Advanced Optical Microscopy for Biomedical Research
Dr Alex Corbett
FAST FOCUSING 3D MICROSCOPY
3D images of biological samples are captured by acquiring 2D images at different focal depths within the sample. Remote focusing microscopy is a method that allows this refocusing to be achieved quickly (~40 Hz) across large volumes (~400 μm cube). We have employed this method to capture electrical activity in the anterior neural plexus of the sea worm larvae Platynereis dumerilii.
Spinning-disk remote focusing
Remote focusing is an optical technique that forms a 3D image of the specimen using a second microscope objective. This remote image can be then be scanned in the same way that the specimen would be, but without the risk of causing agitation that might affect the behaviour being observed.
Remote focusing is powerful does not provide any inherent optical sectioning that makes it possible to construct a 3D image of a sample. In this project the remote focusing unit was coupled to a spinning disk system that provided the optical sectioning. This enabled 3D images to be acquired rapidly and without interrupting the spontaneous animal behaviour.
Sketch outlining the main components of the SD-RF system
In the first demonstration we imaged several developmental stages of the Platynereis dumerilii sea worm larvae. In particular we imaged the anterior neural plexus to record spontaneous nerual activity
Four different depths within the neural plexus of the sea worm larvae imaged in real time. The coloured boxes highlight specific neurons firing.
A three-dimensional image of a sea worm larvae at 3 days post-fertilisation. The fluorescent staining indicate the nervous system (red) and cilial bands (green).
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Progress in scientific research is currently being undermined by the lack of repeatability of long, complex experiments. Parallelisation of imaging experiments can be used to reduce the impact of natural specimen variability and increase the statistical power. Running multiple experiments concurrently increases time efficiency as well as greatly increasing confidence in the conclusions of a study.
HIGH THROUGHPUT IMAGING
The Multiscope / RAP microscope
In this project we developed a parallelised bright field microscope (‘Multiscope’) which can capture in quick succession multiple fields of view from individual wells of a standard 96-well plate. Image processing metrics derived from brightfield images can be used to infer animal behaviour. The Multiscope was used to demonstrate neuro-peptide modulated feeding behaviour in Platynereis dumerilii.
The Multiscope concept. By illuminating each red LED in turn (left), it is possible to image each of the samples (green) onto a single camera (right).
The raw data captured from by the Multiscope camera is shown below. To make sense of it we have to de-interlace the data to extract videos of the 9 separate wells in which the larvae are moving.
Videos from each individual well can then be automatically processed to uniquely identify and track larvae. It is then possible to determine larvae orientation, speed and total distance travelled. These image metrics can then be mapped onto specific behaviours, with significant differences in behaviour being clearly identifiable.
Data below show differences in larvae speed (green and purple bars) and total distance travelled (blue and orange bars) after the addition of food or the addition of a neuropeptide which can modulate appetite.
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The reproducibility of biomedical studies using microscopy images can be greatly improved by appropriate calibration of the microscope itself. This requires multiple information sources such as the illumination uniformity, colour channel alignment and the lateral and axial resolution.
Creating standards using direct laser writing
We have contributed to this effort in two ways: (i) creation of the first fluorescent calibration standards created by direct laser writing (www.psfcheck.com) and (ii) writing the first fully automated image analysis software than can determine instrument resolution (PyCalibrate). Since they were first introduced in 2019, these tools have been successfully employed by over 150 laboratories worldwide.
The PSFcheck slide and patterns
Direct laser writing can create fluorescent features in polymers that can be used to measure the imaging performance of a wide range of optical microscopes (widefield, confocal, multiphoton, single molecule localisation). The animation shows nine fluorescent features written with a 10 micrometre spacing.