Optical Tweezers Raman Spectroscopy

The study and understanding of biochemical changes inside single living cells has a huge potential in many fields of research and industry. However, the current techniques used in cell and molecular biology are not applicable to a single cell and are unable to monitor changes in situ. Non biochemical methods such as fluorescence or confocal microscopy require the use of fluorophores and dyes, visualising only a small number of organelles at any one time.

Raman spectroscopy has the capability of identifying different biochemical constituents, such as nucleic acids, proteins and lipids, with the advantage of acquiring all this information concurrently. The natural environment for many cells (such as blood cells) is to be in suspension. Optical Tweezers permits one to constrain a floating cell allowing one to measure its Raman Spectrum (OTRS). This technique is non destructive, non invasive, (and therefore sterile), and little or no sample preparation is required. The detection of glutamate in a single nerve terminal and the study of the effect of alcohol solution on single human red blood cells were some successful biological applications of OTRS.

We succeeded in keeping a single yeast cell alive in the trap for up to three hours, while studying its living processes, one of which was the detection of a hyperosmotic stress response. We also used this time-resolved information to investigate the cell-cycle of a single yeast cell.

In many cases it may not be just desirable to have information about when biochemical changes take place inside a cell but also to find out where these changes occur. Raman images show the distribution of chemicals within the cell. They can be more easily interpreted than spectra alone by biomedical researchers already familiar with imaging techniques and also contain more information than conventional microscopy images.

In a single beam trap a cell continues to move and rotate to a certain degree due to Brownian motion and organelle motility, hence during the acquisition time one measures time-averaged Raman signals. However in this case Raman imaging cannot be performed. To facilitate imaging we proposed using multiple trapping beams around the periphery of the cell to immobilise the cell. This not only allows for immobilising asymmetric cells, but also distributes the optical trapping power more evenly throughout the volume, limiting photodamage. Holographic Optical Tweezers (HOT), produced by a Spatial Light Modulator (SLM), are used to generate such multiple tweezing sites and to scan a floating cell back and forth across the focus of a stationary Raman excitation beam. In this way an image of the entire cell is built up. We demonstrated the first example of a Raman image of a cancer cell in suspension, with movement completely controlled by HOT.

OTRS makes it possible to acquire previously inaccessible time- and space-resolved information of cells in suspension. Biomedical interest in areas such as drug delivery systems, cell-cell interactions and cell signalling means that this is a technique which has great future potential.