Detectability of unresolved particles in off-axis digital holographic microscopy.

Off-axis digital holographic microscopy (DHM) provides both amplitude and phase images, and so it may be used for label-free 3D tracking of micro- and nano-sized particles of different compositions, including biological cells, strongly absorbing particles, and strongly scattering particles. Contrast is provided by differences in either the real or imaginary parts of the refractive index (phase contrast and absorption) and/or by scattering. While numerous studies have focused on phase contrast and improving resolution in DHM, particularly axial resolution, absent have been studies quantifying the limits of detection for unresolved particles. This limit has important implications for microbial detection, including in life-detection missions for space flight. Here we examine the limits of detection of nanosized particles as a function of particle optical properties, microscope optics (including camera well depth and substrate), and data processing techniques and find that DHM provides contrast in both amplitude and phase for unresolved spheres, in rough agreement with Mie theory scattering cross-sections. Amplitude reconstructions are more useful than phase for low-index spheres and should not be neglected in DHM analysis.

The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates.

The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800 nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM enabled discrimination of SYTO 9 labelled cells from background, but that the lifetime of SYTO 9 was shorter in cells on minerals than in pure culture under our conditions. Photosynthetic microorganisms were easily observed using both FLIM and confocal. Unlabelled, nonpigmented bacteria showed weak signals that were difficult to distinguish from background when minerals were present, though cellular autofluorescence consistent with NAD(P)H could be seen in pure cultures, and phasor analysis permitted detection on rocks. Gypsum and marble samples showed similar autofluorescence profiles, with little autofluorescence in the yellow-to-red range. Lifetime or time-gated imaging may prove a useful tool for environmental microbiology. LAY DESCRIPTION: The standard method of bacterial enumeration is to label the cells with a fluorescent dye and count them under high-power fluorescence microscopy. However, this can be difficult when the cells are embedded in soil and rock due to fluorescence from the surrounding minerals and dye binding to ambiguous features of the substrate. The use of fluorescence lifetime imaging (FLIM) can disambiguate these signals and allow for improved detection of bacteria in environmental samples.

Recent advances in experimental design and data analysis to characterize prokaryotic motility.

Bacterial motility plays a key role in important cell processes such as chemotaxis and biofilm formation, but is challenging to quantify due to the small size of the individual microorganisms and the complex interplay of biological and physical factors that influence motility phenotypes. Swimming, the first type of motility described in bacteria, still remains largely unquantified. Light microscopy has enabled qualitative characterization of swimming patterns seen in different strains, such as run and tumble, run-reverse-flick, run and slow, stop and coil, and push and pull, which has allowed for elucidation of the underlying physics. However, quantifying these behaviors (e.g., identifying run distances and speeds, turn angles and behavior by surfaces or cell-cell interactions) remains a challenging task. A qualitative and quantitative understanding of bacterial motility is needed to bridge the gap between experimentation, omics analysis, and bacterial motility theory. In this review, we discuss the strengths and limitations of how phase contrast microscopy, fluorescence microscopy, and digital holographic microscopy have been used to quantify bacterial motility. Approaches to automated software analysis, including cell recognition, tracking, and track analysis, are also discussed with a view to providing a guide for experimenters to setting up the appropriate imaging and analysis system for their needs.

Quantification of Motility in Bacillus subtilis at Temperatures Up to 84°C Using a Submersible Volumetric Microscope and Automated Tracking.

We describe a system for high-temperature investigations of bacterial motility using a digital holographic microscope completely submerged in heated water. Temperatures above 90°C could be achieved, with a constant 5°C offset between the sample temperature and the surrounding water bath. Using this system, we observed active motility in Bacillus subtilis up to 66°C. As temperatures rose, most cells became immobilized on the surface, but a fraction of cells remained highly motile at distances of >100 µm above the surface. Suspended non-motile cells showed Brownian motion that scaled consistently with temperature and viscosity. A novel open-source automated tracking package was used to obtain 2D tracks of motile cells and quantify motility parameters, showing that swimming speed increased with temperature until ∼40°C, then plateaued. These findings are consistent with the observed heterogeneity of B. subtilis populations, and represent the highest reported temperature for swimming in this species. This technique is a simple, low-cost method for quantifying motility at high temperatures and could be useful for investigation of many different cell types, including thermophilic archaea.

Using the Gouy phase anomaly to localize and track bacteria in digital holographic microscopy 4D images.

Described over 100 years ago, the Gouy phase anomaly refers to the additional π phase shift that is accumulated as a wave passes through focus. It is potentially useful in analyzing any type of phase-sensitive imaging; in light microscopy, digital holographic microscopy (DHM) provides phase information in the encoded hologram. One limitation of DHM is the weak contrast generated by many biological cells, especially unpigmented bacteria. We demonstrate here that the Gouy phase anomaly may be detected directly in the phase image using the z-derivative of the phase, allowing for precise localization of unlabeled, micrometer-sized bacteria. The use of dyes that increase phase contrast does not improve detectability. This approach is less computationally intensive than other procedures such as deconvolution and is relatively insensitive to reconstruction parameters. The software is implemented in an open-source FIJI plug-in.

Genetically Encoded Phase Contrast Agents for Digital Holographic Microscopy.

Quantitative phase imaging and digital holographic microscopy have shown great promise for visualizing the motion, structure, and physiology of microorganisms and mammalian cells in three dimensions. However, these imaging techniques currently lack molecular contrast agents analogous to the fluorescent dyes and proteins that have revolutionized fluorescence microscopy. Here we introduce the first genetically encodable phase contrast agents based on gas vesicles. The relatively low index of refraction of the air-filled core of gas vesicles results in optical phase advancement relative to aqueous media, making them a "positive" phase contrast agent easily distinguished from organelles, dyes, or microminerals. We demonstrate this capability by identifying and tracking the motion of gas vesicles and gas vesicle-expressing bacteria using digital holographic microscopy, and by imaging the uptake of engineered gas vesicles by mammalian cells. These results give phase imaging a biomolecular contrast agent, expanding the capabilities of this powerful technology for three-dimensional biological imaging.

Characterization of Retinol Stabilized in Phosphatidylcholine Vesicles with and without Antioxidants.

Retinol stability has been reported to be improved by encapsulation in liposomes, both with and without cholesterol. However, this improvement is limited because of lipid peroxidation. In this study, we compare the stability of retinol in phosphatidylcholine liposomes under ultraviolet (UV) light or standard room air, with and without the addition of antioxidants. Both butylated hydroxytoluene (BHT) and a proprietary mix (StoppOx) improved the shelf stability from <10 to over 30 d. The addition of cholesterol had no effect. Fluorescence imaging showed a heterogeneous distribution of retinol within the vesicles, including within the aqueous layer. Fluorescence lifetimes were equally heterogeneous. Under UV irradiation, StoppOx protected retinol for significantly longer than BHT and via different mechanisms. This suggests that natural antioxidants work well to improve the retinol stability, but that further work to determine the optimal vesicle structure remains to be performed.

Scintillation Yield Estimates of Colloidal Cerium-Doped LaF3 Nanoparticles and Potential for "Deep PDT".

A hybrid of radiotherapy and photodynamic therapy (PDT) has been proposed in previously reported studies. This approach utilizes scintillating nanoparticles to transfer energy to attached photosensitizers, thus generating singlet oxygen for local killing of malignant cells. Its effectiveness strongly depends upon the scintillation yield of the nanoparticles. Using a liquid scintillator as a reference standard, we estimated the scintillation yield of Ce0.1La0.9F3/LaF3 core/shell nanoparticles at 28.9 mg/ml in water to be 350 photons/MeV under orthovoltage X-ray irradiation. The subsequent singlet oxygen production for a 60 Gy cumulative dose to cells was estimated to be four orders of magnitude lower than the "Niedre killing dose," used as a target value for effective cell killing. Without significant improvements in the radioluminescence properties of the nanoparticles, this approach to "deep PDT" is likely to be ineffective. Additional considerations and alternatives to singlet oxygen are discussed.

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM).

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

Digital Holographic Microscopy, a Method for Detection of Microorganisms in Plume Samples from Enceladus and Other Icy Worlds.

Detection of extant microbial life on Earth and elsewhere in the Solar System requires the ability to identify and enumerate micrometer-scale, essentially featureless cells. On Earth, bacteria are usually enumerated by culture plating or epifluorescence microscopy. Culture plates require long incubation times and can only count culturable strains, and epifluorescence microscopy requires extensive staining and concentration of the sample and instrumentation that is not readily miniaturized for space. Digital holographic microscopy (DHM) represents an alternative technique with no moving parts and higher throughput than traditional microscopy, making it potentially useful in space for detection of extant microorganisms provided that sufficient numbers of cells can be collected. Because sample collection is expected to be the limiting factor for space missions, especially to outer planets, it is important to quantify the limits of detection of any proposed technique for extant life detection. Here we use both laboratory and field samples to measure the limits of detection of an off-axis digital holographic microscope (DHM). A statistical model is used to estimate any instrument's probability of detection at various bacterial concentrations based on the optical performance characteristics of the instrument, as well as estimate the confidence interval of detection. This statistical model agrees well with the limit of detection of 103 cells/mL that was found experimentally with laboratory samples. In environmental samples, active cells were immediately evident at concentrations of 104 cells/mL. Published estimates of cell densities for Enceladus plumes yield up to 104 cells/mL, which are well within the off-axis DHM's limits of detection to confidence intervals greater than or equal to 95%, assuming sufficient sample volumes can be collected. The quantitative phase imaging provided by DHM allowed minerals to be distinguished from cells. Off-axis DHM's ability for rapid low-level bacterial detection and counting shows its viability as a technique for detection of extant microbial life provided that the cells can be captured intact and delivered to the sample chamber in a sufficient volume of liquid for imaging. Key Words: In situ life detection-Extant microorganisms-Holographic microscopy-Ocean Worlds-Enceladus-Imaging. Astrobiology 17, 913-925.

Improved Tracking and Resolution of Bacteria in Holographic Microscopy Using Dye and Fluorescent Protein Labeling.

Digital holographic microscopy (DHM) is an emerging imaging technique that permits instantaneous capture of a relatively large sample volume. However, large volumes usually come at the expense of lower spatial resolution, and the technique has rarely been used with prokaryotic cells due to their small size and low contrast. In this paper we demonstrate the use of a Mach-Zehnder dual-beam instrument for imaging of labeled and unlabeled bacteria and microalgae. Spatial resolution of 0.3 µm is achieved, providing a sampling of several pixels across a typical prokaryotic cell. Both cellular motility and morphology are readily recorded. The use of dyes provides both amplitude and phase contrast improvement and is of use to identify cells in dense samples.

Real-Time Detection of Staphylococcus Aureus Using Whispering Gallery Mode Optical Microdisks.

Whispering Gallery Mode (WGM) microresonators have recently been studied as a means to achieve real-time label-free detection of biological targets such as virus particles, specific DNA sequences, or proteins. Due to their high quality (Q) factors, WGM resonators can be highly sensitive. A biosensor also needs to be selective, requiring proper functionalization of its surface with the appropriate ligand that will attach the biomolecule of interest. In this paper, WGM microdisks are used as biosensors for detection of Staphylococcus aureus. The microdisks are functionalized with LysK, a phage protein specific for staphylococci at the genus level. A binding event on the surface shifts the resonance peak of the microdisk resonator towards longer wavelengths. This reactive shift can be used to estimate the surface density of bacteria that bind to the surface of the resonator. The limit of detection of a microdisk with a Q-factor around 104 is on the order of 5 pg/mL, corresponding to 20 cells. No binding of Escherichia coli to the resonators is seen, supporting the specificity of the functionalization scheme.

A Submersible, Off-Axis Holographic Microscope for Detection of Microbial Motility and Morphology in Aqueous and Icy Environments.

Sea ice is an analog environment for several of astrobiology's near-term targets: Mars, Europa, Enceladus, and perhaps other Jovian or Saturnian moons. Microorganisms, both eukaryotic and prokaryotic, remain active within brine channels inside the ice, making it unnecessary to penetrate through to liquid water below in order to detect life. We have developed a submersible digital holographic microscope (DHM) that is capable of resolving individual bacterial cells, and demonstrated its utility for immediately imaging samples taken directly from sea ice at several locations near Nuuk, Greenland. In all samples, the appearance and motility of eukaryotes were conclusive signs of life. The appearance of prokaryotic cells alone was not sufficient to confirm life, but when prokaryotic motility occurred, it was rapid and conclusive. Warming the samples to above-freezing temperatures or supplementing with serine increased the number of motile cells and the speed of motility; supplementing with serine also stimulated chemotaxis. These results show that DHM is a useful technique for detection of active organisms in extreme environments, and that motility may be used as a biosignature in the liquid brines that persist in ice. These findings have important implications for the design of missions to icy environments and suggest ways in which DHM imaging may be integrated with chemical life-detection suites in order to create more conclusive life detection packages.

Use of dyes to increase phase contrast for biological holographic microscopy.

Holographic microscopy is an emerging biological technique that provides amplitude and quantitative phase imaging, though the contrast provided by many cell types and organelles is low, and until now no dyes were known that increased contrast. Here we show that the metallocorrole Ga(tpfc)(SO3)2, which has a strong Soret band absorption, increases contrast in both amplitude and phase and facilitates tracking of Escherichia coli with minimal toxicity. The change in phase contrast may be calculated from the dye-absorbance spectrum using the Kramers-Kronig relations, and represents a general principle that may be applied to any dye or cell type. This enables the use of holographic microscopy for all applications in which specific labeling is desired.

Initial photophysical characterization of the proteorhodopsin optical proton sensor (PROPS).

Fluorescence is not frequently used as a tool for investigating the photocycles of rhodopsins, largely because of the low quantum yield of the retinal chromophore. However, a new class of genetically encoded voltage sensors is based upon rhodopsins and their fluorescence. The first such sensor reported in the literature was the proteorhodopsin optical proton sensor (PROPS), which is capable of indicating membrane voltage changes in bacteria by means of changes in fluorescence. However, the properties of this fluorescence, such as its lifetime decay components and its origin in the protein photocycle, remain unknown. This paper reports steady-state and nanosecond time-resolved emission of this protein expressed in two strains of Escherichia coli, before and after membrane depolarization. The voltage-dependence of a particularly long lifetime component is established. Additional work to improve quantum yields and improve the general utility of PROPS is suggested.

Differential toxicity of gold-doxorubicin in cancer cells vs. cardiomyocytes as measured by real-time growth assays and fluorescence lifetime imaging microscopy (FLIM).

The kinetics of toxicity of doxorubicin (Dox) and gold nanoparticle-conjugated doxorubicin (Au-Dox) were investigated in cultured B16 melanoma cells and cardiomyocytes using real-time cell-growth imaging. Both bolus exposure and continuous exposure were used. Modeling of the growth curve dynamics suggested patterns of uptake and/or expulsion of the drug that were different for the different cell lines and exposures. Dox alone in B16 cells fit to a model of slow drug buildup, whereas Au-Dox fit to a pattern of initial high drug efficacy followed by a decrease. In cardiomyocytes, the best fit was to a model of increasing drug concentration which then began to decrease, consistent with breakdown of the doxorubicin in solution. Cardiomyocytes were more sensitive than B16 cells to Dox alone (IC50 123 ± 2 nM vs. 270 ± 2 nM with continuous exposure), but were dramatically less sensitive to Au-Dox (IC50 1 ± 0.1 µM vs. 58 ± 5 nM with continuous exposure). Bolus exposure for 40 min led to significant cell death in B16 cells but not in cardiomyocytes. Fluorescence lifetime imaging (FLIM) showed different patterns of uptake of Au-Dox in the two cell types that explained the differential toxicity. While Au-Dox concentrated in the nuclei of B16 cells, it remained endosomal in cardiomyocytes. These results suggest that stable conjugates of nanoparticles to doxorubicin may be useful for treating resistant cancers while sparing healthy tissue.

Targeting B16 tumors in vivo with peptide-conjugated gold nanoparticles.

This study examines the effects of polyethylene glycol (PEG) and peptide conjugation on the biodistribution of ultrasmall (2.7 nm) gold nanoparticles in mice bearing B16 melanoma allografts. Nanoparticles were delivered intravenously, and biodistribution was measured at specific timepoints by organ digestion and inductively coupled plasma mass spectrometry. All major organs were examined. Two peptides were tested: the cyclic RGD peptide (cRGD, which targets integrins); and a recently described peptide derived from the myxoma virus. We found the greatest specific tumor delivery using the myxoma peptide, with or without PEGylation. Un-PEGylated cRGD performed poorly, but PEGylated RGD showed a significant transient collection in the tumor. Liver and kidney were the primary targets of all constructs. None of the particles were able to cross the blood-brain barrier. Although it was able to deliver Au to B16 cells, the myxoma peptide did not show any cytotoxic activity against these cells, in contrast to previous reports. These results indicate that the effect of passive targeting by PEGylation and active targeting by peptides can be independent or combined, and that they should be evaluated on a case-by-case basis when designing new nanosystems for targeted therapies. Both myxoma peptide and cRGD should be considered for specific targeting to melanoma, but a thorough investigation of the cytotoxicity of the myxoma peptide to different cell lines remains to be performed.

Determination of biodistribution of ultrasmall, near-infrared emitting gold nanoparticles by photoacoustic and fluorescence imaging.

This study compares fluorescence and photoacoustic (PA) imaging of ex vivo tumors and organs from tumor-bearing mice injected intravenously with ultrasmall (<3 nm ) tiopronin-capped Au nanoparticles and compares the data with inductively coupled plasma mass spectrometry (ICP-MS). Good agreement is seen in particle distributions and concentrations at the organ level. The spatial resolution from the imaging techniques allows for localization of the particles within organ structures. Although the particles do not have a plasmon peak, their absorbance in the near-infrared (NIR) is sufficient for PA excitation. PA imaging shows an increase of signal as particle concentrations increase, with changes in spectrum if particles aggregate. Fluorescence imaging using the particles' native NIR emission shows agreement in general intensity in each organ, though quenching of emission can be seen at very high concentrations. Both of these imaging techniques are noninvasive and labor-saving alternatives to organ digestion and ICP-MS and may provide insight into cellular distribution of particles. The simple construct avoids the use of toxic semiconductor materials or dyes, relying upon the gold itself for both the fluorescence and PA signal. This provides a useful alternative to more complex approaches to multimodal imaging and one that is readily translatable to the clinic.

Intratumoral gold-doxorubicin is effective in treating melanoma in mice.

Intratumoral injection of ultra-small gold nanoparticles (AuNPs) conjugated to doxorubicin (Au-Dox) is effective against both murine B16 and human SK-MEL-28 tumors in mice. Au-Dox suppresses growth of B16 tumors in immunocompetent mice by >70% for at least 19 days. In SK-MEL-28 xenografts, Au-Dox suppresses tumor growth almost completely for >13 weeks, while tumors treated with Dox alone demonstrate accelerated growth after 10 weeks. Histological analysis shows significant apoptosis and necrosis in Au-Dox treated tumors. Intratumoral injection is significantly more effective than intravenous injection, which leads to significant accumulation in liver and kidney with sub-therapeutic concentrations of Au-Dox. However, IV injection does not lead to significant damage in non-target organs, so improved targeting should permit this mode of delivery with little risk of systemic toxicity. The current construct is suitable for tumors accessible to intratumoral injection and represents a viable approach doxorubicin-resistant solid tumors. FROM THE CLINICAL EDITOR: Drug resistance is a significant problem in the fight against cancer. The authors describe a new approach in combating drug resistance in tumor cells by conjugating ultrasmall gold nanoparticles to doxorubicin. They tested the efficacy in in-vivo models using two melanoma cell lines. The promising results obtained from intra-tumoral injections contribute a way in future drug designs showing that conjugation to nanoparticles could lead to more effective and synergistic killing of tumor cells.

Nuclear uptake of ultrasmall gold-doxorubicin conjugates imaged by fluorescence lifetime imaging microscopy (FLIM) and electron microscopy.

Fluorescence lifetime imaging microscopy (FLIM) has been used to image free and encapsulated doxorubicin (Dox) uptake into cells, since interaction of Dox with DNA leads to a characteristic lifetime change. However, none of the reported Dox conjugates were able to enter cell nuclei. In this work, we use FLIM to show nuclear uptake of 2.7 nm mean diameter Au nanoparticles conjugated to Dox. The pattern of labelling differed substantially from what was seen with free Dox, with slower nuclear entry and stronger cytoplasmic labelling at all time points. As the cells died, the pattern of labelling changed further as intracellular structures disintegrated, consistent with association of Au-Dox to membranes. The patterns of Au distribution and intracellular structure changes were confirmed using electron microscopy, and indicate different mechanisms of cytotoxicity with stable Au-Dox conjugates compared to Dox alone. Such conjugates are promising tools for overcoming resistance in Dox-resistant cancers.