C10600 10b orca r2

Telomere distribution was assessed by fluorescence in situ hybridization (FISH) using a biotin-labeled pan-telomere probe (Cambio) following the manufacturer’s protocol. Then, 2D fluorescence microscopy was carried out at magnification of 100× using a BX61 Olympus microscope equipped with a CCD camera (C10600-10B—ORCA-R2, Hamamatsu) and appropriate filters. Images of at least 100 sperm nuclei per patient were captured using SmartCapture3 (Digital Scientific). Next, 3D fluorescence microscopy was carried out using an Olympus IX71 microscope. After identification of the best plane of focus for the center of the nucleus, 61 images were captured in 0.1 µm increments throughout the nucleus (30 images below and 30 above the central plane) using Metamorph software.
Telomeres are the only parts of the sperm nucleus that remain histone bound and not protamine bound [72 (link)], so the cells were not swollen prior to FISH. This had the benefit of allowing for morphological analysis without extreme distortion of the nuclear shape, while retaining accessibility of the FISH probes to the majority of the telomeres.

The microscope slides containing the stained samples were imaged using an UPLFLN 40x oil immersion objective (Olympus America, Center Valley, PA) and digital CCD camera ORCA-R2 C10600-10B (Hamamatsu Photonics, Shizuoka Prefecture, Japan) mounted on an IX-83 inverted motorized microscope (Olympus America, Center Valley, PA). Fluorescence images of ten randomly selected fields of view per slide were taken at 40x magnification (6.22 pixels/μm resolution) for each sample.

Live-cell imaging was carried out with an upright microscope (BX61WI, Olympus, Tokyo, Japan) connected to a digital CCD camera (ORCA-R2, C10600-10B, Hamamatsu Photonics K.K., Hamamatsu, Japan) and a xenon arc light source (MT20-E, Olympus). Fluorescence and bright field images were taken with a 10× water-immersion objective (UMPLFLN10XW, Olympus) and acquired with xcellence software (Olympus). For calcium imaging, time-lapse videos were sampled at a frequency of 2 Hz with a green filter set (FITC, excitation 485/20, emission 521 nm). Red filter set (CY3, excitation 560/25 nm, emission 607 nm) was used for imaging of nls-dTomato or calcein red-orange signals. Electrophysiological and optical recordings were synchronized with TTL pulses delivered via a microcontroller board (Leonardo, Arduino, Boston, Massachusetts, United States).

The samples were mounted at 90° and imaged with an IX-83 inverted motorized microscope (Olympus America, Center Valley, PA, USA). Images were taken using an UPLFLN 40x oil immersion objective (Olympus America, Center Valley, PA, USA) and a digital CCD camera ORCA-R2 C10600-10B (Hamamatsu Photonics, Shizuoka Prefecture, Japan). Ten fields of views randomly selected for each sample and imaged at 40x magnification (6.22 µm/pixel). Connexin 43 images were obtained with the Olympus Fluoview FV3000 microscope (Olympus America, Center Valley, PA, USA) also using a UPLSAPO 40x silicone oil immersion objective (Olympus America, Center Valley, PA, USA).

smFISH probes were designed against daf-21 sequences utilizing the Stellaris FISH Probe Designer (Biosearch Technologies, Inc; (www.biosearchtech/com/stellarisdesigner). Probe sets of 44 probes of 22 nucleotides each labeled with TAMRA dye (Biosearch Technologies, Inc.) were used. At least 10–20 1d and 4d old adult animals per strain were fixed using 4% paraformaldehyde and resuspended in 70% ethanol at 4°C for approximately 24 hours. Samples were then hybridized with the daf-21 Stellaris FISH Probe set following the manufacturer’s instructions (www.biosearchtech.com/stellarisprotocols). For quantification of puncta, images were acquired on an Axio Observer A1 inverted microscope (Zeiss) using a 63X oil objective and a digital CCD camera (Orca-R2 C10600-10B, Hamamatsu). All samples were imaged under identical settings. Mean pixel intensities in the regions containing ASH/ASI neuronal cell bodies were measured using ImageJ (NIH).