Raman tweezers sorting of single microbial cells

Abstract

We have selectively isolated microbial cells by identifying and then manipulating cells using a combination of Raman microspectroscopy and optical trapping. The criterion for cell discrimination is based on spectral peak shifts within the Raman spectrum of individual cells. A specific shift in the phenylalanine peak position from 1001 rel. cm(-1) to 965 rel. cm(-1) is utilized to indicate the uptake of (13) C within the cell that utilized (13) C-substrate. Cells were captured and manipulated using an infrared (1064 nm) laser while Raman spectra were acquired over shorter timescales (30 s) using a co-aligned 514.5 nm laser beam. Selected cells were manoeuvred to a clean part of a capillary tube and the tubes were cleaved to physically separate the cells. The technique was tested for cell viability and cross-contamination effects using 70 single yeast cells (Saccharomyces cerevisia). Following these tests, 58 single bacterial cells (Escherichia coli DH5α, and Pseudomonas fluorescens SBW25::Km-RFP) that exhibited (13) C uptake were sorted from bacterial populations. Among those isolated cells, 11 out of 18 yeast cells and 7 out of 18 single SBW25::Km-RFP cells were recovered by incubation; 2 out of 7 sorted yeast cells and 3 out of 8 sorted bacterial cells (single SBW25::Km-RFP) were genome amplified correctly. We show that the Raman tweezers approach has the potential to open a new frontier to study unculturable microorganisms, which account for more than 99% microbes in natural environment.

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

“…BONCAT-FISH allows the activity screening of thousands of cells within a few hours, rather than a few dozen per day as achieved by isotope labeling techniques. Both bioorthogonal (this study) and nondestructive isotope labeling (10,60) approaches further allow the sorting of individual cells and aggregates from complex samples, although sorting throughput for BONCAT-FACS is much higher. Sorted cells can be subjected to whole-genome amplification (WGA) and sequencing, thus allowing direct access to the genetic potential of cells functionally important under defined conditions.…”

“…With a few notable exceptions (10,11,39,(60)(61)(62), single-cell genome sequencing efforts so far have been "target-blind"; they did not select for a specific taxon of interest or focus on metabolically active cells that could be considered key species for ecosystem functioning. The power of these techniques to investigate microbial partnerships involved in AOM was recently demonstrated by the separation of FISH-identified ANME consortia from methane seep sediments via immunomagnetic capture [magneto-FISH (39, 63)] and fluorescent-activated cell sorting [FISH-FACS (61,64)].…”

Immobilized Monolayer Nanoparticles in a Microfluidic Device for Surface Enhanced Raman Scattering Measurement

“…The C-COO − vibrational stretch for 12 C-lactate reaches its peak at 854 cm −1 , whereas this peak shifts to (126) 843 cm −1 for 13 C-lactate. Due to the extra neutron in the 13 C nucleus compared to 12 C, the vibrational energy of 13 C is smaller and its peak position shifts to the left [26]. Figure 4(b) shows the Raman spectrum of 10-mM ATP.…”

Coupling FACS and Genomic Methods for the Characterization of Uncultivated Symbionts

“…Other methods such as serial dilution (Zhang, et al, 2006), micromanipulation (Kvist, et al, 2007;Hongoh, et al, 2 2008), laser capture microdissection (Navin, et al, 2011), Raman tweezers (Brehm-Stecher & Johnson, 2004;Huang, et al, 2009) and microfluidics (Marcy, et al, 2007;Blainey, et al, 2011) have also been used effectively to separate cells prior to genetic or genomic analysis. However, for characterizing uncultivated symbionts these methods fall short of FACS in a number of ways.…”