Among various optical methods, fluorescence imaging has been the most widely exploited thanks to its superior sensitivity and specificity, but the resolvable colors are restricted to 2-5 colors because of the intrinsically broad and featureless spectra. Recently, this fluorescent “color barrier” was broken and super-multiplex optical imaging became possible taking advantage of well-designed Raman probes. However, the acquisition of the super-multiplex images is still relatively slow which impedes wider applications. Here, we demonstrate fast super-multiplex organelle imaging with high-speed color switching and acquisition, which accelerates the imaging speed by 2 orders of magnitude. We applied it in imaging cytometry, tracing mitosis and fast organelle motions in live cells. We anticipate that high-speed supermultiplex optical imaging can expand to a much wider field of biological researches.
Worldwide, there has been an increase in the number of cases of non-Hodgkin lymphoma (NHL). Burkitt lymphoma comprises of 30-40% of pediatric NHL cases and is a rapidly growing tumor. Access to efficient diagnostic paradigms are therefore crucial for quick therapeutic intervention. Currently, the identification of Burkitt lymphoma and other NHL involves histologic and genetic testing which can be costly and slow. Also, the process of fixing tissue and staining biopsy samples can lead to inconsistent results. Recently, Raman spectroscopy has exposed potential biomarkers in B-cells that could be indicative of cancer. However, slow acquisition speed limits the viability of adapting Raman spectroscopy in a clinical setting. Here we demonstrate a high-speed method to visualize Burkitt lymphoma cells and non-malignant B-cells which does not involve chemical alteration or destruction of cells. Preliminary results indicate higher collection of lipid droplets in malignant B-cells compared to normal B-cells. Using a support-vector machine learning algorithm, we were able to exploit these chemical differences and classify malignant cells from non-malignant cells with a sensitivity of 80% and specificity of 81.2%. Further work into refining this process can lead towards faster identification of cells and could potentially provide deeper insights into the chemical processes that occur within malignant blood cells.
Simultaneous localization of multiple cellular components related to the cellular activities, e.g. metabolism of small molecules, is not well understood due to the intrinsic limitations of fluorescence imaging technologies. The broad fluorescence emission often limits the available color number to ~4. Additionally, staining of small metabolic precursors is still difficult using fluorophores because the relatively large size of fluorophores will affect the regular metabolism of small molecules. Here, we apply our newly developed high-speed multicolor stimulated Raman and fluorescence imaging platform to observe and investigate lipid metabolism in live HeLa cells. Metabolic products generated from the deuterated palmitic acid were imaged in the Raman silent region using stimulated Raman scattering microscopy; meanwhile four kinds of organelles were imaged using fast-tunable confocal fluorescence microscopy. By taking advantages of both stimulated Raman imaging and fluorescence imaging, it enables the localization of multiple components up to five during cellular metabolism in live cells, which can be a helpful method to research complex biomedical processes.
Polarization-resolved stimulated Raman scattering spectroscopies and microscopies have been utilized to investigate the symmetry and orientation of molecular vibrational modes and to provide extra spectral signatures, while the polarization modulation introduced additional complexity and the successive measurement on different polarization states limits the imaging speed. Here we demonstrate dual-polarization hyperspectral stimulated Raman scattering microscopy which enables detailed imaging measurement in two orthogonal polarization states simultaneously at video-rate speed. Two polarized Raman images can be obtained within ~0.03s, while the Raman shift is scanned in the CH stretching region in 3 s by virtue of rapid wavelength tunability of laser pulses. We observed different kinds of polymer beads and liquid, the results of which prove the ability to measure the symmetry of vibrational modes and to distinguish the overlapped peaks. Moreover, HeLa cells were imaged to prove the applicability to biological samples and show additional spectral signatures in perpendicular spectra. This novel method endows fast yet detailed imaging analysis of biomolecules in live specimens to research on drug delivery, electric stimulation, metabolic engineering etc.