Advanced Light Microscopy

Advanced Microscopy Techniques

To enhance our understanding of cancer biology at multiple scales, we have dedicated substantial efforts to optimizing and expanding access to a broad range of optical microscopy techniques and applications, including:

• Molecular Mobility:
  Optogenetics, Photoactivation/Photoswitching, Fluorescence Loss in Photobleaching (FLIP), Fluorescence Recovery After Photobleaching (FRAP)
• Microinjection/Micromanipulation
• Non-Linear Microscopy:
  Two-Photon Excitation (2PE), Second Harmonic Generation (SHG)
• Protein Interactions:
  Colocalization, Fluorescence Resonance Energy Transfer (FRET), Fluorescence Lifetime Imaging
• Laser-Induced Stimulation:
  DNA Damage Response, Laser Ablation
• Mechanobiology
• Nanoscopy:
  Super-Resolution Microscopy (STED, STORM, PALM)
• Correlative Light Electron Microscopy (CLEM)

Sample Preparation Support

We provide guidance on sample preparation and staining, for both live-cell and fixed-cell experiments. In particular, we offer:

• protocols for immunofluorescence and live-cell staining
• protocols for clarification of organoids and tissues
• Reagent supplies (commonly used antibodies, Dapi for nuclei staining, conjugated antibodies for STED microscopy)
• Troubleshooting for improved sample preparation and acquisition

Image Analysis & Data Processing

With approximately 180 custom analysis protocols developed annually, our team provides support immediately after the collection of the data, from image processing to data analysis and interpretation. All the plugins and pipelines can be developed in open-source software (Fiji, CellProfiler, QuPath) or commercial platforms (Matlab, Arivis, Harmony, Prism). Examples of implemented tools include:

• Cell, Nuclei, and Particle Detection:
  Segmentation and classification using classical methods or machine learning tools (2D and 3D).
• Cell and Particle Tracking:
  Pipelines for single-cell tracking, Particle Image Velocimetry (PIV), Wound Healing Assays, and particle trafficking.
• Molecular Mobility:
  FRAP analysis.
• Label-free imaging analysis:
  Machine learning tools for collagen fiber orientation analysis.
• Protein Interactions:
  Quantification of colocalization, foci formation (2D and 3D), and FRET analysis.
• Immunohistochemistry imaging analysis:
  Object detection pipelines using color threshold segmentation or machine learning (pre-trained or custom-trained models), applicable to whole-slide and single-image analyses.
• AI-Assisted Image Analysis
  Integration of artificial intelligence and machine learning tools for enhanced segmentation, tracking, and quantitative microscopy workflows.

Training & Support

We provide comprehensive support tailored to both senior researchers and early-career scientists, offering personalized training to foster their professional development. Our team ensures that users gain the necessary expertise to fully exploit the advanced microscopy resources available at IFOM.

Available Microscopes

• Widefield microscopes(4 upright, 1 inverted, 1 stereo) for fluorescence and/or transmitted light microscopy
• IXplore (Evident): Widefield Microscope for live-cell imaging or fixed samples
• Thunder (Leica): Widefield Microscope with computational clearing for live-cell imaging or fixed samples
• DeltaVision (GE Healthcare): Wide Field Microscope with Deconvolution for live-cell imaging or fixed samples
• TIRFM(Leica): Total internal reflection fluorescence microscope, for live-cell imaging or fixed samples.
• Spinning Disk CSU (Evident): Widefield Microscopy, spinning-disk confocal Microscopy, Photomanipulation for live-cell imaging or fixed samples
• TCS SP2 AOBS (Leica): Confocal Microscopy
• TCS SP5 Inverted (Leica): Confocal Microscopy, non-linear microscopy, Photo manipulation for fixed samples or live cell imaging
• TCS SP5 Upright (Leica):Confocal Microscopy, non-linear microscopy for fixed samples or live cell imaging, Intravital microscopy
• TCS SP8 STED (Leica): Confocal Microscopy, Super Resolution microscopy, FLIM (Fluorescence Lifetime Imaging Microscopy), Photomanipulation, for fixed samples or live-cell imaging
• TCS SP8 DLS (Leica): Confocal Microscopy for fixed samples, Digital Light-Sheet microscopy for live-cell imaging or fixed samples
• Stellaris 8 DIVE (Leica): Confocal Microscopy, non-linear microscopy, FLIM, Photomanipulation, for live-cell imaging or fixed samples, Intravital microscopy

More information

• Evident Scietific
• Leica Microsystems

Other equipment

• Envision (Revvity): Micro Plate reader
• AIS2: Microinjection System
• CellASIC ONIX2 (Merck): Microfluidic System

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