The largest database of trusted experimental protocols

25 protocols using Imspector software

Unless stated in the figure legend, image raw data were not deconvolved, but smoothed with a low‐pass filter using the Imspector Software (Abberior Instruments). When deconvolution was applied, we relied on the Richardson‐Lucy algorithm and the Imspector Software (Abberior Instruments).
In all images, the color tables were adapted for optimal contrast. Background subtraction was usually below 5% of the maximum signal intensity.
+ Open protocol
+ Expand
sSTED-FCS measurements were performed on a Abberior Instrument Expert Line STED super-resolution microscope (Abberior Instruments GmbH, Göttingen, Germany) placed in a biosafety level 3 environment, using 485- and 640-nm pulsed excitation laser sources and a pulsed STED laser operating at 775 nm and an 80-MHz repetition rate. The fluorescence excitation and collection were performed using a 100×/1.40 numerical aperture (NA) UPlanSApo oil immersion objective (Olympus, Southend-on-Sea, UK). All acquisition operations were controlled by Imspector software (Abberior Instruments GmbH, Germany). Point FCS data for the estimation of observation spot size were recorded using a hardware correlator (Flex02-08D, correlator.com, Newark, NJ, USA operated by the company’s software) [10 (link)].
+ Open protocol
+ Expand
Samples were prepared according to the protocol for nanobody staining of U2OS-Nup96-GFP samples and were imaged on an Abberior STED/RESOLFT microscope (Abberior Instruments; Expert Line) running the Imspector software (Abberior Instruments). The microscope comprises of an IX83 stage (Olympus) in combination with a UPlan-S Apochromat 100x / NA 1.40 oil objective (Olympus). Pixel size was set to 15 nm in x, y direction. Super-resolved images were acquired by donut-shaped depletion using a 775 nm pulsed laser along with a 640 nm pulsed laser, exciting STAR 635P tagged Nup96-mEGFP. A single plane of the lower side of the nucleus was imaged. Emission was collected through a 685/70 nm bandpass filter. We used a depletion power of approx. 150 mW in the sample. Higher depletion powers could in principle increase the resolution further, but in our case lead to strong bleaching and high noise for these rather dim samples.
+ Open protocol
+ Expand
STED images of MEFs were acquired with a two-color Abberior STED 775 QUAD scanning nanoscope (Abberior Instruments, Goettingen Germany) using a 485 nm excitation laser and a pulsed 595 nm STED laser (PicoGreen labeling) or a 640 nm excitation laser together with a 775 nm STED laser (Antibody labeling) and a 100x oil immersion objective lens (NA 1.4). Image analysis was performed manually using Imspector Software, by measuring the diameter of nucleoids at full width at half maximum (Abberior Instruments, Goettingen Germany).
+ Open protocol
+ Expand
sSTED-FCS measurements were performed on the Abberior Instrument Expert Line STED super-resolution microscope (Abberior Instruments GmbH, Göttingen, Germany) using 485- and 640-nm pulsed excitation laser sources and a pulsed STED laser operating at 775 nm and an 80-MHz repetition rate. The fluorescence excitation and collection were performed using a 100×/1.40 numerical aperture (NA) UPlanSApo oil immersion objective (Olympus Industrial, Southend-on-Sea, UK). All acquisition operations were controlled by Imspector software (Abberior Instruments GmbH), and point FCS data for the calibration of the observation spot sizes were recorded using a hardware correlator (Flex02-08D, correlator.com, operated by the company’s software).
Confocal microscope FCS measurements were performed on a Zeiss LSM 780 (Zeiss, Iena, Germany) using a HeNe 633-nm laser as the excitation source. Fluorescence excitation and collection were performed using a 63×/1.40-NA PlanApo oil immersion objective (Zeiss). Acquisition operations were controlled by the Zeiss Zen software. Point FCS was used to calibrate the observation spot with the LSM 780 internal hardware correlator.
+ Open protocol
+ Expand
After performing immunostaining of differentiated pHBE cells on intact supports with the primary antibodies, secondary immunostaining was performed for 1 h at 22 °C using anti-mouse STAR 635P (Abberior, 1:1000) and anti-rabbit STAR 580 (Abberior, 1:1000) antibodies, followed by mounting with Abberior Mount Solid (Abberior) or 2,2'-thiodiethanol (Sigma-Aldrich). STED imaging was performed at 100x (NA 1.4) magnification using an Abberior Instruments Quad Scanning STED microscope with 594 and 640 nm excitation lasers and a 1.5 W, 775 nm depletion laser. Data was acquired using Imspector software (Abberior Instruments).
+ Open protocol
+ Expand
For STED microscopy, affinity-purified secondary antibodies (Dianova, Germany) were coupled with NHS carbonate-StarRED or NHS carbonate-StarOrange (Abberior, Germany). After immune staining, the tissue was cleared using increasing concentrations of TDE (2,2’-Thiodiethanol; end concentration 60%) and mounted. Images were acquired using Facility Line (Abberior Instruments, Germany) with Olympus IX83 microscope (Germany) and Imspector software (Abberior Instruments, Germany). The images were deconvolved using Huygens Professional Software (version 20.10, Scientific Volume Imaging B.V., Netherlands). The classic maximum likelihood estimation (CMLE) algorithm for deconvolution were performed using standard settings, except for quality change threshold. Deconvolved images were loaded in Fiji ImageJ and difference of Gaussian filter were performed (smoothing effect Gaussian radius 1: 0.5, local background subtraction size Gaussian radius 2: 5, local background subtraction strength: 50, prevent binning artefacts factor: 1–2).
+ Open protocol
+ Expand
For high-resolution STED microscopy, fixed and stained cells on cover slips (12 mm radial cover slips, 0.17± 0.005 mm) were visualized with use of an Abberior 3D STED 2-Channel Super Resolution- and resolft microscope (Abberior Instruments GmbH, Göttingen, Germany). Images were acquired with the Imspector software (Abberior GmbH, Göttingen, Germany).
+ Open protocol
+ Expand
STED imaging was performed with a λ = 775 nm STED system (Abberior Instruments GmbH, Göttingen, Germany), containing an easy 3D optic module (Abberior Instruments) and the = 640 nm excitation laser line using a 100× Olympus UPlanSApo (NA 1.4) oil immersion objective. Images were acquired using the 590‐ and 640‐nm excitation laser lines.
Deconvolution of STED images was performed in Imspector software (Abberior Instruments) via the linear deconvolution tool.
Images were further processed in Fiji‐Imagej (Schindelin et al, 2012) by using the Gaussian‐Blur Filter and setting the sigma radius to 1 to further increase signal/noise ratio. Different analyzed excitation channels from the same sample were also merged into one image by using this software.
+ Open protocol
+ Expand
Images were acquired using Facility Line (Abberior Instruments, Göttingen, Germany) with Olympus IX83 microscope (Hamburg, Germany) and Imspector software (Abberior Instruments, Göttingen, Germany). Images were de-convoluted using Huygens Professional Software (version 20.10, Scientific Volume Imaging B.V., Hilversum, The Netherlands). CMLE algorithm was performed using a quality change threshold of 0.1 and a maximum of 40 cycles. Co-localization was analyzed using a colocalization analyzer of Huygens software. The mean values of the Pearson correlation coefficient (PCC) were evaluated statistically.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!