Customer Journeys Part 2 – TILT Light Sheet

By 27th March 2020Cairn News, Customer Journeys, News

Customer Journeys Part 2 - TILT Light Sheet Microscopy

In this new series we are highlighting some of the challenges our customers have been faced with and what solutions our technical sales team have provided

Introduction - Customer Research Challenge

Imaging living specimens over an extended timeframe is challenging, owing to the increased risk of phototoxicity as well as the bleaching of fluorescent reporters. In conventional epifluorescence microscopy where the excitation light passes through the entire specimen, these risks are exacerbated; moreover, the visualisation of features of interest in the focal plane may be obscured by the excitation of fluorophores above and below it, known as ‘out of focus’ fluorescence.

The group of Dr Kikuё Tachibana are interested in furthering understanding of how an oocyte transforms into a zygote following fertilisation. Insight into the mechanisms within the zygote that establish totipotency are also of great interest to the group, with chromatin architecture forming one area of focus. In order to gain such insights, the group relies on several advanced imaging techniques, including confocal microscopy. Oocytes and zygotes are difficult to image over an extended timeframe because they are extremely photosensitive; too much excitation light will arrest development. Therefore, a balance is essential. Images need to be acquired for a long enough time to capture sufficient signal and at a suitable frequency to ‘see’ and track the processes of interest, but not so frequently that the light dosage is such that it causes irreparable damage. For the reasons discussed above, this is particularly challenging using epifluorescence microscopy and the group therefore wanted to identify a new imaging modality better suited to their requirements.

 

Cairn Research Solution

The ‘Tilt’ light sheet illumination system creates a thin sheet of excitation light, ensuring that only a ‘slice’ of the sample is ever illuminated at any given time. Fluorophores above and below the focal plane are no longer excited, minimising out of focus fluorescence and reducing phototoxicity and photobleaching, helping to ensure that living specimens can be imaged for longer. Invented by Dr Paul Maddox and Tanner Fadero (University of North Carolina, USA) and designed and manufactured by Cairn Research on their behalf, the Tilt module can be coupled to a range of commercially available inverted microscopes and is unique in that it can be used with a very wide variety of objective lenses, including high numerical aperture (NA) oil immersion lenses. Both of these features were important to the Tachibana group because they wanted to upgrade an existing Zeiss Axiovert 200M microscope frame whilst still using several immersion objective lenses already used on that frame. It is often desirable to use a high NA objective lens wherever possible to maximise capture of emitted fluorescence and to provide improved spatial resolution compared to a lens with a lower NA. Another useful design feature of the Tilt module is that oocytes mounted on the system can also be imaged using transmitted illumination, permitting the end user to capture simple brightfield (or differential interference contrast and phase contrast) images as well as fluorescence images during multichannel acquisition series.

The ‘Tilt’ module replaces the existing stage of a microscope and can be easily attached to most commercially available inverted frames. Here it is attached to the Tachibana group’s Zeiss Axiovert 200M. The Tilt module is an optomechanical assembly that creates a light sheet (via a cylindrical lens and photomask) and enables it to be precisely positioned within the working distance of a wide variety of objective lenses. The Tilt also contains an integrated motorised x, y stage as well as a z-axis piezo device. A Prime BSI CMOS camera (Teledyne Photometrics), an UNO stage top incubator (okolab), a C-Flex laser combiner (Cobolt) with 488nm (100mW) and 561nm (100mW) laser heads were also installed as part of the microscope upgrade. The system was configured by Cairn Research to be driven by MicroManager software. As the C-Flex does not have dedicated drivers for MicroManager, an interface solution was supplied by Cairn Research to control on/off shuttering and the intensity of each laser from within the software.

Conclusion

Based on an assessment of the needs of the group, an existing microscope frame was upgraded, adding a light sheet imaging capability, permitting the end user to monitor the dynamic processes inside an oocyte or early stage embryo with improved spatiotemporal resolution. As a result of the addition of the Tilt module, these processes can be imaged with greater frequency whilst minimising detrimental impact upon viability, permitting new insights to be gleaned.

“With the Mizar Tilt system, we are finally able to follow the delicate events of mammalian embryogenesis with unprecedented spatiotemporal resolution" Imre Gaspar

Imre Gaspar