To visualize the membrane structures, 1 ml of exponentially growing bacterial culture was stained by Nile Red (10 μg/ml) or by FM4–64 (1 μg/ml), washed twice with 1× PBS and re-suspended in 1× PBS containing 1 μM SYTOX green. The sample was then immediately either spotted onto a poly-l -lysine glass slide and covered with a poly-l -lysine coverslip (GLG method or P-GLG method when pressure ~80 kPa was applied for 10 s) or spotted on a 1× PBS agarose pad covered with a non-coated coverslip (GAG method). Pictures were obtained at the indicated time points (t = 0 is the start of microscopy, typically 30 s after coverslip addition) using a Olympus BX63 fluorescence microscope equipped with a Andor Zyla 5.5 sCMOS camera (alternatively, an Olympus IX81 microscope equipped with Hamamatsu Orca/ER camera was also used). Olympus CellP imaging software or Olympus Image-Pro Plus 6.0 software was used for image acquisition and analysis.
5.5 scmos camera
Manufactured by Olympus
The 5.5 sCMOS camera is a scientific-grade imaging device that utilizes a state-of-the-art sCMOS sensor. It offers a resolution of 2048 x 2048 pixels and a high-speed data readout. The camera is designed for demanding scientific and industrial applications that require high-resolution, low-noise imaging.
Automatically generated - may contain errors
Lab products found in correlation
Mvx10 zoom body, by Olympus (2 mentions)
Imspector software, by Miltenyi Biotec (2 mentions)
Cellp imaging software, by Olympus (1 mentions)
Ix81 microscope, by Hamamatsu Photonics (1 mentions)
Image pro plus 6, by Olympus (1 mentions)
Bx63 fluorescence microscope, by Oxford Instruments (1 mentions)
Orca er camera, by Hamamatsu Photonics (1 mentions)
Saphyr platform, by Bionano Genomics (1 mentions)
Nanobind disk, by Bionano Genomics (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Mvplapo 2 objective, by Olympus (1 mentions)
Dmi 6000 tcs sp5, by Leica (1 mentions)
4 protocols using 5.5 scmos camera
1
Bacterial Membrane Visualization Protocol
2
Nanochannels-based DNA Sizing in Saphyr
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The
DNA extracted in the device (sans heat
concentration) and using the Nanobind disk were sized in a research-grade
version of the Saphyr platform developed by Bionano Genomics Inc.26 (link) The DNA from either extraction method was suspended
in 1× Bionano flow buffer along with dithiothreitol at a concentration
of 0.1 M and YOYO-1 dsDNA stain at a concentration of 0.5 μM.
For each sizing experiment, 8 and 11 μL of this DNA mix was
loaded in the reservoirs labeled as inlet and outlet on the Saphyr
flow cell, respectively. The DNA were electrokinetically loaded into
the roughly 34 nm wide and 1 mm long square nanochannels using a custom
electrophoresis script.27 (link) The fluorescently
labeled backbone of the stretched DNA molecules was illuminated using
an OBIS (488 nm, 150 mW, Coherent) laser and imaged using an Andor
Zyla 5.5 sCMOS camera with a 60× air objective (NA = 0.90), mounted
on an Olympus IX-71 microscope. The images were processed using the
Bionano image processing algorithm (available from Bionano Genomics),
and the DNA sizes calculated using a 1 pixel = 366 bp conversion factor.
DNA extracted in the device (sans heat
concentration) and using the Nanobind disk were sized in a research-grade
version of the Saphyr platform developed by Bionano Genomics Inc.26 (link) The DNA from either extraction method was suspended
in 1× Bionano flow buffer along with dithiothreitol at a concentration
of 0.1 M and YOYO-1 dsDNA stain at a concentration of 0.5 μM.
For each sizing experiment, 8 and 11 μL of this DNA mix was
loaded in the reservoirs labeled as inlet and outlet on the Saphyr
flow cell, respectively. The DNA were electrokinetically loaded into
the roughly 34 nm wide and 1 mm long square nanochannels using a custom
electrophoresis script.27 (link) The fluorescently
labeled backbone of the stretched DNA molecules was illuminated using
an OBIS (488 nm, 150 mW, Coherent) laser and imaged using an Andor
Zyla 5.5 sCMOS camera with a 60× air objective (NA = 0.90), mounted
on an Olympus IX-71 microscope. The images were processed using the
Bionano image processing algorithm (available from Bionano Genomics),
and the DNA sizes calculated using a 1 pixel = 366 bp conversion factor.
3
Volumetric Imaging of Lung Sections
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Volumetric imaging of lung sections was performed using Mylteny Biotech LaVision UltraMicroscope II with an Andor Zyla 5.5 sCMOS Camera, an Olympus MVX-10 Zoom Body (magnification range: 0.63–6.3×), and an Olympus MVPLAPO 2× objective (NA = 0.5). Z-stacks were recorded in 16-bit TIFF image format using LaVision Bio-Tec ImSpector Software (v7.0.127.0) with 2 µm step size either and 1× magnification for entire sample overview, or 4× for higher-resolution imaging of selected regions. The light sheet was set to NA 0.156 with 100% light sheet width. Chromatic correction was applied for each fluorescence channel.
4
Multi-modal Imaging of Biological Samples
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For light sheet microscopy, a LaVision UltraMicroscope II with an Andor Zyla 5.5 sCMOS Camera, an Olympus MVX-10 Zoom Body (magnification range: 0.63–6.3x), and an Olympus MVPLAPO 2 × objective (NA = 0.5) equipped with a dipping cap was used. Z-stacks were acquired with a light sheet NA of 0.156, a light sheet width of 100%, and a z-step size of 2 µm. LaVision BioTec ImSpector Software (v7.0.127.0) was used for image acquisition.
Confocal laser scanning microscopy was performed with a Leica DMI 6000 TCS SP5 equipped with a HC PL APO CS2 40× water immersion objective (NA = 1.1; Leica, Germany; #15506360), using the Leica Application Suite Advanced Fluorescence software (V. 2.7.3.9723) and a z-step size of 0.5 µm.
Confocal laser scanning microscopy was performed with a Leica DMI 6000 TCS SP5 equipped with a HC PL APO CS2 40× water immersion objective (NA = 1.1; Leica, Germany; #15506360), using the Leica Application Suite Advanced Fluorescence software (V. 2.7.3.9723) and a z-step size of 0.5 µm.
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