The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is ...expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ca
channels are essential to cell birth, life, and death. They can be externally activated by optogenetic tools, but this requires robust introduction of exogenous optogenetic genes for expression of ...photosensitive proteins in biological systems. Here we present femtoSOC, a method for direct control of Ca
channels solely by ultrafast laser without the need for optogenetic tools or any other exogenous reagents. Specifically, by focusing and scanning wavelength-tuned low-power femtosecond laser pulses on the plasma membrane for multiphoton excitation, we directly induced Ca
influx in cultured cells. Mechanistic study reveals that photoexcited flavins covalently bind cysteine residues in Orai1 via thioether bonds, which facilitates Orai1 polymerization to form store-operated calcium channels (SOCs) independently of STIM1, a protein generally participating in SOC formation, enabling all-optical activation of Ca
influx and downstream signaling pathways. Moreover, we used femtoSOC to demonstrate direct neural activation both in brain slices in vitro and in intact brains of living mice in vivo in a spatiotemporal-specific manner, indicating potential utility of femtoSOC.
High-speed optical imaging is an indispensable technology for blur-free observation of fast transient dynamics in virtually all areas including science, industry, defense, energy, and medicine. High ...temporal resolution is particularly important for microscopy as even a slow event appears to occur “fast” in a small field of view. Unfortunately, the shutter speed and frame rate of conventional cameras based on electronic image sensors are significantly constrained by their electrical operation and limited storage. Over the recent years, several unique and unconventional approaches to high-speed optical imaging have been reported to circumvent these technical challenges and achieve a frame rate and shutter speed far beyond what can be reached with the conventional image sensors. In this article, we review the concepts and principles of such ultrafast optical imaging methods, compare their advantages and disadvantages, and discuss an entirely new class of applications that are possible using them.
Microalga-based biomaterial production has attracted attention as a new source of drugs, foods, and biofuels. For enhancing the production efficiency, it is essential to understand its differences ...between heterogeneous microalgal subpopulations. However, existing techniques are not adequate to address the need due to the lack of single-cell resolution or the inability to perform large-scale analysis and detect small molecules. Here we demonstrated large-scale single-cell analysis of
(a unicellular microalgal species that produces paramylon as a potential drug for HIV and colon cancer) with our recently developed high-throughput broadband Raman flow cytometer at a throughput of >1,000 cells/s. Specifically, we characterized the intracellular content of paramylon from single-cell Raman spectra of 10,000
cells cultured under five different conditions and found that paramylon contents in
cells cultured in an identical condition is given by a log-normal distribution, which is a good model for describing the number of chemicals in a reaction network. The capability of characterizing distribution functions in a label-free manner is an important basis for isolating specific cell populations for synthetic biology via directed evolution based on the intracellular content of metabolites.
Organelle positioning in cells is associated with various metabolic functions and signaling in unicellular organisms. Specifically, the microalga Chlamydomonas reinhardtii repositions its ...mitochondria, depending on the levels of inorganic carbon. Mitochondria are typically randomly distributed in the Chlamydomonas cytoplasm, but relocate toward the cell periphery at low inorganic carbon levels. This mitochondrial relocation is linked with the carbon‐concentrating mechanism, but its significance is not yet thoroughly understood. A genotypic understanding of this relocation would require a high‐throughput method to isolate rare mutant cells not exhibiting this relocation. However, this task is technically challenging due to the complex intracellular morphological difference between mutant and wild‐type cells, rendering conventional non‐image‐based high‐event‐rate methods unsuitable. Here, we report our demonstration of intelligent image‐activated cell sorting by mitochondrial localization. Specifically, we applied an intelligent image‐activated cell sorting system to sort for C. reinhardtii cells displaying no mitochondrial relocation. We trained a convolutional neural network (CNN) to distinguish the cell types based on the complex morphology of their mitochondria. The CNN was employed to perform image‐activated sorting for the mutant cell type at 180 events per second, which is 1–2 orders of magnitude faster than automated microscopy with robotic pipetting, resulting in an enhancement of the concentration from 5% to 56.5% corresponding to an enrichment factor of 11.3. These results show the potential of image‐activated cell sorting for connecting genotype–phenotype relations for rare‐cell populations, which require a high throughput and could lead to a better understanding of metabolic functions in cells.
Intelligent image‐activated cell sorting (iIACS) based on the complex morphology of the mitochondria in C. reinhardtii. Using a CNN with a 5 ms processing time, we achieved enrichment of C. reinhardtii populations from 50% to 80.5% and from 5% to 56.5%, resulting in an enrichment factor of 1.6, and 11.3 respectively. These results show the capability iIACS for high event‐rate sorting based on complex intracellular morphologies.
We report high-throughput optical coherence tomography (OCT) that offers 1,000 times higher axial scan rate than conventional OCT in the 800 nm spectral range. This is made possible by employing ...photonic time-stretch for chirping a pulse train and transforming it into a passive swept source. We demonstrate a record high axial scan rate of 90.9 MHz. To show the utility of our method, we also demonstrate real-time observation of laser ablation dynamics. Our high-throughput OCT is expected to be useful for industrial applications where the speed of conventional OCT falls short.
High-speed isolation of microparticles (
e.g.
, microplastics, heavy metal particles, microbes, cells) from heterogeneous populations is the key element of high-throughput sorting instruments for ...chemical, biological, industrial and medical applications. Unfortunately, the performance of continuous microparticle isolation or so-called sorting is fundamentally limited by the trade-off between throughput, purity, and yield. For example, at a given throughput, high-purity sorting needs to sacrifice yield, or
vice versa
. This is due to Poisson statistics of events (
i.e.
, microparticles, microparticle clusters, microparticle debris) in which the interval between successive events is stochastic and can be very short. Here we demonstrate an on-chip microparticle sorter with an ultrashort switching window in both time (10 μs) and space (10 μm) at a high flow speed of 1 m s
−1
, thereby overcoming the Poisson trade-off. This is made possible by using femtosecond laser pulses that can produce highly localized transient cavitation bubbles in a microchannel to kick target microparticles from an acoustically focused, densely aligned, bumper-to-bumper stream of microparticles. Our method is important for rare-microparticle sorting applications where both high purity and high yield are required to avoid missing rare microparticles.
We demonstrate an on-chip microparticle sorter with an ultrashort switching window using femtosecond laser pulses to overcome the fundamental limitation of the sorting performance described by Poisson statistics.
Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to ...differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm
) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of
.
Valproic acid (VPA) is known to induce hepatic steatosis due to mitochondrial toxicity in rodents and humans. In the present study, we administered VPA to SD rats for 3 or 14 days at 250 and 500 ...mg/kg and then performed lipidomics analysis to reveal VPA-induced alteration of the hepatic lipid profile and its association with the plasma lipid profile. VPA induced hepatic steatosis at the high dose level without any degenerative changes in the liver on day 4 (after 3 days dosing) and at the low dose level on day 15 (after 14 days dosing). We compared the plasma and hepatic lipid profiles obtained on day 4 between the VPA-treated and control rats using a multivariate analysis to determine differences between the two groups. In total, 36 species of plasma lipids and 24 species of hepatic lipids were identified as altered in the VPA-treated group. Of these lipid species, ether-phosphatidylcholines (ePCs), including PC(16:0e/22:4) and PC(16:0e/22:6), were decreased in both the plasma and liver from the low dose level on day 4, however, neither an increase in hepatic TG level nor histopathological hepatic steatosis was observed at either dose level on day 4. Hepatic mRNA levels of glycerone-phosphate O-acyltransferase (Gnpat), which is a key enzyme for biosynthesis of ePC, was also decreased by treatment with VPA along with the decrease in ePCs. In conclusion, the changes in ePCs, (PC16:0e/22:4 and PC16:0e/22:6), have potential utility as predictive biomarkers for VPA-induced hepatic steatosis.
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