Axioskop epifluorescence microscope

Manufactured by Zeiss

Sourced in Germany

The Axioskop epifluorescence microscope is a high-performance laboratory microscope designed for fluorescence imaging applications. It features a sturdy, ergonomic design and advanced optics to provide clear, high-quality images. The microscope is equipped with a range of illumination options, including LED and mercury vapor lamps, to accommodate various fluorescent labeling techniques. Its modular design allows for easy customization and upgrading to meet the specific needs of researchers and scientists.

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Axioskop 2 phase-contrast/epifluorescence microscope Axioskop 2 Plus epifluorescence microscope Axioskop 2 epifluorescence microscope Axioskop 2 upright epifluorescence microscope Axioskop epifluorescence microscope filter I Axioskop microscope Epifluorescence microscope Axioskop

23 protocols using axioskop epifluorescence microscope

Quinacrine-based Yeast Viability Assay

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Approximately 1 × 10⁸ cells were harvested at indicated time points after induction of expression and washed with 500 μL of YEPD containing 100 mM HEPES (pH 7.6). After centrifugation, the pellet was resuspended in 500 μL of YEPD with 100 mM HEPES (pH 7.6) and 400 μM quinacrine. After incubation for 10 min at 28°C and 145 rpm, samples were incubated 5 min on ice. After centrifugation, cells were washed twice in 500 μL ice-cold HEPES buffer supplemented with 2% D-glucose. For PI co-staining, 500 μL of HEPES buffer with 2% D-glucose and 100 μg/L PI was added and incubated 10 min in the dark on ice. Cells were analyzed using a Zeiss Axioskop epifluorescence microscope. For quantification, 300–700 cells per genotype and experiment were manually counted.

Aufschnaiter A., Habernig L., Kohler V., Diessl J., Carmona-Gutierrez D., Eisenberg T., Keller W, & Büttner S. (2017). The Coordinated Action of Calcineurin and Cathepsin D Protects Against α-Synuclein Toxicity. Frontiers in Molecular Neuroscience, 10, 207.

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FISH-based microbial community analysis

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Selected activated sludge samples were obtained from the aerobic tanks of full-scale WWTPs as part of the MiDAS FISH survey [4 (link)]. Sample fixation and FISH were performed essentially as described by Daims et al. [16 ]. Probes were applied, with recommended competitors and helpers, at the stringency conditions given in Table 1or their original publications. The NON-EUB nonsense probe was used as a negative hybridization control [17 (link)]. Both the 3’ and 5’ ends of oligonucleotide probes were labeled, with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS) or with the sulfoindocyanine dyes (Cy3 and Cy5) (DOPE-FISH [18 (link)]), in order to improve the in situ fluorescent signal to background ratio. Microscopic analysis was performed with either an Axioskop epifluorescence microscope (Carl Zeiss, Oberkochen, Germany) or a LSM510 Meta laser scanning confocal microscope (Carl Zeiss).

McIlroy S.J., Awata T., Nierychlo M., Albertsen M., Kindaichi T, & Nielsen P.H. (2015). Characterization of the In Situ Ecophysiology of Novel Phylotypes in Nutrient Removal Activated Sludge Treatment Plants. PLoS ONE, 10(9), e0136424.

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Microscopic Observation of Phytoplankton Diversity

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Cells for microscopy observations were collected directly from the CTD at the same or hydrographically similar depths as water collected for DNA samples, although at different times. The microscopy samples were always collected at local noon, while the CTD casts for the nucleic acid samples varied from early morning until late afternoon. The entire contents of the Niskin were gravity filtered onto a 47 mm diameter Poretics (Millipore) membrane filter with a pore size of 10 μm. Gravity-filtration time varied from 30 min to 2 h. If the filter clogged by 2 h, the remaining volume in the Niskin was measured using a graduated cylinder, and the volume filtered was noted. The filter was mounted onto an oversize microscope slide (75 mm × 50 mm × 1 mm) and examined at 400X under a Zeiss Axioskop Epifluorescence microscope (Zeiss, Berlin, Germany). Phycoerythrin and chlorophyll a (Chl a) was used to identify the symbiotic Richelia and the phytoplankton community by epifluorescence microscopy using green (510–560 nm) and blue (450–490) nm excitation wavelengths, respectively. The hosts of the DDAs were identified based on cell morphology. Qualitative observations of cell integrity for the DDAs, and general composition of the phytoplankton community were recorded.

Stenegren M., Berg C., Padilla C.C., David S.S., Montoya J.P., Yager P.L, & Foster R.A. (2017). Piecewise Structural Equation Model (SEM) Disentangles the Environmental Conditions Favoring Diatom Diazotroph Associations (DDAs) in the Western Tropical North Atlantic (WTNA). Frontiers in Microbiology, 8, 810.

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Optimizing Multicolor FISH Conditions

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FISH was performed as previously described (43 ). Optimal formamide concentration for FISH probes was determined after performing hybridization at different formamide concentrations in the range of 0 to 70% (with 5% increments). The intensity of at least 50 cells was measured using ImageJ (64 (link)) software. Optimal hybridization conditions are described in Table S1 in the supplemental material. EUBmix (65 (link), 66 (link)) was used to target all bacteria, and NON-EUB (67 (link)) was used as a negative control for sequence-independent probe binding. Quantitative FISH (qFISH) biovolume fractions of individual genera were calculated as a percentage area of the total biovolume, hybridizing with both EUBmix probes and a specific probe. qFISH analyses, performed using the Daime image analysis software (68 (link)), were based on 30 fields of view taken at ×63 magnification. Microscopic analysis was performed with an Axioskop epifluorescence microscope (Carl Zeiss, Germany) equipped with a Leica DFC7000 T charge-coupled device (CCD) camera or a white light laser confocal microscope (TCS SP8 X; Leica). Multicolor FISH was performed as described by Lukumbuzya et al. (69 (link)). Raman microspectroscopy combined with FISH was used to detect intracellular storage polymers (polyP, PHA, and glycogen) in probe-defined species and was performed as previously described (70 (link)).

Petriglieri F., Singleton C.M., Kondrotaite Z., Dueholm M.K., McDaniel E.A., McMahon K.D, & Nielsen P.H. (2022). Reevaluation of the Phylogenetic Diversity and Global Distribution of the Genus “Candidatus Accumulibacter”. mSystems, 7(3), e00016-22.

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Microscopic Monitoring of Jellyfish Metamorphosis

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Induction of metamorphosis to strobilation was monitored visually under a Leica MZ125 (Leica Microsystems) stereomicroscope. In order to monitor for the presence of symbionts inside the various stages of the animals, observations were carried out under a Zeiss Axioskop epifluorescence microscope with a rhodamine filter. Larvae, scyphistomae, or strobilae were previously anesthetized by 10 min incubations with 10% MgCl₂ in filtered seawater at 25 ± 2 °C, and then placed on the microscope slides for the observations.

Cabrales-Arellano P., Islas-Flores T., Thomé P.E, & Villanueva M.A. (2017). Indomethacin reproducibly induces metamorphosis in Cassiopea xamachana scyphistomae. PeerJ, 5, e2979.

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Quantifying Cryptosporidium Oocysts by Microscopy

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Enumeration of Cryptosporidium oocysts by microscopy was conducted in duplicate for a subset of samples (n = 8) by Australian Laboratory Services (Scoresby, Vic). To quantify recovery efficiency, each individual faecal composite or homogenate was seeded with ColorSeed (Biotechnology Frontiers Ltd. [BTF], Sydney, Australia). Cryptosporidium oocysts were purified from faecal samples using immunomagnetic separation (IMS) employing the Dynal GC Combo kit (Dynal, Oslo, Norway) as described by Cox et al., (2005) [44 (link)]. Oocysts were stained with Easystain and 4’,6’,-diamidino-2-phenylindole (DAPI; 0.8 μg.ml^-1) (Biotechnology Frontiers Ltd. [BTF], Sydney, Australia) and examined with an Axioskop epifluorescence microscope (Zeiss, Germany) using filter set 09 (blue light excitation) for Easystain (BTF), filter set 02 (UV light excitation) for DAPI staining, and filter set 15 (green light excitation) for ColorSeed (BTF). The identification criteria described in U.S. EPA method 1623 [45 ] were used for Easystain-labeled and DAPI-stained objects.

Zahedi A., Monis P., Aucote S., King B., Paparini A., Jian F., Yang R., Oskam C., Ball A., Robertson I, & Ryan U. (2016). Zoonotic Cryptosporidium Species in Animals Inhabiting Sydney Water Catchments. PLoS ONE, 11(12), e0168169.

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Tryptophan-Immobilized PLLA Fluorescence

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The fluorescence emissions of the pristine PLLA and the tryptophan-immobilized PLLA surfaces were analyzed using a Zeiss Axioskop epifluorescence microscope (Jena, Germany).

Sánchez-Bodón J., Diaz-Galbarriatu M., Sola-Llano R., Ruiz-Rubio L., Vilas-Vilela J.L, & Moreno-Benitez I. (2024). Catalyst-Free Amino-Yne Click Reaction: An Efficient Way for Immobilizing Amoxicillin onto Polymeric Surfaces. Polymers, 16(2), 246.

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Caspase Activity Assay Protocol

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A distinctive feature of PCD is the activation of caspase enzymes. Caspase activity was assayed with Image-iT™ LIVE Green Poly Caspases Detection Kit (Invitrogen, Carlsbad, CA USA), which is based on a fluorescent inhibitor of caspases (FLICA™). Cells were harvested by centrifugation at 1000 × g for 5 min, resuspended in 1X FLICA working reagent, and incubated for 60 min at room temperature in darkness. Cells were washed twice with wash buffer and fixed before analyzing under a Zeiss Axioskop epifluorescence microscope outfitted with a FITC filter set (excitation wavelength 470/40 nm and emission of 525/50 nm). Measurements from each sample were made from three 1 mL aliquots.

Choi C.J., Brosnahan M.L., Sehein T.R., Anderson D.M, & Erdner D.L. (2017). Insights into the loss factors of phytoplankton blooms: The role of cell mortality in the decline of two inshore Alexandrium blooms. Limnology and oceanography, 62(4), 1742-1753.

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Quantifying Retinal Ciliary Protein Localization

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To quantify EYS localization near the connecting ciliary region, EYS-positive puncta associated with the basal end of acetylated α-tubulin immunoreactivity, EYS-positive puncta not associated with the basal end of acetylated α-tubulin immunoreactivity, and acetylated α-tubulin immunoreactivity not associated with EYS puncta, were counted from confocal images captured from EYS and acetylated α-tubulin immunostained retinal sections. Data were analyzed by ANOVA. Post hoc comparisons were done by Student’s t-test with Bonferroni correction.
To quantify GNAT2 and 1D4 immunofluorescence reactivity, immunofluorescence was visualized with a Zeiss Axioskop epifluorescence microscope. Epifluorescence images were captured with a mono 12-bit camera and QCapture Pro 6.0 (QImaging) from the dorsal retina near the optic nerve head. Fluorescence intensity in the outer segment layer was measured with ImageJ. Fluorescence intensity in areas with no tissue was considered background and was subtracted. Data were analyzed by Student’s t-test.

Liu Y., Rittershaus J.M., Yu M., Sager R, & Hu H. (2022). Deletion of POMT2 in Zebrafish Causes Degeneration of Photoreceptors. International Journal of Molecular Sciences, 23(23), 14809.

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Measuring Intracellular Reactive Oxygen Levels

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Levels of intracellular reactive oxygen species (ROS), known mediators for PCD, were measured using the oxidation-sensitive fluorescent probe, carboxy-H₂DCFDA [5-(and 6)- carboxy-2’,7’-dichlorodihydrofluorescein diacetate] (Invitrogen, Carlsbad, CA USA). Aliquots (1 mL) of samples were centrifuged at 1000 × g for 5 min. The supernatant was carefully discarded, and the pellet was resuspended in 1 mL of 10 mM PBS buffer, pH 7.5 containing a final concentration of 5 μM H₂DCFDA and incubated for 60 min at 20°C in darkness. Cells were then washed twice with PBS. Green DCF fluorescence of cells was observed with a Zeiss Axioskop epifluorescence microscope equipped with a FITC filter set (excitation wavelength 470/40 nm and emission of 525/50 nm).

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