The Platform

The platform is equipped with up-right and inverted fluorescent microscopes, and analysis workstations.

Imaging applications include:

  • Real-time visualization of dynamic cellular processes (signaling, intracellular transport, organization of organelles and subcellular structures)
  • Single-cell analysis, to visualise cell heterogenity within a population
  • Recording of cellular morphological changes or cell death (in response to particular stimuli, damage, stress conditions)
  • Measurements of cell migration
  • High definition analysis of subcellular structures (5 fluorescence excitation channels, simultaneous visualization of 4 stainings, deconvolution)
  • Proximity ligation assays for in situ protein interactions and in situ post-translational modifications.

Equipment

Nikon Eclipse 90i upright microscope

The Nikon Eclipse 90i is an up-right microscope for the analysis of fixed samples (fluorescence and bright-field)

  • Digital head, to perform all microscope-associated operations (shutter, filter change, objective change) both in manual and digital mode
  • motorized along the z-axis, to acquire stacks of images on different focal planes
  • acquisition software Nis-Elements AR
  • Intensilight fluorescence lamp
  • Objectives: Plan Fluor Nikon 20x (N.A. 0.5), 40x (N.A. 0.75), 100x (oil, N.A. 1.3)
  • Filter cubes: DAPI (Ex 360/40; BS 400LP; EM 460/50), GFP (Ex 470/40; BS 495LP; EM 525/50), Cy3 (Ex 545/30; BS 570LP; EM 610/75), Texas Red/Cy3.5 (Ex 560/40; BS 595LP; EM 630/60), Cy5 (Ex 620/60; BS 660LP; EM 700/75)
  • Digital camera QiCAM Fast 1394 (QImaging): monochrome cooled camera; 1392 x 1040 pixels; 4.65µm x 4.65µm pixel size

Nikon Eclipse Ti inverted microscope

The Nikon Eclipse Ti is an inverted motorized microscope indicated for time-lapse recording of live samples, using DIC (differential interference contrast), Phase Contrast and fluorescence

  • completely motorised to perform all functions in automated mode (z-axis, xy stage movement, microscope camera and lamp functions)
  • integrated Perfect Focus System (PFS): corrects focus drifts during long-term observations.
  • Acquisition software Nis-Elements AR with JOBS module: allows personalized experimental settings, e.g., acquisition intervals, choice of observation fields (multipoint acquisition), fluorescent channels, time exposure, z-axis. Automated and conditioned acquisition workflows can be easily executed
  • Okolab stage incubator, allowing prolonged observations (up to 5-6 days)
  • Intensilight fluorescence lamp, with six discrete levels of light intensity
  • Set of objectives for DIC, Ph, fluorescence
  • Filter cubes: DAPI (BFP-A, Semrock), FITC (EX 480/30, BS 505, EM 535/40), TRITC (EX 535/50, BS 575, EM 590LP)
  • Digital camera Clara (Andor): cooled monochrome, 1392 x 1040 pixels; 6,45 µm x 6,45 µm pixel size

Workstations for data analysis

Nis-Elements HC software with:

  • deconvolution
  • creation of image projections from z-stacks using Maximum Intensity Projection, Minimum Intensity Projection, Extended Depth of Focus functions
  • 3D visualisation of z-stacks
  • manual or automatic (binarization) measures and counting of: geometric features (distances, lengths, areas, etc.), volume measurements, signal intensity (mean, sum, profile), number of objects, colocalization module, measures over time.
  • bio-analyses: wound healing assays, proliferation assays, cell tracking, cell count
  • elaboration of quantitative data: automatically generated tables and histograms of several measured values, with statistics, easily exported to excel worksheets;
  • qualitative elaboration of analysis using LUTs
  • easy annotation on images (e.g., associated measured data, labels, scalebars)
© 2021, this site is WIP. See the repo