Supervisors: Prof Kees Weijer and Dr Mike MacDonald
This project involves conducting research in high speed lightsheet fluorescence microscopy to study cell behaviours in live embryonic tissues entailing training in lightsheet microscopy, developmental cell biology, experimental instrumentation and advanced high volume data processing and image analysis.
The work undertaken so far has involved further development of the microscope optical setup and the software. This has led to a much higher frame rate of images acquisition. It is now possible to acquire at more than 50 frames per second. The main reason for the implementation of higher frame rates is to allow the acquisition of information from around 90 – 95% of the surface of a 4mm chick embryo at the beginning of development, instead of the approximately 45% of the embryo’s surface captured in current experiments. With a single scan (see Fig. 1a) it is possible to image the middle part of the embryo’s surface (approximately 45% of embryo’s surface). By performing a dual scan (Fig. 1b) almost the complete surface can be imaged. This dual scan is performed by first imaging the bottom half of the embryo’s surface and then the top half, or vice-versa. Then both halve data sets are merged together.
An early stage chick embryo is typically 4-5 mm in diameter. To obtain an image of the embryo’s surface as shown in figure 1, approximately 2000 cross section images taken at a 45 angle are acquired and combined into a 3D data stack, after which the surface is calculated. The requirements for the implementation of a dual scan mode are:
- The same image quality as in the single scan mode;
- The time required per embryos surface acquisition should be the same or shorter.
The image quality is assured by: the laser power, the scanner parameters and the camera capabilities. In the dual scan mode the laser power utilized and the scanner parameters in use are adjusted such that the changes implemented do not affect the images and remain within the device limits. The camera is capable of acquiring images at 100 frames per second using all 2560 x 2160 pixels on the chip, giving a minimum exposure time of 10 milliseconds. For a single scan approximately 2000 cross section images at 12 – 14 frames/sec are acquired over a 2 – 3 minutes time interval. Increasing the frame rate two or three folds leads to a single scan being performed in half of the time. Hence, with this level of speed-up, the dual scan can be performed in the same 2 – 3 minutes as the previous single scans at a lower speed.
The dual scan acquisition mode allows imaging and observation of almost the complete embryo surface dynamics during the early stage development. This is key as we are imaging the onset of gastrulation, the primitive streak formation, the location of which cannot be determined before the experiment commences.
Journal and Conference Papers and Posters
[November 2017] Gaussian vs Bellel light sheets: performance analysis in live large sample imaging. Sascha L Reidt, Ricardo Bango da Cunha Correia, Antti Karjalainen, Manli Chuai, Michael P. MacDonald and Cornelis J. Weijer, in revision for Nat.Sci Rep.
[October 2016] Bessel and Gaussian beam illumination modes on LSM imaging chick embryo development – Migration of mesoderm cells. Poster presented at School of Life Sciences Poster Session, Dundee, Scotland, 14th October 2016.
[September 2016] Characterisation of Bessel and Gaussian beam illumination modes on LSM imaging quality of early chick embryo development. Oral presentation at 3rd Light Sheet Fluorescence Microscopy International Conference (LSFM 2016), Sheffield, UK, 3rd September 2016.
[August 2016] Characterisation of Bessel and Gaussian beam illumination modes on LSM imaging quality of early chick embryo development. Poster presented at the Biophotonic approaches: From molecules to living systems conference, Dundee, Scotland, 23rd August 2016.