3 Gafchromic film irradiated with 6 MV photons and 6 MeV electrons using reflective mode scanners

We explore in our study the effects of electrons and X-rays irradiations on the newest version of the Gafchromic EBT3 film. Experiments are performed using the Varian “TrueBeam 1.6” medical accelerator delivering 6 MV X-ray photons and 6 MeV electron beams as desired. The main interest is to compare the responses of EBT3 films exposed to two separate beams of electrons and photons, for radiation doses ranging up to 500 cGy. The analysis is done on a flatbed EPSON 10000 XL scanner and cross checked on a HP Scanjet 4850 scanner. Both scanners are used in reflection mode taking into account landscape and portrait scanning positions. After thorough verifications, the reflective scanning method can be used on EBT3 as an economic alternative to the transmission method which was also one of the goals of this study. A comparison is also done between single scan configuration including all samples in a single A4 (HP) or A3 (EPSON) format area and multiple scan procedure where each sample is scanned separately on its own. The images analyses are done using the ImageJ software. Results show significant influence of the scanning configuration but no significant differences between electron and photon irradiations for both single and multiple scan configurations. In conclusion, the film provides a reliable relative dose measurement method for electrons and photons irradiations in the medical field applications.