Composition-tunable nanocrystals are fluorescent nanoparticles with a uniform particle size and with adjustable optical
characteristics. When used for optical labeling of biomolecular targets these and other nanotechnology solutions have
enabled new approaches which are possible because of the high optical output, narrow spectral signal, consistent
quantum efficiency across a broad emission range and long lived fluorescent behavior of the nanocrystals. When
coupled with spectral imaging the full potential of multiplexing multiple probes in a complex matrix can be realized.
Spectral imaging can be used to improve sensitivity of narrowband fluorophores through application of chemometric
image processing techniques used to reduce the influence of autofluorescence background.
Composition-tunable nanocrystals can be complexed together to form nanoclusters which have the advantage of
significantly stronger signal and therefore a higher sensitivity. These nanoclusters can be targeted in biomolecular
systems using standard live-cell labeling and immunohistochemistry based techniques. Composition-tunable
nanocrystals and nanoclusters have comparable mass and brightness across a wide emission range. This enables the
production of nanocrystal-based probes that have comparable reactivity and sensitivity over a large color range.
We present spectral imaging results of antibody targeted nanocrystal cluster labeling of target proteins in cultured cells
and a Western blot experiment. The combination of spectral imaging with the use of clusters of nanocrystals further
improves the sensitivity over either of the approaches independently.
Apoptosis, also known as programmed cell death, is a process in which cells initiate a series of events to trigger their
own demise. Normal cells use this mechanism in the regulation of their life cycle. On the contrary, abnormal or cancer
cells have lost the ability to regulate themselves by this process. Because of this, there is much interest in the study of
the apoptotic process. Currently, there are many commercial assays available to detect apoptosis in cells, most of which
are fluorescence based. Limitations of such fluorescent assays lead to arbitrary or inclusive results.
Raman spectroscopy is a powerful technique that yields specific molecular information on samples under study. The
Raman spectra obtained from cell samples are very complex, yet the differences in the complex Raman spectra analyzed
using chemometric techniques can identify chemical and physiological information about cells. Furthermore, Raman
spectroscopy is a sensitive, rapid, reagentless, low-cost technique, making it a superior alternative to traditional
fluorescence based apoptosis assays.
In this study, we have employed Raman spectroscopy and Raman chemical imaging, along with chemometric
techniques, to distinguish apoptotic cells from non-apoptotic cells in two prostate cancer cell lines, PC3 and LnCAP.
Initial results indicate that Raman spectra of apoptotic and non-apoptotic cells are different in both cell lines.
Furthermore, chemometric analysis of the data shows that the spectra separate into two distinct populations, apoptotic
and non-apoptotic. Traditional fluorescence based apoptotic assays confirm the results. This work provides ample
evidence that Raman spectroscopy is a valuable tool in biomedical imaging.
Raman spectroscopy is a powerful technique for rapid, non-invasive and reagentless analysis of materials, including
biological cells and tissues. Raman Molecular Imaging combines high molecular information content Raman
spectroscopy and digital full field imaging to enable the investigation of cells and tissues. We have conducted widefield
imaging using a new class of birefringent liquid crystal tunable filter that provides high throughput over an extended
wavelength range. This tool has been applied to investigate the linkage between reagentless spectral imaging in tissue
and cells and standard reagent based approaches. In this report, we describe Raman imaging data on a clinical tissue
sample and cultured cells. The results demonstrate the sensitivity of Raman Molecular Imaging and fluorescence
spectral imaging to molecular differences in biological systems laying the foundation for the eventual use of this
approach as a biological research and clinical diagnostic tool.