Confocal and advanced optical microscopy

Team

Operational Manager Dalia  El Arawi (dalia.el-arawi@inserm.fr)

Scientific manager Marcel Tawk (marcel.tawk@inserm.fr)

Confocal microscope: SP8 (Leica)

Precision imaging at the heart of life sciences.

The Leica SP8 system is an essential tool for modern biological research. Using laser scanning technology, it produces high-resolution optical sections by eliminating out-of-focus signals. It is the gold standard for 3D imaging and the tracking of specific molecules in both fixed and live samples.

This microscope offers total flexibility for live-cell imaging thanks to its incubation chamber, high-sensitivity hybrid detectors, and its ability to reconstruct large 3D volumes via a motorized stage.

Expertises and Applications
  • Spectral confocal: Precise multiplexing and spectral unmixing.
  • Dynamic Imaging: Real-time tracking (Time-lapse) and molecular mobility (FRAP).
  • 3D Reconstruction : Multi-layer acquisition (Z-stack) for 3D modeling.
  • Mosaic and Stitching: Automated large-sample scanning with seamless image stitching.
  • Optogenetics : Spatiotemporal illumination control and uncaging.
  • Multi-scale Analysis: From sub-cellular structures to tissue-level studies
Configuration
1. Stand and Optics
  • Model: Leica DMI6000 inverted and motorized
  • Objectives (Magnification / Numerical Aperture / Immersion / Working Distance):
    • 10x / 0.30 Air (WD 11000 µm)
    • 20x / 0.75 Multi-Immersion: Water, Oil, Glycerol (WD 660 µm)
    • 40x / 1.10 Water (WD 650 µm)
    • 63x / 1.20 Water (WD 300 µm)
    • 63x / 1.40 Oil (WD 140 µm)
2. Epifluorescence Mode (Non-confocal)
  • Source: Leica EL 6000 (>2000h lifespan), intensity adjustable in 5 levels.
  • Filter Cubes:
    • A (UV): Exc. 340-380 / Dichr. 400 / Em. LP 425 (DAPI, Hoechst).
    • I3 (Blue): Exc. 450-490 / Dichr. 510 / Em. LP 515 (GFP, FITC, Alexa 488).
    • N2.1 (Green): Exc. 515-560 / Dichr. 580 / Em. LP 590 (TRITC, Cy3, Alexa 546/555/568).
3. Confocal System and Detection
  • Laser Sources: 405 nm, Argon (458, 476, 488, 496, 514 nm), 561 nm, and 633 nm.
  • Detectors:
    • Spectral: 2 hybrid detectors (HyD) + 2 photomultipliers (PMT).
    • Range: Fine selection between 400 and 800 nm.
    • Transmission: 1 dedicated channel (Brightfield).
  • Digitization: Image dynamics in 8, 12, or 16 bits.
4. Automation and Environment
  • Motorized XY Stage: Mosaic acquisition (Stitching) and multi-positioning. High-precision repositioning for Time-lapse imaging.
  • Holders: Slides, Petri dishes, and multi-well plates.
  • Incubation System: Stand placed under temperature regulation and CO2 circulation.
5. Acquisition Modes
  • Dimensional: xyz, xzy
  • Temporal: xt, xyt, xzt, xzyt
  • Spectral: xyλ, xzλ, xyλz
  • Combined: xyλt, xzλt, xyzλt

Application examples on Leica SP8

Red fluorescence at the cell body and growth cones reflecting the presence of an interaction between two proteins FKBP52 and Tau (distance <40nm). Interaction detected by a Duolink “Proximity Ligation Assay” approach (PLA, red fluorescence) on differentiated PC12 cells.

Lysosomal clustering (red) in a perinuclear area visualized in SH-SY5Y cells capable of inducibly producing tau protein and expressing FKBP52-GFP and Lamp1-RFP. t = 145 min to t = 300 min after tau induction. 

This video shows the increase in FKBP52-GFP concentration around lysosome clusters (arrows).

Confocal microscope: ConfoCor2 (Zeiss)

Resp. Abdel Ghoumari.

The ConfoCor 2 system is a confocal laser scanning microscope designed for fluorescence correlation spectroscopy (FCS) and related techniques. It offers highly sensitive detection and analysis of molecular dynamics and interactions in a small, defined viewing volume.

Confocal spinning disk microscope: TCS4D (Leica)

This microscope (Leica-TCS4D confocal laser microscope – UMS44) uses laser excitation and confocal optics to capture high-resolution three-dimensional fluorescence images. The system allows for time-lapse imaging, allowing the observation of dynamic processes in a sample over time. It allows the observation of living samples (cells, organoids, small animals). The spinning-disk uses lasers to illuminate samples across the entire observation area simultaneously. It reduces acquisition time and photobleaching of fluorescent samples as well as phototoxicity from illumination.

More about UMS44

Optical microscopy

Our laboratory has the latest generation of optical microscopes for histological analysis.