CrestOptics will run the X-light V2 spinning disk and super-resolution LiveSR system for the 18th European Light Microscopy Initiative Meeting at the University College Dublin, 5 – 8 June 2018.
Join us for workshops sessions we will have the pleasure to share with Cairn Research and Gataca Systems in the O’brien Centre, room E2.18.
Enhancing fluorescence signal: noise performance in multiwavelength image capture
Simultaneous acquisition of fluorescence signals from multiple fluorophores or multiple spectrally separated channels is critical for many quantitative imaging applications in modern microscopy. Solid-state LED and laser light sources can provide high-speed switching options that allow tight control of the timing of illumination that can be used to gain improvements in signal: noise of temporal recordings.
In conjunction with multi-band filter sets and imaging relays that separate the multiple channels to different detectors or detector regions, we will demonstrate the advantages and drawbacks of different approaches to achieving high-speed multi-wavelength imaging. This includes a novel approach to enhancement of image quality using a high-speed filter wheel to reduce or eliminate cross-talk using multi-band filter sets.
High-speed fluorescence sectioning approaches
Localisation of fluorescence signal to discrete layers of a thick sample is essential for monitoring processes within cells and tissues. Traditionally the dominant approach to achieving reliable signal recordings over a thin plane has involved laser-scanning confocal microscopy. However, applying this raster scanning approach to fast dynamic recording requires very high instantaneous illumination point intensity and can be phototoxic to biological systems. By switching to parallel scanning or thin plane illumination approaches such as spinning disk, TIRF or lightsheet microscopy there are reductions in energy density at the sample that bring advantages in both temporal resolution and biological validity.
We will use an affordable spinning disk system that can be implemented as a modular upgrade to a standard research microscope to demonstrate the performance benefits of a spinning disk confocal for high-speed imaging. Further enhancement of the sectioning capabilities offered by laser techniques will be shown using a scanning TIRF head that provides fine control of the sample penetration depth on the fly. We will also show a high NA lightsheet system and present extended time-lapse recordings to show the low phototoxicity.
Live super-resolution imaging from multiple channels
Scientific demand for ever-increasing spatial resolution has pushed fluorescence imaging beyond the diffraction limit and resulted in the emergence of a range of approaches to achieve super-resolution imaging. The compromise between speed of image capture and ultimate spatial resolution is key to the success of experiments in this regime so we present here two approaches with different ultimate resolutions. We will use a stochastic approach with a scanning TIRF head to achieve resolutions in the 10’s nm range and compare this to an optical demodulation approach using a spinning disk confocal. The feasibility of acquisition of images at resolutions of under 110nm in real time will be demonstrated.
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