Huh-7 cells were infected with HCV strains JFH1 or cell culture adapted DBN3a (MOI = 0.1). Infected cells were kept in culture and when they were passaged, approximately 5×104 cells were seeded on glass coverslips. The day after, cells were fixed with 3% PFA, transferred out of the BSL3 laboratory and immunostained as described above using antibodies to NS5A and Pex14. Image stacks of cells of interest were acquired using an inverted Eclipse Ti inverted confocal microscope (Nikon) equipped with a CSU-W1 spinning-disk (Yokogawa, Roper Scientific). A live-SR module (Gataca Systems) was added to the system to improve the obtained resolutions. Observations were done with a 60× oil immersion objective (Nikon Plan Apo 60× NA 1.4). Signals were sequentially collected by using single fluorescence excitation and acquisition settings to avoid crossover. Image stacks were then processed using the Imaris software to model a 3D-image of peroxisomes. Objects smaller than 0.1 μm were filtered out and the volume of each object was measured, enabling the volume of each peroxisome and the average number of peroxisomes per cell to be calculated. For each measurement, 30 cells from 3 independent infections were analyzed. Statistical differences were determined with a Kruskall-Wallis test.
Planapo 60 na1
Manufactured by Nikon
Sourced in Italy
The Nikon PlanApo 60× NA1.40 is a high-magnification, high-numerical aperture objective lens designed for laboratory and research applications. It provides a large working distance and is optimized for use with immersion oil. The lens offers a numerical aperture of 1.40, enabling high-resolution imaging.
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Lab products found in correlation
Eclipse ti inverted confocal microscope, by Nikon (1 mentions)
Ls785, by Teledyne (1 mentions)
Pixis 400br, by Teledyne (1 mentions)
Bc43 spinning disk confocal system, by Oxford Instruments (1 mentions)
Lsm 710 nlo confocal microscope, by Zeiss (1 mentions)
C1 te2000 u laser scanning, by Nikon (1 mentions)
Cytospin 4 centrifuge, by Thermo Fisher Scientific (1 mentions)
May gr nwald s stain solution, by Muto Pure Chemicals (1 mentions)
Giemsa s stain solution, by Muto Pure Chemicals (1 mentions)
Filipin 3, by Santa Cruz Biotechnology (1 mentions)
Bz x 810 digital microscope, by Keyence (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
7 protocols using planapo 60 na1
1
Peroxisome analysis in HCV-infected cells
2
Single-Cell Raman Tweezers Imaging System
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The Raman tweezers system used in this work was described previously [17, 24] . Briefly, a laser beam at 780 nm is introduced into an inverted microscope (Nikon, TiS) that contains an external phase contrast system and an immersion objective (Plan Apo 60×, NA1.4) to form a single-beam optical trap. A spore or growing cell in an aqueous medium can be trapped ~10 µm above the bottom quartz coverslip. Backward Raman scattering light from the trapped cell exceited by the same laser was collected and focused on the entrance slit of a spectrograph (Princeton Instruments, LS-785) equipped with a back-illuminated deep depletion charge coupled device (Princeton Instruments, PIXIS 400BR).
The live-cell imaging of hundreds of individual cells was performed with phase contrast microscopy incorporated in Raman tweezers [17] . Phase contrast images were captured with a digital CCD camera (1,392 x1,040 pixels) at a rate of 1 frame per 15 s for up to 10 hours. A homemade auto-focus system was developed to actively lock in the focus of the objective, in which a diode laser at 650 nm was introduced to detect the distance change between the objective and the surface of the sample coverslip. The feedback electronic signal was added to a piezo attached on the objective to lock its position. The measured long-term stability of the focus locking along the z direction was ~10 nm.
The live-cell imaging of hundreds of individual cells was performed with phase contrast microscopy incorporated in Raman tweezers [17] . Phase contrast images were captured with a digital CCD camera (1,392 x1,040 pixels) at a rate of 1 frame per 15 s for up to 10 hours. A homemade auto-focus system was developed to actively lock in the focus of the objective, in which a diode laser at 650 nm was introduced to detect the distance change between the objective and the surface of the sample coverslip. The feedback electronic signal was added to a piezo attached on the objective to lock its position. The measured long-term stability of the focus locking along the z direction was ~10 nm.
3
Multimodal Confocal Imaging of mPFC
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All slides were scanned using the Zeiss LSM 710 NLO confocal microscope (Zeiss, White Plains, NY). Wavelength absorbance‐emission values were DAPI (410‐449), Adam17 (484‐552), and Aif (599‐670). Using an oil immersion objective, z‐stacks of the mPFC were obtained at 63× magnification (Objective i Plan‐Apochromat 63x/1.4 Oil DIC M27; field of view = 25 mm). Additional images were captured using an Andor BC43 Spinning Disk Confocal system (Andor Technology, Belfast) with an oil immersion objective (Plan Apo 60×, NA 1.4; Nikon). Excitation was achieved using 405 nm, 488 nm, 561 nm, and 640 nm lasers in sequence, and emission light was detected by an Andor sCMOS camera (4.2MP; 6.5 µm pixel size).
4
Mitochondrial Distribution and ROS Analysis
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Oocytes were observed using a Nikon C1/TE2000-U laser scanning confocal microscope (Nikon Instruments, Firenze, Italy) equipped with the objective Nikon Plan Apo 60×/NA 1.40 in oil immersion. A 543 nm helium/neon laser and a G-2A filter were used to detect the MitoTracker Orange CMTM Ros (551 nm excitation and 576 nm emission). A 488 nm argon ion laser and a B-2A filter were used to detect dichlofluorescein (DCF) (495 nm excitation and 519 nm emission). To allow 3D distribution analysis, oocytes were observed in 25 optical sections with a step size of 0.45 µm. The mitochondrial distribution pattern was evaluated using previously reported criteria: (1) finely granular, typical of immature oocytes; (2) perinuclear and subcortical (P/S), indicating cytoplasmic maturity; and (3) abnormal, displaying irregular distribution of mitochondria [18 (link)]. Oocytes with intracellular ROS diffused throughout the cytoplasm, together with areas/sites of mitochondria/ROS overlapping, were considered viable.
5
Microscopic analysis of cancer cell morphology
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Briefly, cells (BxPC-3, 4×105 cells/well; KP-4, 4×105 cells/well; PANC-1, 3×105 cells/well) were seeded and cultured for 24 h in 60 mm-dishes, followed by treatment with lapatinib (10 µM) and FTY720 (10 or 15 µM) at 37°C for 24 h. To detect adherent and detached cells, floating cells in culture medium and trypsin-treated adherent cells were collected. Then, cells were spread onto glass slides using a Cytospin 4 centrifuge (Thermo Fisher Scientific, Inc.) and air-dried. These cells were stained with May-Grünwald's stain solution (Muto Pure Chemicals Co., Ltd.) for 3 min at room temperature. The glass slides were allowed to stand for another 3 min after adding the same phosphate-buffered saline (PBS) volume. After removing the May-Grünwald's stain solution, the cells were stained with diluted (1:20 with water) Giemsa's stain solution (Muto Pure Chemicals Co., Ltd.) for 20 min at room temperature. After washing and drying, the cells were observed using a BZ-X810 digital light microscope (Keyence Corporation) featuring PlanApo 60× NA1.40 (Nikon Corporation).
6
Cholesterol Visualization in KP-4 Cells
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Briefly, KP-4 cells (2×104 cells/well) were seeded into CELLview 35-mm glass-bottom cell culture dishes with four compartments for 24 h, and the cells were treated in the presence (1 µM) or absence of U18666A at 37°C for 24 h. Next, cells were washed with PBS and fixed with 3% paraformaldehyde for 1 h at room temperature, followed by incubation with 20 mM glycine for 10 min to quench the paraformaldehyde. For cholesterol staining, the cells were incubated with 25 µg/ml filipin III (Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. After washing with PBS, the cells were observed using a BZ-X810 digital microscope (Keyence Corporation) featuring PlanApo 60× NA1.40 (Nikon Corporation).
7
Monitoring Protein Dynamics with ANAP
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Yeast cells were grown to logarithmic phase (OD600≈0.5) in synthetic dropout media containing 2% raffinose. Cells were pelleted by low-speed centrifugation at 1000 g for 5 min, resuspended in low fluorescence synthetic drop out medium (YNB-Folic Acid-Riboflavin medium) supplemented with 2% galactose and 0.4 mM ANAP and growth was continued for 7 hours in the dark. Cells were washed 4 times with phosphate buffered saline plus 2% glucose to remove residual ANAP. Microscopy images were acquired with a Nikon A1R confocal microscope equipped with a Nikon Plan Apo 60× NA 1.40 oil immersion objective lens and a 32-channel spectrum detector. ANAP was excited at 405 nm, and the emitted light was detected with a wavelength resolution of 6 nm per channel to monitor fluorescence emission between 420 and 610 nm. After image acquisition, regions of interest within cells were selected and fluorescence emission spectra were analyzed.
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