In this review we describe label-free optical spectroscopy techniques which are able to non-invasively measure the (bio)chemistry in biological systems. Raman spectroscopy uses visible or near-infrared light to measure a spectrum of vibrational bonds in seconds. Coherent anti-Stokes Raman (CARS) microscopy and stimulated Raman loss (SRL) microscopy are orders of magnitude more efficient than Raman spectroscopy, and are able to acquire high quality chemically-specific images in seconds. We discuss the benefits and limitations of all techniques, with particular emphasis on applications in biomedicine—both in vivo (using fiber endoscopes) and in vitro (in optical microscopes).

Finite element electromagnetic simulations of scanning probe microscopy tips and substrates are presented. The enhancement of the scattered light intensity is found to be as high as 10(12) for a 20 nm radius gold tip, and tip-substrate separation of 1 nm. Molecular resolution imaging (< 1 nm) is achievable, even with a relatively large radius tip (20 nm). We also make predictions for imaging in aqueous environments, noting a sizable red shift of the spectral peaks. Finally, we discuss signal levels, and predict that high-speed Raman mapping should be possible with gold substrates and a small tip-substrate separation (< 4 nm).

The characterisation of stem cells is of vital importance to regenerative medicine. Failure to separate out all stem cells from differentiated cells before therapies can result in teratomastumours of multiple cell types. Typically, characterisation is performed in a destructive manner with fluorescent assays. A truly non-invasive method of characterisation would be a major breakthrough in stem cell-based therapies. Raman spectroscopy has revealed that DNA and RNA levels drop when a stem cell differentiates into other cell types, which we link to a change in the relative sizes of the nucleus and cytoplasm. We also used Raman spectroscopy to investigate the biochemistry within an early embryo, or blastocyst, which differs greatly from colonies of embryonic stem cells. Certain cell types that differentiate from stem cells can be identified by directly imaging the biochemistry with CARS microscopy; examples presented are hydroxyapatite -a precursor to bone, and lipids in adipocytes.

Finite element simulations of laser-induced heating in scanning probe microscopy are presented. The electromagnetic field is first simulated for a variety of tip and substrate materials, and for air and aqueous environments. This electromagnetic field, in the end of the tip and substrate under the tip, produces Joule heating. Using this Joule heat source, steady state thermal simulations are performed. As a result of the large enhancement of optical power by the tip-substrate cavity, predicted temperature rises can be over 3 orders of magnitude higher than the values predicted without a tip present, but the optical signal can be enhanced by over 10 orders. Gold tips and substrates are predicted to give the highest optical signal for a given temperature increase.