Raman cell sorting for single-cell research.

Cells constitute the fundamental units of living organisms. Investigating individual differences at the single-cell level facilitates an understanding of cell differentiation, development, gene expression, and cellular characteristics, unveiling the underlying laws governing life activities in depth. In recent years, the integration of single-cell manipulation and recognition technologies into detection and sorting systems has emerged as a powerful tool for advancing single-cell research. Raman cell sorting technology has garnered attention owing to its non-labeling, non-destructive detection features and the capability to analyze samples containing water. In addition, this technology can provide live cells for subsequent genomics analysis and gene sequencing. This paper emphasizes the importance of single-cell research, describes the single-cell research methods that currently exist, including single-cell manipulation and single-cell identification techniques, and highlights the advantages of Raman spectroscopy in the field of single-cell analysis by comparing it with the fluorescence-activated cell sorting (FACS) technique. It describes various existing Raman cell sorting techniques and introduces their respective advantages and disadvantages. The above techniques were compared and analyzed, considering a variety of factors. The current bottlenecks include weak single-cell spontaneous Raman signals and the requirement for a prolonged total cell exposure time, significantly constraining Raman cell sorting technology's detection speed, efficiency, and throughput. This paper provides an overview of current methods for enhancing weak spontaneous Raman signals and their associated advantages and disadvantages. Finally, the paper outlines the detailed information related to the Raman cell sorting technology mentioned in this paper and discusses the development trends and direction of Raman cell sorting.

Precision isolation and cultivation of single cells by vortex and flat-top laser ejection.

Single-cell isolation stands as a critical step in single-cell studies, and single-cell ejection technology based on laser induced forward transfer technology (LIFT) is considered one of the most promising methods in this regard for its ability of visible isolating single cell from complex samples. In this study, we improve the LIFT technology and introduce optical vortex laser-induced forward transfer (OV-LIFT) and flat-top laser-induced forward transfer (FT-LIFT) by utilizing spatial light modulator (SLM), aiming to enhance the precision of single-cell sorting and the cell's viability after ejection. Experimental results demonstrate that applying vortex and flat-top beams during the sorting and collection process enables precise retrieval of single cells within diameter ranges of 50 µm and 100 µm, respectively. The recovery rates of Saccharomyces cerevisiae and Escherichia coli DH5α single cell ejected by vortex beam are 89 and 78%, by flat-top beam are 85 and 57%. When employing Gaussian beam sorting, the receiving range extends to 400 µm, with cultivation success rates of S. cerevisiae and E. coli DH5α single cell are 48 and 19%, respectively. This marks the first application of different mode beams in the ejection and cultivation of single cells, providing a novel and effective approach for the precise isolation and improving the viability of single cells.

Rapid detection of six Oceanobacillus species in Daqu starter using single-cell Raman spectroscopy combined with machine learning.

Many traditional fermented foods and beverages industries around the world request the addition of multi-species starter cultures. However, the microbial community in starter cultures is subject to fluctuations due to their exposure to an open environment during fermentation. A rapid detection approach to identify the microbial composition of starter culture is essential to ensure the quality of the final products. Here, we applied single-cell Raman spectroscopy (SCRS) combined with machine learning to monitor Oceanobacillus species in Daqu starter, which plays crucial roles in the process of Chinese baijiu. First, a total of six Oceanobacillus species (O. caeni, O. kimchii, O. iheyensis, O. sojae, O. oncorhynchi subsp. Oncorhynchi and O. profundus) were detected in 44 Daqu samples by amplicon sequencing and isolated by pure culture. Then, we created a reference database of these Oceanobacillus strains which correlated their taxonomic data and single-cell Raman spectra (SCRS). Based on the SCRS dataset, five machine-learning algorithms were used to classify Oceanobacillus strains, among which support vector machine (SVM) showed the highest rate of accuracy. For validation of SVM-based model, we employed a synthetic microbial community composed of varying proportions of Oceanobacillus species and demonstrated a remarkable accuracy, with a mean error was less than 1% between the predicted result and the expected value. The relative abundance of six different Oceanobacillus species during Daqu fermentation was predicted within 60 min using this method, and the reliability of the method was proved by correlating the Raman spectrum with the amplicon sequencing profiles by partial least squares regression. Our study provides a rapid, non-destructive and label-free approach for rapid identification of Oceanobacillus species in Daqu starter culture, contributing to real-time monitoring of fermentation process and ensuring high-quality products.

Deep learning-based ultra-fast identification of Raman spectra with low signal-to-noise ratio.

Ensuring the correct use of cell lines is crucial to obtaining reliable experimental results and avoiding unnecessary waste of resources. Raman spectroscopy has been confirmed to be able to identify cell lines, but the collection time is usually 10-30 s. In this study, we acquired Raman spectra of five cell lines with integration times of 0.1 and 8 s, respectively, and the average accuracy of using long-short memory neural network to identify the spectra of 0.1 s was 95%, and the average accuracy of identifying the spectra of 8 s was 99.8%. At the same time, we performed data enhancement of 0.1 s spectral data by real-valued non-volume preserving method, and the recognition average accuracy of long-short memory neural networks recognition of the enhanced spectral data was improved to 96.2%. With this method, we shorten the acquisition time of Raman spectra to 1/80 of the original one, which greatly improves the efficiency of cell identification.

Stable SERS Detection of Lactobacillus fermentum Using Optical Tweezers in a Microfluidic Environment.

Rapid identification of fermented lactic acid bacteria has long been a challenge in the brewing industry. This study combined label-free surface-enhanced Raman scattering (SERS) and optical tweezer technology to construct a test platform within a microfluidic environment. Six kinds of lactic acid bacteria common in industry were tested to prove the stability of the SERS spectra. The results demonstrated that the utilization of optical tweezers to securely hold the bacteria significantly enhanced the stability of the SERS spectra. Furthermore, SVM and XGBoost machine learning algorithms were utilized to analyze the obtained Raman spectra for identification, and the identification accuracies exceeded 95% for all tested lactic acid bacteria. The findings of this study highlight the crucial role of optical tweezers in improving the stability of SERS spectra by capturing bacteria in a microfluidic environment, prove that this technology could be used in the rapid identification of lactic acid bacteria, and show great significance in expanding the applicability of the SERS technique for other bacterial testing purposes.

Building an ensemble learning model for gastric cancer cell line classification via rapid raman spectroscopy.

Cell misuse and cross-contamination can affect the accuracy of cell research results and result in wasted time, manpower and material resources. Thus, cell line identification is important and necessary. At present, the commonly used cell line identification methods need cell staining and culturing. There is therefore a need to develop a new method for the rapid and automated identification of cell lines. Raman spectroscopy has become one of the emerging techniques in the field of microbial identification, with the advantages of being rapid and noninvasive and providing molecular information for biological samples, which is beneficial in the identification of cell lines. In this study, we built a library of Raman spectra for gastric mucosal epithelial cell lines GES-1 and gastric cancer cell lines, such as AGS, BGC-823, HGC-27, MKN-45, MKN-74 and SNU-16. Five spectral datasets were constructed using spectral data and included the full spectrum, fingerprint region, high-wavelength number region and Raman background of Raman spectra. A stacking ensemble learning model, SL-Raman, was built for different datasets, and gastric cancer cell identification was achieved. For the gastric cancer cells we studied, the differentiation accuracy of SL-Raman was 100% for one of the gastric cancer cells and 100% for six of the gastric cancer cells. Additionally, the separation accuracy for two gastric cancer cells with different degrees of differentiation was 100%. These results demonstrate that Raman spectroscopy combined with SL-Raman may be a new method for the rapid and accurate identification of gastric cancer. In addition, the accuracy of 94.38% for classifying Raman spectral background data using machine learning demonstrates that the Raman spectral background contains some useful spectral features. These data have been overlooked in previous studies.

Classification of pathogenic bacteria by Raman spectroscopy combined with variational auto-encoder and deep learning.

Rapid and early identification of pathogens is critical to guide antibiotic therapy. Raman spectroscopy as a noninvasive diagnostic technique provides rapid and accurate detection of pathogens. Raman spectrum of single cells serves as the "fingerprint" of the cell, revealing its metabolic characteristics. Rapid identification of pathogens can be achieved by combining Raman spectroscopy and deep learning. Traditional classification techniques frequently require lots of data for training, which is time costing to collect Raman spectra. For trace samples and strains that are difficult to culture, it is difficult to provide an accurate classification model. In order to reduce the number of samples collected and improve the accuracy of the classification model, a new pathogen detection method integrating Raman spectroscopy, variational auto-encoder (VAE), and long short-term memory network (LSTM) is proposed in this paper. We collect the Raman signals of pathogens and input them to VAE for training. VAE will generate a large number of Raman spectral data that cannot be distinguished from the real spectrum, and the signal-to-noise ratio is higher than that of the real spectrum. These spectra are input into the LSTM together with the real spectrum for training, and a good classification model is obtained. The results of the experiments reveal that this method not only improves the average accuracy of pathogen classification to 96.9% but also reduces the number of Raman spectra collected from 1000 to 200. With this technology, the number of Raman spectra collected can be greatly reduced, so that strains that are difficult to culture or trace can be rapidly identified.

Multi-point scanning confocal Raman spectroscopy for accurate identification of microorganisms at the single-cell level.

Raman spectroscopy has been widely used for microbial analysis due to its exceptional qualities as a rapid, simple, non-invasive, reproducible, and real-time monitoring tool. The Raman spectrum of a cell is a superposition of the spectral information of all biochemical components in the laser focus. In the case where the microbial size is larger than the laser spot size, the Raman spectrum measured from a single-point within a cell cannot capture all biochemical information due to the spatial heterogeneity of microorganisms. In this work, we have proposed a method for the accurate identification of microorganisms using multi-point scanning confocal Raman spectroscopy. Through an image recognition algorithm and the control of a high-precision motorized stage, Raman spectra can be integrated at one time to measure the multi-point biochemical information of microorganisms. This solves the problem that the measured single microbial cells are of different sizes, and the laser spot of the confocal Raman system is not easy to change. Here, the single-cell Raman spectra of three Escherichia coli and seven Lactobacillus species were measured separately. The commonly used supervised classification method, support vector machine (SVM), was applied to compare the data based on the single-point spectra and multi-point scanning spectra. Multi-point spectra showed superior performance in terms of their accuracy and recall rates compared with single-point spectra. The results show that multi-point scanning confocal Raman spectra can be used for more accurate species classification at different taxonomic levels, which is of great importance in species identification.

Rapid detection of beer spoilage bacteria based on label-free SERS technology.

Beer spoilage bacteria have been a headache for major breweries. In order to rapidly identify spoilage bacteria and improve the sensitivity and signal-to-noise ratio of bacterial SERS detection, the label-free SERS technique was used as a starting point, and we found eight bacteria species that led to beer spoilage. The impact of AgNP concentration and AgNP and bacterial binding time on the final results were thoroughly investigated. To maximize the increase in the SERS signal, an aluminized chip was created. We merged the t-SNE reduced dimensional analysis algorithm, and SVM, KNN, and LDA machine learning algorithms to further investigate the effect of the approach on the final identification rate. The results demonstrate that SERS spectra had an increased intensity and signal-to-noise ratio. The machine learning classification accuracy rates were all above 90%, indicating that the bacteria were correctly classified and identified.

Laser induced forward transfer isolating complex-shaped cell by beam shaping.

Beam shaping techniques have been widely used in holographic optical tweezers to accurately manipulate tiny particles and hologram optimization algorithms have also been widely reported to improve the optical trapping performance. In this paper, we presented a beam shaping laser induced forward transfer (BS-LIFT) technique to isolate complex-shaped cells. To do this, we built up a BS-LIFT instrument which combined beam shaping methods and laser induced forward transfer using liquid-crystal-on-silicon spatial light modulator. The laser beam was modulated into multiple desired points at the focal plane employing the Gerchberg-Saxton (GS) algorithm. Feasibility was verified through transferring various samples. To our knowledge, this is the first demonstration of BS-LIFT applied to the transfer complex-shaped cells. We successfully transferred cells whose size ranged from 1 µm to 100 µm. Our design will provide a novel approach for the application of this beam shaping technique and the isolation of single cells with variable shapes.