Photomanipulation Microscope

This Wellcome Trust-funded project is developing a versatile photo-manipulation system that combines holographic optical tweezers, patterned illumination, and photoablation with real-time confocal microscopy. The system is designed to ablate biospecimens, control their behaviour, and quantitatively characterise their properties while simultaneously imaging the sample with a high-end confocal microscope.

Located in the Department of Physiology, Development and Neuroscience, Anatomy building, Optical Development Lab

This project aims to develop a versatile photo-manipulation system to ablate biospecimens, control their behaviours and characterise their properties quantitatively whilst imaging the sample with a high-end confocal microscope.

photomanipulation microscope

Capabilities

  • The complete system is composed of:
    • holographic optical tweezers
    • a bespoke Digital Micromirror Device (DMD) based patterned illumination unit
    • a Ti:Sapphire laser based ablation unit integrated on a commercial Nikon TiE2 frame.
  • The holographic optical tweezers use a NIR laser to trap microspheres for measuring cell’s properties.
  • The patterned illumination unit offers high resolution (1920 x 1200) and high speed for photo-activation or photo-conversion of biospecimens.
  • The ablation unit emits femtosecond laser with the maximum peak power of 300kW for cutting cell membranes or ablating large-scale tissues.
  • The microscope adapts 5 objectives including:
    • 10x, 20x, 40x (Silicon oil), 60x (Oil) and 100x (Oil), for imaging various biospecimens from single cell level to the entire embryo level.
  • The system can acquire two-colour live confocal imaging of biospecimens excited by 405/405/445/470/520/528/555/640 nm lasers.

Technical Specifications

Nikon Ti2 microscope frame

  • Objective-10X; 20X; 40X (Silicon oil); 60X (Oil); 100X (Oil)
  • Temperature control

CrestOptics X-light spinning disk confocal

  • Spinning disk - 50 μm pinhole / 250 μm spacing or 50 μm pinhole / 500 μm spacing
  • Excitation lasers - 405; 445; 470; 520; 528; 555; 640 nm
  • Camera – 2x sCMOS Prime95B
  • Simultaneous dual colour imaging

Optical Tweezers

  • Trapping laser – 1064 nm CW laser
  • Calibrated stiffness range - 5 pN/μm to 200 pN/μm
  • Μulti-spot trapping capability

Patterned Illumination

  • DMD based pattered illumination, (1920 x1200 pixel)
  • Activation light source - 405; 445; 470; 520; 528; 555; 640 nm
  • Simultaneous activation/deactivation of different areas

Applications

Various studies have been undertaken based on this system with the collaboration with biologists, including:

  • membrane tension measurements on HeLa cells,
  • photo-activation and photo-deactivation of zebrafish embryos,
  • photo-conversion of fly embryos,
  • large scale ablation on zebrafish tissues,
  • membrane cutting on fly embryos

Furthermore, our system has the capability to undertake complex bio-experiments without transferring specimens between different microscopes. To illustrate, imaging of live HeLa cells, quantifying the cell membrane tension, studying cell shape governed by actin-mCherry, locally photoactivating a single cell, photo-ablating the membrane of the cell and tracing the variation of membrane tension.

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