Image Acquisition & Tiling

Image Acquisition & Tiling

The Tiling feature included in the INSCOPER Microscopy Imaging Solution is used to image an entire slide (or well) or a large region of interest in the sample. Several images are acquired one after one in a row, called “tiles”, then “stitched” afterward to constitute the whole sample.

This tutorial shows how to set up the acquisition sequence and proceed to the tiling of U2OS cells transfected with ALC1-GFP and labeled with Hoechst at 37°C. The specimen is placed in an 8-chambres glass holder.

 

The steps are as follows:

  1. Select the illumination channel.
  2. Switch the “Positions” dimension to “Tiling”
  3. Define the parameters for tiling imaging:
    1. Select 2 key points, on diagonal;
    2. Set the overlap and define the read mode.
  4. Set the channel in the Multi-Channels dimension.
  5. Select the data processing algorithm.
  6. Choose the storage location for the dataset.
  7. Start the acquisition & confirm.
  8. Monitor the progress of the acquisition.
  9. Visualize your data.
Random Illumination Microscopy – Toulouse, France

Random Illumination Microscopy – Toulouse, France

System description & applications

Random Illumination Microscopy is a new and promising super-resolution technique for live cell imaging.

Random Illumination Microscopy is an innovative SIM-based super-resolution technique for live cell imaging. RIM exploits speckled illuminations and statistical reconstruction to provide super-resolved images down to 90nm. Its high temporal resolution and low phototoxicity make it the ideal solution for live  cell imaging.

RIM is also a robust and low-cost technique thanks to its simplicity. 

microscope device list

  • Microscope Nikon Tie
  • Light Source Lumencor SpectraX
  • 2x Camera Hamamatsu Fusion
  • Laser bench Oxxius LBX-4C (405, 445, 488, 561)
  • Piezo PiezoConcept
  • 1x SLM 4D
FRAP Calibration

FRAP Calibration

For the optimal use of the Photomanipulation Dimension of the INSCOPER Imaging Software, it is recommended to calibrate the laser in order to match up the position of the scanner optical head with the camera screen size. The microscope system is equipped with FRAP module Starscan with two galvanometric mirrors. For this tutorial, we used a blue fluorescent slide (laser 405).
Widefield & Tiling – Paris, France

Widefield & Tiling – Paris, France

System description & applications

This microscopy system automated by INSCOPER is used for wide field fluorescence, bright field imaging with colour camera, high content screening-like, FRET and time lapse experiments.

The microscope is installed at the Plate-Forme IMAG’IC, Institut Cochin in Paris, and is used by several researchers.

 

microscope device list

  • Microscope : Zeiss Axio Observer 7

  • Illumination:  XCite 120PC

  • Filter wheel: Sutter Lambda 10-3

  • Stage: Marzhauser 130*100 STEP & controller SMC2009

  • Camera: Hamamatsu ORCA Flash 4.0 V3

  • Camera: IDS UI-3880CP-C-HQ

Photoluminescence spectra measuring of dynamically moving nanophosphors  – Paris, France

Photoluminescence spectra measuring of dynamically moving nanophosphors – Paris, France

System description & applications

The research team measures photoluminescence spectra of dynamically moving single nanophosphors and their assemblies, from which they study the orientation and rotational behavior of various nano- and bio-systems.

The laser scanning confocal fluorescence microscope is connected with excitation lasers, piezostage, two types of detectors (spectrometer & APD), and multiple dynamic optomechanical units synchronized all together by INSCOPER. The INSCOPER software has saved a considerable amount of time, which allowed the researchers to start the basic experiments they designed.

 

Microscope device list

  • Oxxius LBX-395 laser,
  • Thorlabs KBD101 rotating mount,
  • Physik Instrumente E-727 piezo-stage,
  • Princeton Instruments IsoPlan – SCT-320 spectrometer,
  • Princeton Instruments PIXIS camera,
  • Thorlabs APD440 photodetector.