Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently‐labeled cells in highly autofluorescent tissue

Standard fluorescence microscopy approaches rely on measurements at single excitation and emission bands to identify specific fluorophores and the setting of thresholds to quantify fluorophore intensity. This is often insufficient to reliably resolve and quantify fluorescent labels in tissues due to high autofluorescence. Here we describe the use of hyperspectral analysis techniques to resolve and quantify fluorescently labeled cells in highly autofluorescent lung tissue. This approach allowed accurate detection of green fluorescent protein (GFP) emission spectra, even when GFP intensity was as little as 15% of the autofluorescence intensity. GFP‐expressing cells were readily quantified with zero false positives detected. In contrast, when the same images were analyzed using standard (single‐band) thresholding approaches, either few GFP cells (high thresholds) or substantial false positives (intermediate and low thresholds) were detected. These results demonstrate that hyperspectral analysis approaches uniquely offer accurate and precise detection and quantification of fluorescence signals in highly autofluorescent tissues. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Photoacoustic flow cytometry

Conventional flow cytometry using scattering and fluorescent detection methods has been a fundamental tool of biological discoveries for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents the long-term study of cells in their native environment. Here, we summarize recent advances of new generation flow cytometry for in vivo noninvasive label-free or targeted detection of cells in blood, lymph, bone, cerebral and plant vasculatures using photoacoustic (PA) detection techniques, multispectral high-pulse-repetition-rate lasers, tunable ultrasharp (up to 0.8nm) rainbow plasmonic nanoprobes, positive and negative PA contrasts, in vivo magnetic enrichment, time-of-flight cell velocity measurement, PA spectral analysis, and integration of PA, photothermal (PT), fluorescent, and Raman methods. Unique applications of this tool are reviewed with a focus on ultrasensitive detection of normal blood cells at different functional states (e.g., apoptotic and necrotic) and rare abnormal cells including circulating tumor cells (CTCs), cancer stem cells, pathogens, clots, sickle cells as well as pharmokinetics of nanoparticles, dyes, microbubbles and drug nanocarriers. Using this tool we discovered that palpation, biopsy, or surgery can enhance CTC release from primary tumors, increasing the risk of metastasis. The novel fluctuation flow cytometry provided the opportunity for the dynamic study of blood rheology including red blood cell aggregation and clot formation in different medical conditions (e.g., blood disorders, cancer, or surgery). Theranostics, as a combination of PA diagnosis and PT nanobubble-amplified multiplex therapy, was used for eradication of CTCs, purging of infected blood, and thrombolysis of clots using PA guidance to control therapy efficiency. In vivo flow cytometry using a portable fiber-based devices can provide a breakthrough platform for early diagnosis of cancer, infection and cardiovascular disorders with a potential to inhibit, if not prevent, metastasis, sepsis, and strokes or heart attack by well-timed personalized therapy.     

Laser Scanning Cytometry for selection of green fluorescent protein transgenic mice using small number of blood cells

A Laser Scanning Cytometry-based method was developed for identification of transgenic mice expressing green fluorescent protein (GFP) using minute amounts of peripheral blood. The difference between the autofluorescence of cells not expressing GFP and the fluorescence of GFP expressing cells after excitation with Ar-ion laser (wavelength 488 nm) and detection of emitted fluorescent light in the green channel was high enough for unambiguous identification of the GFP expressing mice. The sensitivity of this method was estimated 1:10(4) for detection of rare GFP expressing cells under the conditions used. This sensitivity should be sufficient for many studies on microchimerism. Because of the possibility for relocation of the cells, this method will be particularly useful for characterizing the cells with high GFP expression using other markers of cell phenotype or conventional morphological analysis.     

Nanotechnology‐based molecular photoacoustic and photothermal flow cytometry platform for in‐vivo detection and killing of circulating cancer stem cells

In‐vivo multicolor photoacoustic (PA) flow cytometry for ultrasensitive molecular detection of the CD44+ circulating tumor cells (CTCs) is demonstrated on a mouse model of human breast cancer. Targeting of CTCs with stem‐like phenotype, which are naturally shed from parent tumors, was performed with functionalized gold and magnetic nanoparticles. Results in vivo were verified in vitro with a multifunctional microscope, which integrates PA, photothermal (PT), fluorescent and transmission modules. Magnet‐induced clustering of magnetic nanoparticles in individual cells significantly amplified PT and PA signals. The novel noninvasive platform, which integrates multispectral PA detection and PT therapy with a potential for multiplex targeting of many cancer biomarkers using multicolor nanoparticles, may prospectively solve grand challenges in cancer research for diagnosis and purging of undetectable yet tumor‐initiating cells in circulation before they form metastasis. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Photoacoustic and photothermal cytometry using photoswitchable proteins and nanoparticles with ultrasharp resonances

Photoswitchable fluorescent proteins (PSFPs) with controllable spectral shifts in emission in response to light have led to breakthroughs in cell biology. Conventional photoswitching, however, is not applicable to weakly fluorescent proteins. As an alternative, photothermal (PT) and photoacoustic (PA) spectroscopy have demonstrated a tremendous potential for studying absorbing nonfluorescent proteins and nanoparticles. However, little progress has been made in the development of switchable PT and PA probes with controllable spectral shifts in absorption. Here, we introduce the concept of photothermally switchable nanoparticles (PTSNs). To prove the concept, we demonstrated fast, reversible magnetic–PT switching of conventional and gold‐coated magnetic nanoparticle clusters in cancer cells in vitro and PT switching of nonlinear ultrasharp plasmonic resonances in gold nanorods molecularly targeted to circulating cells in vivo. We showed that genetically encoded PSFPs with relatively slow switching can serve as triple‐modal fluorescent, PT, and PA probes under static conditions, while PTSNs with ultrafast switching may provide higher PA sensitivity in the near‐infrared window of tissue transparency under dynamic flow conditions. Application of nonlinear phenomena for super‐resolution spectral PT and PA cytometry, microscopy, and spectral burning beyond the diffraction and spectral limits are also proposed. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

One-step split GFP staining for sensitive protein detection and localization in mammalian cells

Although epitope tags are useful to detect intracellular proteins and follow their localization with antibodies, background and nonspecific staining often remain problematic. We describe a simple assay based on the split GFP complementation system. Proteins tagged with the 15-amino acid GFP 11 fragment are detected with a solution of the recombinant nonfluorescent complementary GFP 1-10 fragment to reconstitute a fluorescent GFP. In contrast to antibody-based staining methods, this one-step assay presents high specificity and very low background of fluorescence, thus conferring higher signal-to-noise ratios. We demonstrate that this new application of the split GFP tagging system facilitates detection of proteins displaying various subcellular localizations using flow cytometry and microscopy analysis.     

Photoacoustic and fluorescent effects in multilayer plasmon‐dye interfaces

Progress in understanding the cell biology and diseases depends on advanced imaging and labeling techniques. Here, we address this demand by exploring novel multilayered nanocomposites (MNCs) with plasmonic nanoparticles and absorbing dyes in thin nonabsorbing shells as supercontrast multimodal photoacoustic (PA) and fluorescent agents in the near‐infrared range. The proof of concept was performed with gold nanorods (GNRs) and indocyanine green (ICG) dispersed in a matrix of biodegradable polymers. We demonstrated synergetic PA effects in MNCs with the gold‐ICG interface that could not be achieved with ICG and GNRs alone. We also observed ultrasharp PA and emission peaks that could be associated with nonlinear PA and spaser effects, respectively. Low‐toxicity multimodal MNCs with unique plasmonic, thermal and acoustic properties have the potential to make a breakthrough in PA flow cytometry and near‐infrared spasers in vivo by using the synergetic interaction of plasmonic modes with a nearby absorbing medium.      

Use of DNA-specific anthraquinone dyes to directly reveal cytoplasmic and nuclear boundaries in live and fixed cells

Image-based, high-content screening assays demand solutions for image segmentation and cellular compartment encoding to track critical events — for example those reported by GFP fusions within mitosis, signalling pathways and protein translocations. To meet this need, a series of nuclear/cytoplasmic discriminating probes have been developed: DRAQ5™ and CyTRAK Orange™. These are spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. At their most fundamental they provide a convenient fluorescent emission signature which is spectrally separated from the commonly used reporter proteins (e.g. eGFP, YFP, mRFP) and fluorescent tags such as Alexafluor 488, fluorescein and Cy2. Additionally, they do not excite in the UV and thus avoid the complications of compound UV-autofluorescence in drug discovery whilst limiting the impact of background sample autofluorescence. They provide a convenient means of stoichiometrically labelling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Further developments have permitted the simultaneous and differential labelling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions and most recently compound in vitro toxicology testing.     

Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging

Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 x 10(3) cells for spectral imaging versus 1 x 10(6) cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications.     

Photothermal confocal spectromicroscopy of multiple cellular chromophores and fluorophores

Confocal fluorescence microscopy is a powerful biological tool providing high-resolution, three-dimensional (3D) imaging of fluorescent molecules. Many cellular components are weakly fluorescent, however, and thus their imaging requires additional labeling. As an alternative, label-free imaging can be performed by photothermal (PT) microscopy (PTM), based on nonradiative relaxation of absorbed energy into heat. Previously, little progress has been made in PT spectral identification of cellular chromophores at the 3D microscopic scale. Here, we introduce PTM integrating confocal thermal-lens scanning schematic, time-resolved detection, PT spectral identification, and nonlinear nanobubble-induced signal amplification with a tunable pulsed nanosecond laser. The capabilities of this confocal PTM were demonstrated for high-resolution 3D imaging and spectral identification of up to four chromophores and fluorophores in live cells and Caenorhabditis elegans. Examples include cytochrome c, green fluorescent protein, Mito-Tracker Red, Alexa-488, and natural drug-enhanced or genetically engineered melanin as a PT contrast agent. PTM was able to guide spectral burning of strong absorption background, which masked weakly absorbing chromophores (e.g., cytochromes in the melanin background). PTM provided label-free monitoring of stress-related changes to cytochrome c distribution, in C. elegans at the single-cell level. In nonlinear mode ultrasharp PT spectra from cyt c and the lateral resolution of 120 nm during calibration with 10-nm gold film were observed, suggesting a potential of PTM to break through the spectral and diffraction limits, respectively. Confocal PT spectromicroscopy could provide a valuable alternative or supplement to fluorescence microscopy for imaging of nonfluorescent chromophores and certain fluorophores.     

In vivo flow cytometry of circulating clots using negative photothermal and photoacoustic contrasts

Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of thromboembolism including ischemia at strokes and myocardial infarction. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red, and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 μm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting themby negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted.     

Sampling efficiency of a single-cell capillary electrophoresis system.

Capillary electrophoresis (CE) combined with a laser-induced fluorescence (LIF) detection scheme is a powerful approach for single-cell analysis. For measurements requiring a high temporal resolution, CE-LIF is often combined with cell lysis systems based on pulsed lasers. Although extremely rapid, laser lysis has raised some concerns about the efficiency at which the cell contents are sampled. We have assembled a single-cell CE-LIF mounted on the stage of a microscope. This system was coupled with a nanosecond pulsed laser for cell lysis. We have analyzed green fluorescent protein (GFP) expressed in single mammalian cells and developed a novel approach to estimate the cell sampling efficiency (SE) based on the use of fluorescent calibration microspheres and flow cytometry. A significant advantage of this method is that it does not require any knowledge or assumption regarding the cell volume. We have evaluated the SE for different laser pulse energies (from 2 to 9 microJ) and two different pulse focal positions in the xy plane (0-10 microm from the center of the cell). We found the maximum SE at the lowest energy (2 microJ), with the pulse focused directly on the cell. We have demonstrated the utility of a novel method to measure the SE of a single-cell CE system. The measurements presented in this study indicate that rapid cell lysis with nanosecond lasers requires careful optimization of pulse parameters for maximum sampling of the cell contents.     

Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny

Several modifications of a wild-type green fluorescent protein (GFP) gene were combined into a single construct, driven by the ubi-1 promoter and intron region, and transformed into maize. Green fluorescence, indicative of GFP expression, was observed in stably transformed callus as well as in leaves and roots of regenerated plants and their progeny. Cell wall autofluorescence made GFP expression difficult to observe in sections of leaves and roots. However, staining sections with toluidine blue allowed detection of GFP in transgenic tissue. Bright GFP fluorescence was observed in approximately 50% of the pollen of transgenic plants. These results suggest that GFP can be used as a reporter gene in transgenic maize; however, further modification, i.e., to alter the emission spectra, would increase its utility. Received: 17 December 1997 / Revision received: 6 March 1998 / Accepted: 20 March 1998     

The green fluorescent protein as a marker to visualize plant mitochondria in vivo

To determine how to utilize the green fluorescent protein (GFP) as a marker for subcellular localization and as a label for plant mitochondria in vivo, transgenic suspension cells and tobacco plants expressing GFP with and without a mitochondrial localization signal were generated. The first GFP form used, GFP1, is easily observable in cells with low autofluorescence, such as suspension cells or trichomes, but masked in green tissue. For the visualization of GFP in cells and tissues with high autofluorescence, such as leaf, the use of a very strong promoter (35S35SAMV), a highly expressed modified mGFP4 coding region and a brighter mutant form of GFP (S65T) was necessary. Confocal or two-photon laser scanning microscopy reveal a distinct subcellular localization of the fluorescence in cells expressing GFP or coxIVGFP. In cells expressing untargeted GFP, fluorescence accumulates in the nucleoplasm but is also distributed throughout the cytoplasm. It is excluded from vacuoles, nucleoli and from round bodies that are likely to be leucoplasts. In contrast, fluorescence is localized specifically to mitochondria in cells expressing coxIVGFP fusion protein as shown by co-localization with a mitochondrial-specific dye. This permits the direct observation of mitochondria and mitochondrial movements in living plant cells and tissues throughout plant development. Three-dimensional reconstruction of individual cells can give additional information about the distribution and numbers of mitochondria.     

Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis

The green fluorescent protein (GFP) has attracted much interest as a reporter for gene expression. In this paper, application of capillary electrophoresis with laser-induced fluorescent (CE-LIF) for quantitation of green fluorescence protein in cellular extracts and single cells is investigated. The S65T mutant form of GFP protein was successfully expressed in human embryonic kidney (HEK293) cells, and its production was confirmed by fluorescence microscopy and CE-LIF. The mass limit of detection for the mutant S65T was 5.3 x 10(-20) mol, which was better than that for the wild-type GFP by a factor of six. Detection of a small amount of GFP is difficult by conventional techniques such as fluorescent microscopy due to interference from cell autofluorescence at low GFP concentrations. The HEK293 cells were transfected with the GFP plasmid that produced S65T-GFP. Transient production of S65T protein was detected 2 h after the transfection and reached a maximum after 48 h. The protein concentration began to decrease significantly after 96 h. Single cell analysis of HEK293 cells after transfection with GFP plasmid indicate a nonuniform production of S65T-GFP protein among cells.     

Determination of symbiotic nodule occupancy in the model Vicia tetrasperma using a fluorescence scanner

Background

Fluorescent tagging of nodule bacteria forming symbioses with legume host plants represents a tool for vital tracking of bacteria inside the symbiotic root nodules and monitoring changes in gene activity. The constitutive expression of heterologous fluorescent proteins, such as green fluorescent protein (GFP), also allows screening for nodule occupancy by a particular strain. Imaging of the fluorescence signal on a macro-scale is associated with technical problems due to the robustness of nodule tissues and a high level of autofluorescence.

Scope

These limitations can be reduced by the use of a model species with a fine root system, such as Vicia tetrasperma. Further increases in the sensitivity and specificity of the detection and in image resolution can be attained by the use of a fluorescence scanner. Compared with the standard CCD-type cameras, the availability of a laser source of a specified excitation wavelength decreases non-specific autofluorescence while the photomultiplier tubes in emission detection significantly increase sensitivity. The large scanning area combined with a high resolution allow us to visualize individual nodules during the scan of whole root systems. Using a fluorescence scanner with excitation wavelength of 488 nm, a band-pass specific emission channel of 532 nm and a long-pass background channel of 555 nm, it was possible to distinguish nodules occupied by a rhizobial strain marked with one copy of cycle3 GFP from nodules colonized by the wild-type strain.

Conclusions

The main limitation of the current plant model and GFP with the wild-type emission peak at 409 nm is a sharp increase in root autofluorescence below 550 nm. The selectivity of the technique can be enhanced by the use of red-shifted fluorophores and the contrasting labelling of the variants, provided that the excitation (482 nm) and emission (737 nm) maxima corresponding to root chlorophyll are respected.     

TwinGFP, a marker for cell cycle analysis in transiently transfected cells

Green fluorescent protein (GFP) is widely used as a marker to identify transfected cells either by fluorescence microscopy or flow cytometry. However, cell cycle analysis with propidium iodide typically employs ethanol for cell permeabilization. During this treatment, soluble GFPs generally leak out of cells, probably due to their small size. We have now significantly improved cellular retention by creating an in-frame fusion of two GFP DNA sequences, thereby generating a double-sized GFP (TwinGFP, 57 kDa). Permeabilized HeLa cells transfected with pTwinGFP showed a strong green fluorescent signal localized throughout the cells that could easily be detected by fluorescence microscopy and flow cytometry, in contrast to cells transfected with a standard single GFP construct. The experiment indicates that protein size constitutes the major determinant of the loss of fluorescence in permeabilized cells. As a proof of principle, pTwinGFP was cotransfected with the p53 tumor suppressor gene into HeLa cells, and cells transiently expressing p53 could be identified and phenotypically characterized by flow cytometry.     

sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.     

Observer-based online compensation of inner filter effect in monitoring fluorescence of GFP-expressing plant cell cultures

The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria is a very useful reporter for real-time bioprocess sensing. GFP culture fluorescence is a composite signal that can be influenced by factors such as culture autofluorescence, inner filter effect (IFE), and photobleaching. These factors complicate accurate estimation of GFP concentrations from the culture fluorescence. IFE is especially problematic when using GFP in monitoring transgenic plant cell suspension cultures, due to the aggregated nature of the cells and the high biomass concentration in these culture systems. Reported approaches for online compensation of IFE in monitoring culture NADH fluorescence or bioluminescence require online measurement of biomass density or culture turbidity/optical density, in addition to fluorescence/bioluminescence measurement. In this study, culture GFP fluorescence was used successfully to estimate GFP concentration and other important states in bioreactor culture of transgenic tobacco cells, while the influences of IFE and culture autofluorescence were rectified without the need for an additional biomass sensor. This was achieved by setting up a novel model-based state observer. First, we developed an improved model for a backscatter fluorescence probe that takes into account the influence of IFE and autofluorescence on reporting culture GFP concentration from online fluorescence. The state observer was then established using the extended Kalman filter (EKF), based on the fluorescence probe model, a dynamic state model of the plant cell bioreactor, and online GFP fluorescence measurement. Several versions of the observer were introduced to address practical requirements associated with monitoring GFP fluorescence of plant cell cultures. The proposed approach offers an effective means for online compensation of IFE to enable quantitative interpretation of the culture fluorescence signals for accurate reporting of GFP or GFP-fusion protein expression.     

Nanoscopy in a living multicellular organism expressing GFP

We report superresolution fluorescence microscopy in an intact living organism, namely Caenorhabditis elegans nematodes expressing green fluorescent protein (GFP)-fusion proteins. We also superresolve, by stimulated emission depletion (STED) microscopy, living cultured cells, demonstrating that STED microscopy with GFP can be widely applied. STED with GFP can be performed with both pulsed and continuous-wave lasers spanning a wide wavelength range from at least 556–592 nm. Acquiring subdiffraction resolution images within seconds enables the recording of movies revealing structural dynamics. These results demonstrate that numerous microscopy studies of live samples employing GFP as the marker can be performed at subdiffraction resolution.