Fluorescence microscopy was carried out on one of the following systems: (1) an inverted AxioObserver.Z1 microscope with a × 100/1.3 oil EC Plan-Neofluar objective (Zeiss, Thornwood, NY), an Orca ER cooled CCD (charge-coupled device) camera (Hamamatsu, Bridgewater, NJ), a metal-halide lamp and an light-emitting diode (LED) Colibri system (Zeiss) including LED wavelengths at 365 and 470 nm; (2) a Nikon A1R MP laser scanning confocal attachment on a Nikon Ti Eclipse inverted microscope using a × 100/1.45 Plan Apo Lambda oil-immersion objective and standard lasers and filters (Nikon Instruments, Melville, NY); (3) a spinning-disk confocal microscope combining a CSU-X1 spinning disk attachment (Yokogawa Electronic Corporation, Tokyo, Japan) on a Nikon Ti Eclipse inverted microscope (Nikon) equipped with an electron multiplying CCD camera (Evolve, Photometrics, Tucson, AZ), 50 mW lasers at 488 and 561 nm, standard emission filters and a CFI Plan Apo × 100 1.45 numerical aperture oil objective (Nikon). System (1) was controlled by ZEN software (Zeiss). Systems (2) and (3) were controlled by NIS Elements Advanced Research software (Nikon). Details are given within the sections below describing each experiment.
100 1.3 oil ec plan neofluar objective
Manufactured by Zeiss
Sourced in Germany
The × 100/1.3 oil EC Plan-Neofluar objective is a high-performance objective lens designed for use in microscopy applications. It features a magnification of 100x and a numerical aperture of 1.3, which allows for high-resolution imaging. The objective is corrected for chromatic and spherical aberrations, and is suitable for use with immersion oil.
Automatically generated - may contain errors
Lab products found in correlation
Zen software, by Zeiss (1 mentions)
Nis elements advanced research software, by Nikon (1 mentions)
Colibri system, by Zeiss (1 mentions)
A1r mp, by Nikon (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Axiovision, by Zeiss (1 mentions)
Orca er cooled ccd camera, by Hamamatsu Photonics (1 mentions)
Axiocam ccd camera, by Zeiss (1 mentions)
Orca er cooled charge coupled device camera, by Hamamatsu Photonics (1 mentions)
Axiovert 135, by Zeiss (1 mentions)
Ec plan neofluar 40x 0.75 objective, by Zeiss (1 mentions)
Axio imager m1 epifluorescence microscope, by Zeiss (1 mentions)
Whatman, by Cytiva (1 mentions)
4 protocols using 100 1.3 oil ec plan neofluar objective
1
Fluorescence Microscopy Systems Comparison
2
Wide-Field Imaging of Fluorescent Proteins
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All wide-field imaging was performed as described previously (Garcia-Rodriguez et al., 2009 (link)) on one of the following microscope systems: an Axiovert 200M microscope with 100×/1.4 Plan-Apochromat objective (Zeiss, Thornwood, NY) and Orca ER cooled charge-coupled device (CCD) camera (Hamamatsu, Bridgewater, NJ); an Axioskop 2 microscope with 100×/1.4 Plan-Apochromat objective (Zeiss, Thornwood, NY) and an Orca 1 cooled CCD camera (Hamamatsu) or an Axiocam CCD camera (Zeiss); or an inverted AxioObserver.Z1 microscope with a 100×/1.3 oil EC Plan-Neofluar objective (Zeiss) and Orca ER cooled CCD camera (Hamamatsu). For visualization of GFP and mCherry, fluorophores were excited by a mercury or metal halide lamp and imaged through standard fluorescein isothiocyanate and rhodamine filter sets. Hardware was controlled by Openlab (PerkinElmer-Cetus, Waltham, MA), Volocity (PerkinElmer-Cetus), Axiovision (Zeiss), or ZEN (Zeiss) software.
3
Microscopic Phototrophic Organism Enumeration
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Fixed samples were sub-sampled three times and were diluted 1:10 in tap water. One ml was transferred to an Uthermol's chamber for microscopic analysis. An inverted microscope (Zeiss Axiovert 135) was used to identify and count (Zeiss EC Plan-Neofluar 40x/0.75 objective, Zeiss EC Plan-Neofluar 100 × 1.3 Oil objective, if necessary) phototrophic organisms at the level of genera and, if possible, species within three fields of vision. For samples obtained from laboratory experiments, semi-quantitative abundance was described as dominant (‘5', >30 of the cell number counted), frequent (‘4', 10–30%), regular (‘3', 3–10%), scarce (‘2', 1–3%) and sporadic (‘1', <1%) based on two taxonomic references and the recommendations by the Swiss Federal Office for the Environment33 34 35 . For field samples, presence-absence data were documented.
4
Enumeration of Bacterial Cells in Filtrates
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Formaldehyde-fixed filtrate was processed within 24 h after harvest. First, 1 mL of the sample was diluted with 10 mL sterile Milli-Q water to ensure a homogeneous distribution during subsequent gentle vacuum filtration on a Whatman (Florham Park, NY, USA) Cyclopore track etched membrane filter (0.2 µm pore size). A cellulose nitrate filter (0.8 µm pore size) served as support filter; a hand pump was used to keep vacuum at a maximum of −20 kPa. The membrane filters were then air dried for 15 min and stored at −20 °C until further investigation. For bacterial counts, filters were thawed, mounted on a slide, and the ready-to-use VECTASHIELD mounting medium with DAPI H-1200 (Newark, USA) was applied for staining (approximately 25 µL per slide). After putting a cover slip onto the filter, random fields of view were counted with a minimum of 100 cells per filter using a Zeiss AXIO Imager M1 Epifluorescence microscope (Zeiss EC Plan-Neofluar 100×/1.3 oil objective, Oberkochen, Germany; at least 10 squares per filter). Cell number was then extrapolated to the total filter area.
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