Dolor crónico y Sistema Nervioso Central (SNC)

3.6.3 Raman optical tweezers

Although a Raman tweezers system is not employed in the course of this work, these systems are often used in similar studies, and for this reason, a brief description is included here. Ra-man tweezers are based on combining the principle of RaRa-man micro-spectroscopy with optical trapping (52; 53), as illustrated in Figure 3.6. Optical trapping, or optical tweezing, is a tech-nique that utilises a high NA MO, usually configured in an inverted microscope, together with a suitable laser, to trap a cell in the waist of focused laser beam. The tightly focused light cre-ates a sharp gradient of intensity which leads to gradient forces trapping the cell. The trapped cell can be suspended in a liquid (or air) environment, and moved in three dimensions by ma-nipulating a component in the optical set-up. By combining Raman micro-spectroscopy with optical trapping, individual cells can be biochemically probed under physiological conditions, or in microfluidic chips.

Figure 3.6: (a) Schematic of a standard Raman optical tweezers set-up, (b) schematic of a dual beam Raman tweezers set-up. HL, halogen lamp; C, condenser; MO, microscope objective; L, lens; M, mirror; DC, digital camera; DB, dichroic beam splitter; CA, confocal aperture, LP, long-pass filter.

Raman tweezers offer exciting new avenues of research, particularly in the areas of microflu-idics and urine cytology. This technique can be used for investigating the biochemistry of living cells in their natural environment with 3D spatial resolution. (54) It can also be used for diag-nostic classification of bladder and prostate cancer cells, as shown elsewhere (55; 56; 57; 58).

3.6.4 Raman spectroscopy with a fiberoptic probe

Similar to Raman tweezers, a fiberoptic Raman spectroscopy system is not applied within this thesis, but it is discussed as an alternative diagnostic approach. A fiberoptic Raman set-up con-sists of a specialised fiber probe, an excitation source (laser), fiber couplers to deliver the light in and out of the fiber, filters to remove unwanted signals, and a detection system (spectrograph

and cooled CCD camera), as can be seen in Figure 3.7. This fiberoptic probe can be inserted during a cystoscopic diagnostic procedure, and be used to give real-time analysis and classi-fications of the bladder wall. Similar to conventional Raman micro-spectroscopy, the Raman fiberoptic probe can be used to monitor in vitro and ex vivo specimen also. The main advan-tages of this system include the potential to replace the need for multiple biopsies, reductions in the turn-around time for diagnosis, reductions in histological/pathological costs, and improved surgical procedures by using the probe to identify border regions of diseased or damaged tissue.

Figure 3.7: Schematic of a basic Raman fiberoptic probe set-up, which can be applied to bio-logical samples both in vivo and in vitro, with a front view of the design of a fiberoptic probe inset, showing the central delivery fiber which is surrounded by several collection fibers. It is of note that it is often more conventional to integrate the filters into the tip of the fiberoptic probe instead, but for ease of illustration, they have been shown in the positions above.

A Raman probe for cystoscopic or endoscopic diagnostics must be able to fit into the in-strument channel of a standard cystoscope (or endoscope or catheter), be biocompatible, and robust enough to withstand decontamination/disinfection processes. NIR lasers (785–830 nm) are generally used for Raman fiberoptic probes since they minimise thermal damage to the tissue sample being analysed. It is also essential that there are low fluorescence, or photolumi-nescence, signals generated within the probe. The general design of a Raman fiberoptic probe is shown in Figure 3.7; consisting of a central delivery fiber that is surrounded by several col-lection fibers, where the diameter of each fiber is between 100 and 400 µm depending on the system design. Typically a bandpass filter is placed at the tip of the excitation fiber to remove unwanted background signals, and a longpass filter is placed at the tip of the collection fibers to prevent Rayleigh scattered light from entering these fibers. Lenses (such as a ball lens, made from sapphire) at the probe tip or tapered fiber tips can also be used to improve the efficiency of delivering both the excitation light to the tissue and Raman scattered light into the collection fibers (further information is available in Ref. (59)).

3.7. SUMMARY 34

3.7 Summary

Each of the key components of a Raman micro-spectrometer have been discussed here. Impor-tant considerations for the design of a Raman system include the source laser, the microscope body, choice of spectrometer and CCD, and the utilisation of a confocal aperture. As has been highlighted in this chapter, it is particularly important to choose a laser that is suitable for the application involved, such as a laser with a TEM₀₀ Gaussian profile. Other considerations in-volve choosing an NIR laser in order to minimise the possibility of tissue damage, or choosing a laser in the visible wavelength region (e.g. 532 nm) if shorter acquisition times are desired. It is also important to choose a spectrograph and CCD camera that have good quantum efficiencies within the desired acquisition range.

The custom Raman micro-spectrometer that was designed and built in our laboratory has been described in Section 3.6.1, with information given on each of the optical components within the system, such that the design could be replicated in another laboratory. Further details on commercial Raman systems, along with a Raman tweezers set-up and a fiberoptic Raman system have also been provided.

Raman spectroscopy is an opto-electronic technique, resulting in complex spectra that re-quire the development of pre-processing and multivariate statistical algorithms in order to achieve reliable and reproducible spectra, which are necessary for the development of an accurate di-agnostic classifier. Such algorithms involve accurate system calibration (post-acquisition), re-moval of unwanted background signals, and noise reduction, as discussed discussed in further detail in Chapter 4.

Chapter 4