Raman-Activated Cell Counting for Profiling Carbon Dioxide Fixing Microorganisms

Abstract

Raman microspectroscopy is a label-free and nondestructive technique to measure the intrinsic chemical profile of single cells. The naturally weak Raman signals hampered the application of Raman spectroscopy for high-throughput measurements. Nearly all photosynthetic microorganisms contain carotenoids that are active molecules for resonance Raman at a 532 nm excitation wavelength. Hence, the acquisition time for a single photosynthetic microorganism can be as short as 1 ms. The carotenoid bands in Raman spectra of photosynthetic microorganisms utilizing (13)CO(2) shifted when compared to the spectra of cells utilizing (12)CO(2). Here, a mixture of (12)C- and (13)C-cyanobacterial cells were counted using a microfluidic-device-based Raman-activated cell counting procedure to prove the concept that Raman spectroscopy can be used as a high-throughput method to profile a cell population.

Demonstration of Carbon Catabolite Repression in Naphthalene Degrading Soil Bacteria via Raman Spectroscopy Based Stable Isotope Probing

“…11,24 Raman spectroscopy based 13 C stable isotope probing has been effectively used in numerous studies like to identify environmental bacterial communities that fix carbon dioxide, to identify naphthalene degraders and to study predator prey interactions via monitoring the carbon flow. [25][26][27][28][29][30] Monitoring of deuterium labeled carbon sources via Raman spectroscopy has also been used to identify novel mouse cecal microbes 31 and there have been studies to visualize the spatial distribution of deuterium labeled proteins in single human cells. 32 Even, deuterium label probing via CARS imaging has been utilized to identify the presence of exogenous lipids in monocytes.…”

Single cell biotechnology to shed a light on biological ‘dark matter’ in nature

“…It is estimated that over 99% of microorganisms have not as yet been cultivated (Whitman et al ., ). These uncultured microbes are the ‘dark matter’ of the microbiological world and play important roles in natural ecosystems and the human microbiome (Lagier et al ., ; Rinke et al ., ); however, their ecological role and function remain largely elusive (Lasken, ; Li et al ., 2012a,b). Furthermore, these uncultured bacteria represent a significant, yet largely untapped, genetic resource for use in synthetic biology for the provision of novel bioparts or biobricks, in medicine for new drug biosynthesis, in industry for robust biocatalysts and biofuel synthesis, and in environmental bioremediation for new biodegradation genes.…”

“…Among the various single cell sorting techniques (Lasken, ; Li et al ., 2012a,b), an emerging approach is Raman‐activated cell sorting (RACS), which overcomes the requirement for external labelling. Single cell Raman spectra (SCRS) provide a label‐free, non‐invasive and intrinsic phenotypic profile of individual cells which can be used to characterize cell type, physiological state and cell functionalities (Huang et al ., 2004; 2007a,b,c; Harz et al ., ; Li et al ., 2012a,b; Wang et al ., ). A typical SCRS provides an intrinsic chemical ‘fingerprint’ of a single cell, and usually contains multi‐parameter (> 1000 readings) including rich information on nucleic acids, protein, carbohydrates and lipids (Li et al ., 2012a,b).…”

“…Single cell Raman spectra (SCRS) provide a label‐free, non‐invasive and intrinsic phenotypic profile of individual cells which can be used to characterize cell type, physiological state and cell functionalities (Huang et al ., 2004; 2007a,b,c; Harz et al ., ; Li et al ., 2012a,b; Wang et al ., ). A typical SCRS provides an intrinsic chemical ‘fingerprint’ of a single cell, and usually contains multi‐parameter (> 1000 readings) including rich information on nucleic acids, protein, carbohydrates and lipids (Li et al ., 2012a,b). Since SCRS measures the vibration of molecular bonds, it is sensitive to stable isotope compounds and SCRS undergoes Raman shift when cells incorporate stable isotope compounds (e.g.…”

“…C/N metabolism and metabolic activity) and to define cells of interest at a single cell level. A RACS system consists of a SCRS detection system and a cell isolation system that can be optical tweezers (Huang et al ., ), a microfluidic device (Li et al ., 2012a,b) or a single cell ejection system (Wang et al ., ).…”