Mesolab

Optical mesoscopy at the University of Strathclyde

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Introduction

What is the Mesolens?

The Mesolens is a giant microscope objective designed for computer data acquisition rather than the human eye. It arose from a realization in the early days of confocal microscopy that confocal images could not be obtained of large specimens, because the available low magnification objectives had too poor a resolution in depth.

We have created with an unprecedented numerical aperture of nearly 0.5 at a magnification of 4x. This results in a field size of 6 mm, with a working distance of 3 mm, and the possibility to resolve sub-cellular detail throughout this large volume in x, y and z.

Funded by the Medical Research Council 'Next Generation Optical Microscopy Initiative' and supported by the NC3Rs and the Leverhulme Trust, we have created an imaging technology centre around the Mesolens called the Mesolab. If you would like to discuss collaborative research opportunities, please contact us.

About the Mesolens

Features and benefits of the Mesolens in biomedicine

The Mesolens makes possible 3D microscopy with larger specimens than can be imaged with a standard microscope. This allows us to see individual cells in situ, without losing the landmarks at the level of organs, blood vessels or other larger structures. This will help to answer questions such as ‘Are cell certain cells missing when embryonic development is abnormal?’, and ‘Is the cytological structure in the periphery of a metastatic tumour present all around the tumour, or at just a few points that happen to be detected in the tiny field of a normal microscope image?’.

High-resolution images of thin biomedical specimens such as a cell monolayer or tissue section can be obtained using the Mesolens in camera mode. We can record a full-frame brightfield, darkfield or wide-field fluorescence image using a high pixel number (240 MP effective) camera at a rate of around 0.2 Hz, with an increase in speed possible using region of interest recording. A single camera image at the highest resolution has a file size of around 0.5 GB, which can be easily opened and analysed with a standard computer and existing image analysis software.

The ability to record a massively increased amount of data, from a volume at least 100 greater than a convention microscope means that data can be collected using the existing Mesolens systems, archived and then analysed anywhere in the world at any time, post-acquisition. An obvious biomedical advantage is that automatic diagnosis algorithms can then be tested at leisure on the same specimen data.

With unusual combination of large volume of capture with sub-cellular resolution the Mesolens offers the possibility to detect the occurrence of rare events. This includes very sparsely-distributed dividing cells in relatively quiescent tissues. It could show whether an abnormal mitotic index was present, indicating, perhaps, a proliferative tumour.

The Mesolens has a high collection efficiency compared to standard low magnification objective lenses. This makes possible low-light imaging, including the detection of very faint fluorescence emissions. This makes it possible to reduce the optical radiation dose to the specimen during examination. We hesitate to claim no photo-bleaching, but so far we have been unable to photo-bleach any specimen we have studied. If photo-bleaching of fluorescence is a problem in your imaging application we would be delighted to discuss how the Mesolens may be able to help.

We also know from preliminary studies that the Mesolens can show the movement of bioluminescent tumour cells in time-lapse imaging mode. This could provide an understanding of the biology of different types of tumour and rates of metastasis.

The Mesolens can be used to image existing specimens, because no specific fluorophores or biomarkers are required. Any specimen that can be mounted under a type 1.5 coverslip is suitable for imaging with the Mesolens. We have designed simple imaging chambers for imaging thicker specimens, and we make these available for visiting researchers to use with their specimens. The Mesolens is a multi-immersion lens which is designed for water, Type DF oil and glycerol immersion. We can also obtain a lower-resolution image in air, which can be helpful for alignment or a quick specimen check. As well as water, Type DF oil, glycerol and BABB, standard specimen mountants including Vectashield and Histomount are fully compatible with the Mesolens.

A complete Mesolens image sampled at Nyquist resolution over the 6 mm x 6 mm x 3 mm volume generates a 639 GB digital image. This is large, but the capacity and speed of current computers can readily handle data files of this size.
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Technical Details

Magnification: 4x

Numerical aperture: 0.47

Image volume: 6 mm x 6 mm x 3 mm

Measured resolution at 550 nm: 0.7 microns lateral, 7 microns axial

Immersion fluid: water, glycerol, Type DF oil

Imaging modes: camera (brightfield, darkfield, widefield epi-fluorescence), confocal laser scanning (fluorescence, reflection, differential phase contrast)

Excitation wavelengths for fluorescence imaging: LEDs from 365 nm to 660 nm for widefield epi-fluorescence imaging, lasers from 405 nm to 640 nm for confocal laser-scanning mesoscopy

Detectors: high pixel number camera for bright field and widefield epi-fluorescence imaging, photomultipliers (3-channel) for confocal laser-scanning mesoscopy

Other lasers and photodetectors are available upon request.

Images

Interactive pan and zoom images

 

Batrachospermum

Mouse Intestine

Mouse Kidney

Mouse Embryo Slice

Mouse Embryo

Zebra Fish

Widefield camera images

 

Bovine pulmonary artery endothelial cells with MitoTracker Red CMXRos (red), Alexa Fluor 488 Phalloidin (green), and DAPI (blue) - slide commercially available as 'Fluocells slide #1' from ThermoFisher Scientific. Imaged with 20 ms camera exposure in sequential widefield epi-fluorescence mode using a CoolLED pE-4000 illuminator.

Diatom frustules, with the blow-up showing Pinnularia sp. Imaged in brightfield mode with 5 ms camera exposure.

Thin section of mouse embryo stained with H&E and imaged in brightfield mode with 5 ms exposure. Gelatin filters were used to obtain a colour composition.

Zebrafish larvae, one day after hatching. Specimens were fixed and stained with acridine orange, then imaged with 20 ms camera exposure in widefield epi-fluorescence mode using a CoolLED pE-4000 illuminator.

Confocal laser-scanning mesoscopy images

 

Single confocal mesoscopy image of almost half a mouse brain section. This specimen was stained to show (as bright dots) aggregates of SOD1, a protein which shows prion-like misfolding which spreads through the brain and causes amylotrophic lateral sclerosis (Huntingdon's Disease). Specimen prepared by Dr Anne Bertolotti (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK).

A dual-colour optical section of an optically cleared 12.5 day old mouse embryo obtained with the Mesolens in laser scanning confocal mode. The neuronal axons are stained with Alexa 594 (blue) and nuclei with acridine orange (yellow). Scale bar=1 mm. The inset shows a blow-up of the eye region revealing the individual cell nuclei. The image plane of this inset is located 36 microns closer to the specimen surface than in the full image shown. The scale bar on the inset image is 300 microns. We can clearly identify fine structures throughout the embryo such as the developing heart muscle fibers and fine details in the eye such as the corneal endothelium. The arrow in the lower right corner of the blow-up indicates a stained axon.

Series of optical sections through an optically cleared 10 day old mouse embryo stained with acridine orange captured with the Mesolens in laser scanning confocal mode. The axial step difference between optical sections is 10 microns, and the full range of imaging was chosen to image through the entire thickness of the developing heart.

Single confocal mesoscopy image of a mouse kidney section (16 micron thick) with Alexa 488 WGA, Alexa Fluor 568 Phalloidin and DAPI - slide commercially available as 'Fluocells slide #3' from ThermoFisher Scientific.

People

Current staff

Professor Gail McConnell

is the Director of the Centre for Biophotonics and the Mesolab at the University of Strathclyde. Her research group works on the design, development and application of new optical imaging methods, including the Mesolens.

Dr Brad Amos FRS

is a Visiting Professor to the University of Strathclyde from the MRC Laboratory of Molecular Biology and is a Director of Mesolens Ltd. A biologist and designer of optical instruments, together with John White, Mick Fordham and Richard Durbin he developed the point-scanning confocal microscope which is now commonplace in biomedicine.

Dr John Dempster

is a Senior Lecturer at the University of Strathclyde. His primary research involves the development of open source software for acquisition of electrophysiological recordings and imaging data from standard microscopes and the Mesolens.

Mr Lee McCann

is the Imaging Technician at the Mesolab. With an honours degree in Physics from the University of Strathclyde, Lee maintains the Mesolab instrumentation and works with researchers to help obtain the best possible imaging data during their visit.

Mr Es Reid

is a Visiting Scientist to the University of Strathclyde, a Director of Mesolens Ltd and freelance optical designer.

Alumni

Dr Johanna Trägårdh

Dr Rumelo Amor

Contact Us

For more information about the Mesolab, the Mesolens, or to enquire about collaborative research or employment opportunities please contact us on the email address below.