Optiphot

Manufactured by Nikon

Sourced in Japan, China

The Optiphot is a high-performance laboratory microscope designed for a variety of scientific applications. It features a sturdy construction and advanced optical components to provide clear, detailed images. The Optiphot is capable of various observation methods, including brightfield, darkfield, and phase contrast. This versatile instrument is suitable for a wide range of laboratory and research tasks.

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Lab products found in correlation

Dcc1545m gl, by Thorlabs (3 mentions) Eclipse ti e, by Nikon (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Dmrxa, by Leica camera (2 mentions) Lsm 780, by Zeiss (2 mentions) Mab424, by Merck Group (2 mentions) Abc elite kit, by Vector Laboratories (2 mentions) Magnafire, by Optronics (2 mentions) Polypropylene tube, by BD (1 mentions) Toluidine blue, by Beyotime (1 mentions) Insulin transferrin selenium, by Merck Group (1 mentions) Oil red o, by Beyotime (1 mentions) Alizarin red, by Beyotime (1 mentions) 3 isobutyl 1 methylxanthine, by Merck Group (1 mentions) L proline, by Merck Group (1 mentions) Insulin, by Merck Group (1 mentions) Ascorbic acid, by Merck Group (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) β glycerophosphate, by Merck Group (1 mentions) Coolpix l330, by Nikon (1 mentions)

68 protocols using optiphot

Coral Collagen Fiber Visualization

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Fluorescence microscopy—Collagen fibers isolated from the coral were visualized under a fluorescence microscope at 305–450 nm (Optiphot, Nikon, Tokyo, Japan).
Phase contrast microscopy—Live cell cultures were observed under phase contrast microscopy and digital photography (Optiphot, Nikon, Tokyo, Japan).
Scanning electron microscopy (SEM)—For scanning electron microscopy, subsamples were fixed in 4% glutaraldehyde in filtered seawater (0.22 µm FSW), decalcified as described above, dehydrated through a graded series of ethanol up to 100%, and critical point dried with liquid CO₂. The preparations were fractured under a compound microscope, using the tips of fine forceps, and the gastrovascular cavities were then carefully exposed. Next, they were gold-coated and examined under a scanning electron microscope (SEM Jeol-840a, Jeol LTD., Tokyo, Japan). In order to obtain fiber preparations, fibers were isolated as explained above and stored in 70% ethanol. They were then processed, coated with gold-palladium alloy, and examined at high vacuum under an environmental scanning electron microscope (SEM and ESEM, JSM-6700 Field Emission Scanning Electron Microscope, Jeol LTD., Tokyo, Japan). The diameters of collagen fibers and fibrils were obtained from the images, using the ImageJ software.

Benayahu D., Sharabi M., Pomeraniec L., Awad L., Haj-Ali R, & Benayahu Y. (2018). Unique Collagen Fibers for Biomedical Applications. Marine Drugs, 16(4), 102.

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Tethered Cell Assays for Flagellar Rotation

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Cells were prepared for tethered cell-assays by washing several times in motility buffer followed by shearing of the flagella, as described previously (Ford et al., 2017 (link)). Flagellar tethering to beads or coverslips was achieved with a sticky fliC mutation (Scharf et al., 1998 (link)). Cell rotation was imaged and recorded on a Nikon Eclipse Ti-E with a 20× phase objective or a Nikon Optiphot with a 40× phase objective at ∼60 fps with a CCD camera (DCC1545M-GL, Thorlabs Inc). Bead rotation was imaged on a Nikon Optiphot with a 60× phase objective coupled to a photomultiplier setup (Yuan et al., 2010 (link); Ford et al., 2017 (link)).

Ford K.M., Antani J.D., Nagarajan A., Johnson M.M, & Lele P.P. (2018). Switching and Torque Generation in Swarming E. coli. Frontiers in Microbiology, 9, 2197.

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Multilineage Differentiation of ADSCs

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For osteogenic and adipogenic differentiation, ADSCs were seeded on 6-well plates at the density of 1×10⁵ cells/well. Osteogenic induction was performed using differentiation media consisting of DMEM-LG supplemented with 10% FBS, 50 mg/ml of ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone (all Sigma-Aldrich; Merck KGaA). The media was changed every 3 days until day 21.
Adipogenic studies were performed by culturing the cells in differentiation media containing 1 mM dexamethasone, 50 mM 3-isobutyl-1-methylxanthine and 10 mg/ml insulin (all Sigma-Aldrich; Merck KGaA).
For chondrogenesis, 1×10⁵ cells were centrifuged at 500 × g in a polypropylene tube (15 ml; BD Biosciences) for 10 min at 4°C. Aggregates were incubated in chondrogenic media consisting of DMEM supplemented with 10% FBS, 1% insulin-transferrin-selenium, 1 mM sodium pyruvate and 50 mM L-proline (all Sigma-Aldrich; Merck KGaA).
After the differentiation processes were complete, cells were fixed in 4% paraformaldehyde for 30 min at 37°C and stained with Alizarin Red (10 min at 37°C), Oil Red O (10 min at 37°C) and Toluidine Blue (30 min at 37°C; all Beyotime Institute of Biotechnology), respectively. Then, cells were observed with a light microscope (Optiphot; Nikon Corporation) at a high-power magnification of ×100.

Li R., Li Y, & Dong X. (2020). Preconditioning mesenchymal stromal cells with flagellin enhances the anti-inflammatory ability of their secretome against lipopolysaccharide-induced acute lung injury. Molecular Medicine Reports, 22(4), 2753-2766.

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Quantification of Protein Expression and Mitosis

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Proteins (50 μg cell lysates per experimental condition as described previously (7 (link)) were separated using an 8% SDS–PAGE. Antibodies used for immunoblot analysis included antiIGF-1R β-subunit (C-20, sc-713, Santa Cruz, CA).) and furin monoclonal antibody MON-152 (ALX-803–017; ALEXIS). The quantification of Western blot results was performed by using ImageJ developed by NIH. Furin IHC was performed with the same antibody using paraffin embedded normal lung and tumors. P-Histone 3 immunohistochemistry was used to detect cells in the G2-M phases of mitosis. The primary anti phospho-Histo 3 (ser10) rabbit antibody, catalog number 9701, from Cell Signaling (Danvers, MA) was applied to paraffin sections as recommended by the supplier. Once stained, the mitotic index was calculated by counting positively stained p-Histone 3 cells per 200 X field (each field equivalent to 0.32 mm²) in ten to fifteen fields per tumor. A total of 5 control and 5 CMK treated xenotransplants were used per cell line xenografts. All paraffin sections were subjected to a previously published immunostaining protocol using positive and negative controls (8 –9 (link)).All specimens were analyzed and counted using a Nikon Optiphot with a Plan/Apo objective 20X, NA, 0.75, Nikon eyepiece X10, final magnification X200.

Bassi D.E., Zhang J., Renner C, & Klein-Szanto A.J. (2016). Targeting Proprotein Convertases in Furin-Rich Lung Cancer Cells Results in Decreased In vitro and In vivo Growth. Molecular carcinogenesis, 56(3), 1182-1188.

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Comprehensive Motor Activity Assays

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Cells were prepared for motor-assays as discussed elsewhere³². Briefly, cells were grown to an OD₆₀₀~0.5 and then washed several times in motility buffer (0.01 M Phosphate buffer, 0.067 M NaCl, 10⁻⁴ M EDTA, 0.01 M Sodium Lactate and 1 μM Methionine, pH~7.0). Standard glass flow cells were prepared by using double-sided adhesive tapes to stick two glass surfaces together. Cells were sheared and tethered via a sticky filament mutant that adheres to glass and beads^{41 (link)}. Cell-rotation was imaged and recorded on a Nikon microscope (Nikon Eclipse Ti-E) with a 20x phase objective at ~60 fps with a CCD camera (DCC1545M-GL, Thorlabs Inc). Bead-rotation was imaged on a Nikon Optiphot with a 60X phase objective coupled to a photomultiplier setup³². Swarming: Swarm-assays were carried out in an environmental chamber (ETS Model 5472, Electro-Tech Systems, Inc) that allowed a fine control over humidity and temperature. Swarm-plates were imaged ~8 hours after inoculation with a digital camera (Nikon Coolpix L330). Swimming: Cells were grown to an OD₆₀₀~0.5 in TB and then diluted in fresh TB (1:40 dilution). The dilute suspension was observed in a standard flowcell and cell-motion was recorded with the Thorlabs camera at 60 fps.

Chawla R., Ford K.M, & Lele P.P. (2017). Torque, but not FliL, regulates mechanosensitive flagellar motor-function. Scientific Reports, 7, 5565.

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Comprehensive Sperm Motility Analysis

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Total motility and sperm kinetics parameters were assessed by the CASA system (ISAS^® v1, Proiser, R + D S.L., Paterna, Spain) combined with a phase contrast microscope (Nikon Optiphot) equipped with a negative phase contrast 10 × objective and integrated warmer stage and connected to a video camera (Proiser 782M, Proiser R+D).
Semen from CG was evaluated as raw without dilution, and 5 μl of semen pellet obtained after Percoll density gradient centrifugation (HQS fraction) were diluted in 5 μl TALP sperm medium (13 (link)) prewarmed at 37°C. Next, 10 μl of diluted semen were placed on a prewarmed (37°C) Makler^® chamber. During the analysis, the microscope heating stage was maintained at 37°C. Images were relayed, digitized, and analyzed by the ISAS^® v1 software with user-defined settings as follows: frames acquired, 25; frame rate, 20 Hz; minimum particle area 20 microns²; maximum particle areas 70 microns²; progressivity of the straightness 70%. CASA kinetics parameters were total motility (MOT TOT, %), progressive motility (PRG, %), curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity coefficient (LIN, %, = VSL/VCL × 100), amplitude of lateral head displacement (ALH, μm), straightness coefficient (STR, % = VSL/VAP × 100), wobble coefficient (WOB, % = VAP/VCL × 100), and beat cross frequency (BCF, Hz).

Turri F., Capra E., Lazzari B., Cremonesi P., Stella A, & Pizzi F. (2021). A Combined Flow Cytometric Semen Analysis and miRNA Profiling as a Tool to Discriminate Between High- and Low-Fertility Bulls. Frontiers in Veterinary Science, 8, 703101.

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Trypan Blue Cell Viability Assay

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Cell viability was determined by the
trypan blue exclusion assay. At the end of the experiments, the cells
were trypsinized. Trypan blue solution (0.4%) was mixed with cell
suspension in a ratio of 1:4, and the mixtures were kept at room temperature
for 2 min for the cells to be stained with the dye. The number of
viable cells was counted under a microscope (Nikon Optiphot, Tokyo,
Japan). Cells absorbed the dye were counted as dead cells. The cells
that did not absorb the dye were counted as live cells.^{18 (link)}

Huang H., He Y., Cui X.X., Goodin S., Wang H., Du Z.Y., Li D., Zhang K., Tony Kong A.N., DiPaola R.S., Yang C.S., Conney A.H, & Zheng X. (2014). Potent Inhibitory Effect of δ-Tocopherol on Prostate Cancer Cells Cultured in Vitro and Grown As Xenograft Tumors in Vivo. Journal of Agricultural and Food Chemistry, 62(44), 10752-10758.

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Cell Viability Determination via Trypan Blue

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The number of viable cells after each treatment was determined using a hemocytometer under a light microscope (Nikon Optiphot, Tokyo, Japan). Cell viability was determined by the trypan blue exclusion assay, which was performed by mixing 80 µL of cell suspension with 20 µL of 0.04 g/mL trypan blue solution for 2 min. Blue cells were counted as dead cells, whereas undyed cells were counted as live cells.

Li D., Ma Y., Liu W., Ren X., Chen M., Xu X., Sheng Z., Zhang K., Zhou R., Goodin S, & Zheng X. (2020). Celecoxib combined with salirasib strongly inhibits pancreatic cancer cells in 2D and 3D cultures. International Journal of Medical Sciences, 17(12), 1795-1802.

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Visualizing Meiotic Progression in Yeast

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For assessment of meiotic progression, sporulating cells were fixed with ethanol, stained with 4′,6-diamidino-2-phenylindole, and photographed under wet mount using a Nikon Optiphot equipped for epifluorescence as previously described (Krisak et al. 1994 (link)). Sporulation efficiency was analyzed by phase-contrast microscopy, with cells containing two or more spores per ascus scored as positive. For live-cell imaging, it was necessary to use multicopy plasmids to detect the Smk1-GFP and Ssp2-GFP signals. Cells containing an integrated nuclear HTB2-mCherry marker (Table 1) carrying 2µ-based SMK1-GFP or SSP2-GFP plasmids (Table 2) were inoculated from selective medium into YEPA for overnight growth and transferred to sporulation medium as previously described (Tio et al., 2015 (link)). A Leica DM-RXA with oil immersion was used to image 8 μl of sporulating cells, using a 60× lens, at the indicated times. Completion of MII was monitored using the HTB2-mCherry marker and further assayed for GFP fluorescence. The fraction of postmeiotic cells that showed GFP fluorescence varied between trials from 8% to 20% due to plasmid loss. All Smk1-GFP and Ssp2-GFP experiments described in this study were repeated independently at least three times with a minimum of 100 fluorescent cells examined.

Omerza G., Tio C.W., Philips T., Diamond A., Neiman A.M, & Winter E. (2018). The meiosis-specific Cdc20 family-member Ama1 promotes binding of the Ssp2 activator to the Smk1 MAP kinase. Molecular Biology of the Cell, 29(1), 66-74.

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Apoptotic Cell Identification via PI Staining

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Apoptotic
cells were identified
in propidium iodide (PI) stained cells by morphology.^{19 (link)} At the end of the experiment, cells were spined to slides
using a Shandon cytospin centrifuge (Thermo Scientific, Waltham, MA,
U.S.A.) and fixed in a solution containing 50% acetone and 50% methanol.
The cells were then stained with PI at a concentration of 1 μg/mL.
A microscope (Nikon Optiphot, Tokyo, Japan) was used to identified
apoptotic cells according to the morphological changes characteristic
of apoptosis as described earlier.^{19 (link)}

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