Award details

Coherent Anti-Stokes Raman multiplex microscopy for non-invasive imaging of living cells

Reference	BB/D001013/1
Principal Investigator / Supervisor	Professor Paola Borri
Co-Investigators / Co-Supervisors	Professor Wolfgang Langbein
Institution	Cardiff University
Department	School of Biosciences
Funding type	Research
Value (£)	273,639
Status	Completed
Type	Research Grant
Start date	01/03/2006
End date	28/02/2009
Duration	36 months

Abstract

This project has two main aims. 1) The construction of a novel multiplex CARS microscope with improved chemically specificity and sensitivity as compared to previous instruments, and the realization of an economic design for its possible commercial use, 2) The demonstration and optimisation of the novel microscope's capabilities for real-world applications by addressing three biological problems of current interest that are difficult to approach even with state-of-the-art fluorescence microscopy. These examples are a) the change in structure of mitochondria in the pathogenic yeast Candida albicans during apoptosis and in the model yeast Saccharomyces cerevisiae during respiratory oscillations, b) the end-bud growth, development and apoptosis of cells in mammary glands, c) dynamic changes in cell water content and membrane dynamics during development of Dictyostelium discoideum. The main methods to be adopted are as follows. I. The construction of a first version of the microscope for single-frequency CARS, using the ultrafast laser sources available in the Physics Department. This microscope will additionally incorporate confocal and multiphoton fluorescence for a comparative study. Our aims with this set-up are to demonstrate the vibrational contrast, high spatial resolution and fast scanning of the CARS microscopy technique as well as its real time in-vivo imaging capabilities. For these purposes we will investigate model systems, such as polystyrene beads or fixed cells, as well live Candida albicans and Saccharomyces cerevisiae yeast cells. In living cells, CARS microscopy has proven to be particularly successful in imaging of mitochondria by selecting the stretching vibration frequency of the highly dense C-H bonds in the aliphatic chain of lipids. The ability to image volume and structural changes of mithochondria in-vivo and real time, as opposed to electron microscopy, will significantly improve the current understanding of the apoptotic events occurring inthe pathogenic yeast Candida albicans under chemical perturbation which is very important e.g. for the development of antimicrobial compounds for medical applications. Furthermore, our objective is to demonstrate the high depth penetration, still maintaining high image contrast, of the CARS microscope by imaging end bud development in mouse mammary glands and multicellular stages of Dictyostelium development. II. The implementation of the technique for multiplex CARS (M-CARS) microscopy to allow for simultaneous detection of a range of vibrational frequencies, and thus chemical species. In order to obtain a large spectral window for M-CARS and to eliminate the need of complex and expensive multiple laser sources, also in view of the development of a commercial CARS microscope, we propose a new M-CARS method based on using broadband sub-10fs (greater than 1500cm-1 bandwidth) laser pulses from a single laser source. We will develop a novel method to generate Pump and Stokes pulses and a coherent detection scheme to retrieve the full information of the CARS electric field in amplitude and phase, allowing for high sensitivity, selectivity and fast scanning with M-CARS. We propose to use our novel M-CARS microscope to image the membrane dynamics of Dictyostelium cells as they undergo chemotaxis towards cyclic adenosine monophosphate (cAMP). In fact, there is still much to be discovered concerning the mechanisms that underlie chemotaxis, and one particularly under-explored area is the role of membrane dynamics. Using M-CARS it will be possible to observe general vesicle movement within the cell, and examine whether vesicle movement and dynamics are consistent with the membrane flow hypothesis in which plasma membrane is internalized at the rear of the cell and vesicles translocate lipids to the leading edge.

Summary

Our objective is to develop and demonstrate a new microscope generation with improved sensitivity and chemical specificity for real-time studies on living cells. Such an instrument will allow non-invasive, microscopic examination of cells and subcellular structures under physiological conditions with chemical contrast without the need to stain or express tagged proteins, together with three-dimensional imaging capability of high penetration depth in intact tissues. Optical microscopy is an indispensable tool that is driving progress in cell biology, and is still the only practical means of obtaining spatial and temporal resolution within living cells and tissues. However, most cellular constituents have no colour and they are hard to distinguish under a light microscope unless they are stained. Fluorescence microscopy, using antibodies labelled with dyes or fusion of proteins with fluorescent tags has provided a highly sensitive and specific method of visualizing biomolecules. However, when used for real time observations in living cells, these modified biomolecules raise questions if their behaviour is real or artefactual. Furthermore, all fluorescent probes are prone to photo-bleaching that is an irreversible degradation of the fluorescence intensity after excitation with light. Another major difficulty in light microscopy occurs when attempting to image living cells within intact tissues with high axial resolution and long penetration depth. The key idea of this new microscope is to obtain the image contrast via scattering of light with the specific resonances of vibration in chemical bonds (Raman scattering). This phenomenon can be coherently enhanced when using two short laser pulses to resonantly excite the vibrations and generate the so-called Coherent Antistokes Raman Scattering (CARS). Since CARS depends nonlinearly on the exciting light intensity, sufficient intensities for CARS generation are achieved only in the small focal volume where the exciting photons are concentrated resulting in an intrinsic high resolution and sectioning capability similar to multi-photon fluorescence microscopy. The application of CARS microscopy for imaging in cell biology has been recently explored by a few groups but is still at a rather early stage. Significant development effort is needed to improve this technique to a mature state. We propose the construction of a new multiplex CARS microscope able to detect several chemical species in parallel with improved chemical selectivity and sensitivity as compared to what reported in literature up to now. To develop the instrument for the biological context we will collaborate with biologists within the School of Biosciences, who will provide biological samples and interesting problems that are difficult to approach even with state-of-the-art fluorescence microscopy. These problems are: a) the change in structure of mitochondria in the pathogenic yeast Candida albicans during programmed cell death (apoptosis) and in the model yeast Saccharomyces cerevisiae during respiratory oscillations, b) the end-bud growth, development and apoptosis of cells in mouse mammary glands, c) the dynamic changes in cell water content and membrane dynamics during development of the slime mold Dictyostelium discoideum. Besides the collaborators within the School of Biosciences, researchers from both physics and biological disciplines worldwide might benefit from the outcome of this work. The usage of this novel microscopy technique is also likely to be of relevance in medical applications, to improve the diagnostic and treatment of diseases. Additionally, the proposed research contains the realization of an economic design of the multiplex CARS microscope for its possible widespread application, so that microscope manufacturers are likely to be interested in this project.

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Microbiology, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search