Histological classification of atherosclerotic arteries using high-speed confocal Raman microscopy with machine learning

Confocal Raman microscopy is a useful tool to observe composition and constitution of label-free samples at high spatial resolution. However, accurate characterization of microstructure of tissue and its application in diagnostic imaging are challenging due to weak Raman scattering signal and complex chemical composition of tissue. We have developed a method to improve imaging speed, diffraction efficiency, and spectral resolution of confocal Raman microscopy. In addition to the novel imaging technique, the machine learning method enables confocal Raman microscopy to visualize accurate histology of tissue sections. Here, we have demonstrated the performance of the proposed method by measuring histological classification of atherosclerotic arteries and compared the histological confocal Raman images with the conventional staining method. Our new confocal Raman microscopy enables us to comprehend the structure and biochemical composition of tissue and diagnose the buildup of atherosclerotic plaques in the arterial wall without labeling.     

Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array.

Oligonucleotide microarrays, also called "DNA chips," are currently made by a light-directed chemistry that requires a large number of photolithographic masks for each chip. Here we describe a maskless array synthesizer (MAS) that replaces the chrome masks with virtual masks generated on a computer, which are relayed to a digital micromirror array. A 1:1 reflective imaging system forms an ultraviolet image of the virtual mask on the active surface of the glass substrate, which is mounted in a flow cell reaction chamber connected to a DNA synthesizer. Programmed chemical coupling cycles follow light exposure, and these steps are repeated with different virtual masks to grow desired oligonucleotides in a selected pattern. This instrument has been used to synthesize oligonucleotide microarrays containing more than 76,000 features measuring 16 microm 2. The oligonucleotides were synthesized at high repetitive yield and, after hybridization, could readily discriminate single-base pair mismatches. The MAS is adaptable to the fabrication of DNA chips containing probes for thousands of genes, as well as any other solid-phase combinatorial chemistry to be performed in high-density microarrays.     

Nanostructure analysis using spatially modulated illumination microscopy

We describe the usage of the spatially modulated illumination (SMI) microscope to estimate the sizes (and/or positions) of fluorescently labeled cellular nanostructures, including a brief introduction to the instrument and its handling. The principle setup of the SMI microscope will be introduced to explain the measures necessary for a successful nanostructure analysis, before the steps for sample preparation, data acquisition and evaluation are given. The protocol starts with cells already attached to the cover glass. The protocol and duration outlined here are typical for fixed specimens; however, considerably faster data acquisition and in vivo measurements are possible.     

Partial nephrectomy margin imaging using structured illumination microscopy

Partial nephrectomy (PN) is the recommended procedure over radical nephrectomy (RN) for patients with renal masses less than 4 cm in diameter (Stage T1a). Patients with less than 4 cm renal masses can also be treated with PN, but have a higher risk for positive surgical margins (PSM). PSM, when present, are indicative of poor clinical outcomes. The current gold‐standard histopathology method is not well‐suited for the identification of PSM intraoperatively due to processing time and destructive nature. Here, video‐rate structured illumination microscopy (VR‐SIM) was investigated as a potential tool for PSM detection during PN. A clinical image atlas assembled from ex vivo renal biopsies provided diagnostically useful images of benign and malignant kidney, similar to permanent histopathology. VR‐SIM was then used to image entire parenchymal margins of tumor resection covering up to >1800× more margin surface area than standard histology. Aided by the image atlas, the study pathologist correctly classified all parenchymal margins as negative for PSM with VR‐SIM, compared to standard postoperative pathology. The ability to evaluate large surgical margins in a short time frame with VR‐SIM may allow it to be used intraoperatively as a “safety net” for PSM detection, allowing more patients to undergo PN over RN.      

In-situ characterization of the uncrimping process of arterial collagen fibers using two-photon confocal microscopy and digital image correlation

Uncrimping of collagen fibers in the arterial wall is an integral process in regulating the macro-level mechanical response of arteries. Uncrimping of collagen fibers leads to a gradual, but significant strain-stiffening response of the artery at physiological pressures and prevents overdistention at elevated pressures. In this study, we imaged adventitial collagen fibers from fresh primate arteries using two-photon excitation microscopy while subjecting the arteries to physiological inflation pressures and axial stretches. The imaging focal plane was fixed at a constant radial location in the adventitial wall by adjusting the focal distance as the arteries inflated, allowing for the continuously monitoring of the uncrimping process of a single region of collagen fibers. Digital image correlation was then applied to the sequential images to assess and correlate the local displacements to manual traces of selected reference fibers and their engagements. We found that the collagen fibers of interest became fully engaged at a luminal pressure of 20 mmHg, this was then followed by rotation of these fibers as the bulk artery continued to dilate. This technique helps to further the understanding of the uncrimping process of collagen fibers under physiological loads, which can aid in the development of more accurate microstructural constitutive models.     

Visualising fouling of a chromatographic matrix using confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) was used to visualise the spatial location of foulants during the fouling of Q Sepharose FF matrix in finite batch experiments and for examining the subsequent effectiveness of clean-in-place (CIP) treatments in cleaning the heavily fouled beads. Beads were severely fouled with partially clarified E. coli homogenate by contacting the beads with the foulant for contact times of 5 min, 1 or 12 h. The use of two different fluorescent dyes, PicoGreen and Cy5.5, for labelling genomic PicoGreen-labelled dsDNA and protein respectively, allowed the direct observation of the chromatographic beads. The extent of fouling was assessed by measuring the subsequent adsorption of Cy5.5-labelled BSA to the beads. Control studies established that the labelling of BSA did not affect significantly the protein properties. In the control case of contacting the unfouled matrix with Cy5.5-labelled BSA, protein was able to penetrate the entire matrix volume. After fouling, Cy5.5-labelled BSA was unable to penetrate the bead but only to bind near the bead surface where it slowly displaced PicoGreen-conjugated dsDNA, which bound only at the exterior of the beads. Labelled host cell proteins bound throughout the bead interior but considerably less at the core; suggesting that other species might have occupied that space. The gross levels of fouling achieved drastically reduced the binding capacity and maximum Cy5.5-labelled BSA uptake rate. The capacity of the resin was reduced by 2.5-fold when incubated with foulant for up to 1 h. However, when the resin was fouled for a prolonged time of 12 h a further sixfold decrease in capacity was seen. The uptake rate of Cy5.5-labelled BSA decreased with increased fouling time of the resin. Incubating the fouled beads in 1 M NaCl dissociated PicoGreen-labelled dsDNA from the bead exterior within 15 min of incubation but proved ineffective in removing all the foulant protein. Cy5.5-labelled BSA was still unable to bind beyond the outer region of the beads. A harsher CIP treatment of 1 M NaCl dissolved in 1 M NaOH was also ineffective in removing all the foulant protein but did remove PicoGreen-conjugated dsDNA within 15 min of incubation. Cy5.5-labelled BSA was able to bind throughout the bead interior after this more aggressive CIP treatment but at a lower capacity than in the case of fresh beads. The competitive adsorption of BacLight Red-labelled whole cells or cell debris and PicoGreen-conjugated dsDNA was also visualised using CSLM.     

High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device

Optogenetic approaches allow the manipulation of neuronal activity patterns in space and time by light, particularly in small animals such as zebrafish. However, most techniques cannot control neuronal activity independently at different locations. Here we describe equipment and provide a protocol for single-photon patterned optical stimulation of neurons using a digital micromirror device (DMD). This method can create arbitrary spatiotemporal light patterns with spatial and temporal resolutions in the micrometer and submillisecond range, respectively. Different options to integrate a DMD into a multiphoton microscope are presented and compared. We also describe an ex vivo preparation of the adult zebrafish head that greatly facilitates optogenetic and other experiments. After assembly, the initial alignment takes about one day and the zebrafish preparation takes <30 min. The method has previously been used to activate channelrhodopsin-2 and manipulate oscillatory synchrony among spatially distributed neurons in the zebrafish olfactory bulb. It can be adapted easily to a wide range of other species, optogenetic probes and scientific applications.     

Modeling stem cell nucleus mechanics using confocal microscopy

Biomechanics and Modeling in Mechanobiology - Nuclear mechanics is emerging as a key component of stem cell function and differentiation. While changes in nuclear structure can be visually imaged...     

High-throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy

Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high-resolution confocal microscopy, deconvolution and image analysis with NeuronStudio. Recent technical advancements include better preservation of tissue, resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically to identify both optimal resolution and the highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing three experimental groups with eight subjects each can take as little as 1 month if optimized for speed, or approximately 4-5 months if the highest resolution and morphometric detail is sought.     

Insight into pollutant bioavailability and toxicity using Raman confocal microscopy

Raman confocal microscopy was used to discriminate between cultures of Burkholderia xenovorans LB400 exposed to four different common environmental pollutants: phenanthrene, dodecane, 3-chlorobiphenyl and pentachlorophenol. Evidence is presented for the application of Raman spectroscopy as a bioassay for pollutant bioavailability and toxicity.     

A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells

Fluorescent speckle microscopy (FSM) uses a small fraction of fluorescently labeled subunits to give macromolecular assemblies such as the cytoskeleton fluorescence image properties that allow quantitative analysis of movement and subunit turnover. We describe a multispectral microscope system to analyze the dynamics of multiple cellular structures labeled with spectrally distinct fluorophores relative to one another over time in living cells. This required a high-resolution, highly sensitive, low-noise, and stable imaging system to visualize the small number of fluorophores making up each fluorescent speckle, a means by which to switch between excitation wavelengths rapidly, and a computer-based system to integrate image acquisition and illumination functions and to allow a convenient interface for viewing multispectral time-lapse data. To reduce out-of-focus fluorescence that degrades speckle contrast, we incorporated the optical sectioning capabilities of a dual-spinning-disk confocal scanner. The real-time, full-field scanning allows the use of a low-noise, fast, high-dynamic-range, and quantum-efficient cooled charge-coupled device (CCD) as a detector as opposed to the more noisy photomultiplier tubes used in laser-scanning confocal systems. For illumination, our system uses a 2.5-W Kr/Ar laser with 100-300mW of power at several convenient wavelengths for excitation of few fluorophores in dim FSM specimens and a four-channel polychromatic acousto-optical modulator fiberoptically coupled to the confocal to allow switching between illumination wavelengths and intensity control in a few microseconds. We present recent applications of this system for imaging the cytoskeleton in migrating tissue cells and neurons.     

Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy.

BACKGROUND: The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen. METHODS: The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI. RESULTS: Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were determined. The average refractive index calculated for cytoplasmic and nuclear regions was 1.360 +/- 0.004. The uncertainty in the final RI value represents the technique measurement error. CONCLUSIONS: The methodology we describe for viable cell RI measurement with this prototype cell has broad generic application in the study of cell growth and functional responses. The RI value we report may be used in optical analyses of cultured cell structure and morphology.     

Nanoparticles,molecular biosensors,and multispectral confocal microscopy

Complex, multilayered nanoparticles hold great promise for more sophisticated drug/gene delivery systems to single cells. Outermost layers can include cell targeting and cell-entry facilitating molecules. The next layer can include intracellular targeting molecules for precise delivery of the nanoparticle complex inside the cell of interest. Molecular biosensors can be used to confirm the presence of expected molecules (for example, reactive oxygen species (ROS) as a surrogate molecule for signs of infection, or for activation in radiation damage, etc.) prior to delivery of counter-measure molecules such as drugs or gene therapy. They can also be used as a feedback control mechanism to control the proper amount of drug/gene delivery for each cell. Importantly, the full nanoparticle system can be used to prevent any cells from encountering the drug unless that cell is specifically targeted. Thus, if a cell is initially non-specifically targeted, a secondary check for other molecular targets which must also be present inside the target cell of interest can be used to catch initial targeting mistakes and prevent subsequent delivery of treatment molecules to the wrong cells. The precise intracellular location of nanoparticles within specific regions of a cell can be confirmed by 3D multispectral confocal microscopy. These single cell molecular morphology measurements can be extended from individual cells, to other cells in a tissue in tissue monolayers or tissue sections.     

Osmotic water permeability measurements using confocal laser scanning microscopy

 We have developed a method for measurement of plasma membrane water permeability (P _f) in intact cells using laser scanning confocal microscopy. The method is based on confocal recording of the fluorescence intensity emitted by calcein-loaded adherent cells during osmotic shock. P _f is calculated as a function of the time constant in the fluorescence intensity change, the cell surface-to-volume ratio and the fractional content of the osmotically active cell volume. The method has been applied to the measurement of water permeability in MDCK cells. The cells behaved as linear osmometers in the interval from 100 to 350 mosM. About 57% of the total cell volume was found to be osmotically inactive. Water movement across the plasma membrane in intact MDCK cells was highly temperature dependent. HgCl₂ had no effect on water permeability, while amphotericin B and DMSO significantly increased P _f values. The water permeability in MDCK cells transfected with aquaporin 2 was an order of magnitude higher than in the intact MDCK cell line. The water permeability of the nuclear membrane in both cell lines was found to be unlimited. Thus the intranuclear fluid belongs to the osmotically active portion of the cell. We conclude that the use of confocal microscopy provides a sensitive and reproducible method for measurement of water permeability in different types of adherent cells and potentially for coverslip-attached tissue preparations. Received: 12 June 1999 / Revised version: 21 February 2000 / Accepted: 25 February 2000     

Imaging of single fluorescent molecules using video-rate confocal microscopy

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were approximately 10 s for TMR and approximately 2 s for IC5 under the incident laser power of 0.5 W/mm(2). Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm(2). 2D sliding movement of single kinesin molecules along microtubules on glass surface and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.     

Morphological study of TNPO3 and SRSF1 interaction during myogenesis by combining confocal,structured illumination and electron microscopy analysis

Transportin3 (TNPO3) shuttles the SR proteins from the cytoplasm to the nucleus. The SR family includes essential splicing factors, such as SRSF1, that influence alternative splicing, controlling protein diversity in muscle and satellite cell differentiation. Given the importance of alternative splicing in the myogenic process and in the maintenance of healthy muscle, alterations in the splicing mechanism might contribute to the development of muscle disorders. Combining confocal, structured illumination and electron microscopy, we investigated the expression of TNPO3 and SRSF1 during myogenesis, looking at nuclear and cytoplasmic compartments. We investigated TNPO3 and its interaction with SRSF1 and we observed that SRSF1 remained mainly localized in the nucleus, while TNPO3 decreased in the cytoplasm and was strongly clustered in the nuclei of differentiated myotubes. In conclusion, combining different imaging techniques led us to describe the behavior of TNPO3 and SRSF1 during myogenesis, showing that their dynamics follow the myogenic process and could influence the proteomic network necessary during myogenesis. The combination of different high-, super- and ultra-resolution imaging techniques led us to describe the behavior of TNPO3 and its interaction with SRSF1, looking at nuclear and cytoplasmic compartments. These observations represent a first step in understanding the role of TNPO3 and SRFSF1 in complex mechanisms, such as myogenesis.

     

Structured illumination microscopy of a living cell

Due to diffraction, the resolution of imaging emitted light in a fluorescence microscope is limited to about 200 nm in the lateral direction. Resolution improvement by a factor of two can be achieved using structured illumination, where a fine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Here we demonstrate that with the help of a spatial light modulator, this technique can be used for imaging slowly moving structures in living cells. This article has been submitted as a contribution to the Festschrift entitled “Uncovering cellular sub-structures by light microscopy” in honour of Professor Cremer’s 65th birthday.     

Chromatin investigation in the nucleus using a phasor approach to structured illumination microscopy

Chromatin in the nucleus is organized in functional sites at variable level of compaction. Structured illumination microscopy (SIM) can be used to generate three-dimensional super-resolution (SR) imaging of chromatin by changing in phase and in orientation a periodic line illumination pattern. The spatial frequency domain is the natural choice to process SIM raw data and to reconstruct an SR image. Using an alternative approach, we demonstrate that the additional spatial information encoded in the knowledge of the position of the illumination pattern can be efficiently decoded using a generalized version of separation of photon by lifetime tuning (SPLIT) that does not require lifetime measurements. In the resulting SPLIT-SIM, the SR image is obtained by isolating a fraction of the intensity corresponding to the center of the diffraction-limited point spread function. This extends the use of the SPLIT approach from stimulated emission depletion microscopy to SIM. The SPLIT-SIM algorithm is based only on phasor analysis and does not require deconvolution. We show that SPLIT-SIM can be used to generate SR images of chromatin organizational motifs with tunable resolution and can be a valuable tool for the imaging of functional sites in the nucleus.     

In vivo determination of organellar pH using a universal wavelength-based confocal microscopy approach

Many essential cellular processes are affected by transmembrane H(+) gradients and intracellular pH (pHi). The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study.