This work was supported by the National Institute of Health R35GM136223, R01AI141439 to J.X.C.

Comprehensive Protocol for Cell Identification and Metabolic Analysis Using OPTIR-FISH

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence In Situ Hybridization (FISH): Protocols and Applications. , (2010).</li><li>Wang, D. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quick+and+simple+FISH+protocol+with+hybridization-sensitive+fluorescent+linear+oligodeoxynucleotide+probes.">A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes.</a> RNA. 18 (1), 166-175 (2012).</li><li>Greuter, D., Loy, A., Horn, M., Rattei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=probeBase-an+online+resource+for+rRNA-targeted+oligonucleotide+probes+and+primers:+new+features+2016.">probeBase-an online resource for rRNA-targeted oligonucleotide probes and primers: new features 2016.</a> Nucleic Acids Res. 44, D586-D589 (2016).</li><li>Yilmaz, L. S., Parnerkar, S., Noguera, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mathFISH,+a+web+tool+that+uses+thermodynamics-based+mathematical+models+for+in+silico+evaluation+of+oligonucleotide+probes+for+fluorescence+in+situ+hybridization.">mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization.</a> Appl Environ Microbiol. 77 (3), 1118-1122 (2011).</li><li>Lima, C., Muhamadali, H., Xu, Y., Kansiz, M., Goodacre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+isotopically+labeled+bacteria+at+the+single-cell+level+using+high-resolution+optical+infrared+photothermal+spectroscopy.">Imaging isotopically labeled bacteria at the single-cell level using high-resolution optical infrared photothermal spectroscopy.</a> Anal Chem. 93 (6), 3082-3088 (2021).</li><li>Yin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanosecond-resolution+photothermal+dynamic+imaging+via+MHZ+digitization+and+match+filtering.">Nanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering.</a> Nat Commun. 12 (1), 7097 (2021).</li><li>Bai, Y., Yin, J., Cheng, J. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bond-selective+imaging+by+optically+sensing+the+mid-infrared+photothermal+effect.">Bond-selective imaging by optically sensing the mid-infrared photothermal effect.</a> Sci Adv. 7 (20), (2021).</li><li>Xia, Q., Yin, J., Guo, Z., Cheng, J. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mid-infrared+photothermal+microscopy:+principle,+instrumentation,+and+applications.">Mid-infrared photothermal microscopy: principle, instrumentation, and applications.</a> J Phys Chemi B. 126 (43), 8597-8613 (2022).</li><li>Du, J., Wang, H., Wei, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bringing+vibrational+imaging+to+chemical+biology+with+molecular+probes.">Bringing vibrational imaging to chemical biology with molecular probes.</a> ACS Chem Biol. 17 (7), 1621-1637 (2022).</li><li>Miao, Y., Shi, L., Hu, F., Min, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probe+design+for+super-multiplexed+vibrational+imaging.">Probe design for super-multiplexed vibrational imaging.</a> Phys Biol. 16 (4), 041003 (2019).</li><li>Shi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mid-infrared+metabolic+imaging+with+vibrational+probes.">Mid-infrared metabolic imaging with vibrational probes.</a> Nat Methods. 17 (8), 844-851 (2020).</li><li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+mapping+of+lipid+metabolites+using+an+infrared+probe+in+human-derived+model+systems.">Single-cell mapping of lipid metabolites using an infrared probe in human-derived model systems.</a> Nat Commun. 15 (1), 350 (2024).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+tracing+of+deuterium+for+imaging+glucose+metabolism.">Spectral tracing of deuterium for imaging glucose metabolism.</a> Nat Biomed Eng. 3 (5), 402-413 (2019).</li><li>Ge, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRS-FISH:+A+high-throughput+platform+linking+microbiome+metabolism+to+identity+at+the+single-cell+level.">SRS-FISH: A high-throughput platform linking microbiome metabolism to identity at the single-cell level.</a> Proc Natl Acad Sci U S A. 119 (26), e2203519119 (2022).</li><li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+chemical+imaging+by+widefield+photothermal+sensing+of+infrared+absorption.">Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption.</a> Sci Adv. 5 (7), 7127 (2019).</li><li>Zhang, J., Zhao, J., Lin, H., Tan, Y., Cheng, J. -. 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Y.B. is a part-time consultant for Photothermal Spectroscopy Corp.

Here, we described a detailed protocol for applying the OPTIR-FISH platform for simultaneous identification of microbial species and quantification of metabolic activities at the single-cell resolution. The critical steps include culture with stable isotope labeling for studying specific metabolic activities and fluorescence in situ hybridization for identifying target microbial species. Multi-channel fluorescence imaging and OPTIR imaging at selected wavenumbers could be performed sequentially on the same micro...

Cellular metabolism stands as a foundational pillar in cell biology, steering many processes that determine cell health, function, and interaction with the environment. Analyzing metabolism at the individual cell level, particularly within the native environments, provides invaluable insights to reveal the heterogeneous and complex activities in biological systems1. This is especially crucial in the study of microorganisms, as many microbes exhibit unique growth requirements or environmental dependencies that challenge traditional cultivation methods2. For instance, some species may require specific nutrient compositions or symbiotic relationships not easily replicated in a laboratory setting, thus rendering them non-culturable by standard techniques3. Furthermore, the extended periods needed for the cultivation of certain species pose significant challenges for microbiological research, often extending beyond practical time frames for study and analysis. To circumvent these limitations, alternative methods such as polymerase chain reaction and fluorescence in situ hybridization (FISH) allow for the identification of microbial species without necessitating cultivation4, which can achieve a more accurate and holistic view of microbial ecosystems. However, these analytical technologies lack the ability to elucidate cellular metabolism. This gap highlights the ongoing challenges in the microbiology field: the concurrent task of differentiating cellular identity and elucidating metabolism at the single-cell level. Advancements in techniques such as imaging mass spectrometry (IMS) coupled with stable isotopes have emerged as powerful tools for single-cell metabolic analysis5. In these experiments, cells were incubated with substrates containing isotopes such as 13C or 15N. The newly anabolized biomolecules carry these isotopes, making them distinguishable on the m/z spectra. However, IMS suffers from expensive instrumentation, complicated sample preparation, relatively low throughput, and expertise required for analyzing the m/z spectra. When combined with molecular marker-specific fluorescence imaging, advancements have been made in elucidating cellular metabolism with increased specificity6. Nevertheless, challenges persist in bridging these two modalities. The difference in resolution between IMS and fluorescence imaging, combined with different operational setups, makes it difficult to align and correlate findings7.
The integration of vibrational spectroscopic imaging with stable isotope labeling offers a novel solution for the study of single-cell metabolism. The incorporation of heavier isotopes slows down the vibration of chemical bonds, leading to red-shifted peaks in the vibrational spectra8. Notably, vibrational imaging provides a spatial resolution comparable to fluorescence imaging, and both metabolic quantification and cell identification can be performed in a single setup, simplifying the image registration and correlation. Our recent work has demonstrated the combination of an advanced vibrational imaging platform: optical photothermal infrared (OPTIR) with fluorescence in situ hybridization (FISH) to probe glucose metabolism in bacterial communities9 (Figure 1). OPTIR is a vibrational spectroscopic microscopy system that harnesses the photothermal effect of mid-IR absorption by detecting the visible light change, which provides sub-micrometer resolution as in optical microscopy but with the additional vibrational spectroscopic information originating from the mid-IR absorption. FISH is a commonly used technique to determine the microorganism identity at the single-cell level. The sequence of oligonucleotides could be designed to target specific 16S sequences of different taxa, and different fluorophores could be attached. Specific hybridization of the designed oligonucleotide probes with the target rRNA leads to strong fluorescence signals of target species within individual cells, and multi-channel fluorescence imaging could be performed to identify multiple species within the population. As both OPTIR and fluorescence imaging are based on optical detection, combining OPTIR and FISH fluorescence is straightforward to implement. The two modalities share the same optical resolution for single-cell analysis and can be switched conveniently without requiring additional alignment or co-registration.
This protocol presents a detailed guide to leveraging the OPTIR-FISH platform for advanced single-cell structure-function analysis. We used the bacterial samples grown in 13C-glucose-containing media as a test bed and quantified de novo protein synthesis from 13C-glucose. In order to demonstrate the ability of the platform to identify cells, we used bacterial mixtures in which each species was labeled using rRNA-targeted FISH probes. This approach facilitated precise single-cell identification of specific bacterial strains, such as Escherichia coli (E. coli) and Bacteroides thetaiotaomicron (B. theta), and their metabolism. This protocol offers researchers a powerful tool for simultaneous metabolic profiling and species identification at the single-cell level, promising to advance our understanding of cellular interactions, physiology, and their roles in complex environments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>96% Ethanol</td>
			<td>ThermoScientific</td>
			<td>T032021000</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Fluoride</td>
			<td>Crystran</td>
			<td>CAFP10-0.35</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Gluocose (U-13C6, 99%)</td>
			<td>Cambridge Isotopic Laboratories</td>
			<td>CLM-1396-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>E9884-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>formamide</td>
			<td>ThermoScientific</td>
			<td>17899</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria-Bertani broth&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L3522-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>M9 Minimal Salts 5x</td>
			<td>SIGMA</td>
			<td>M6030-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>OPTIR instrument</td>
			<td>Photothermal Spectroscopy Corp.</td>
			<td>mIRage LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraforaldehyde Solution, 4% in PBS</td>
			<td>ThermoScientific</td>
			<td>J19943-K2</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine solution 0.1% (w/v)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P8920-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L3771-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane hydrochloride, 99+%</td>
			<td>ThermoScientific</td>
			<td>A11379.18</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypic Soy Broth</td>
			<td>Sigma-Aldrich</td>
			<td>22092-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

optical photothermal infrared-fluorescence in situ hybridization (optir-fish)

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we present a comprehensive protocol for simultaneous cell identification and metabolic analysis with the OPTIR-FISH platform by combining rRNA-tagged FISH probes and isotope-labeled substrates. Fluorescence imaging provides cell identification by the specific binding of rRNA-tagged FISH probes, while OPTIR imaging provides metabolic activities within single cells by isotope-induced red shift on OPTIR spectra. Using bacteria cultured with 13C-glucose as a test bed, the protocol outlines microbial culture with isotopic labeling, fluorescence in situ hybridization (FISH), sample preparation, optimization of the OPTIR-FISH imaging setup, and data acquisition. We also demonstrate how to perform image analysis and interpret spectral data at the single-cell level with high throughput. This protocol&#39;s standardized and detailed nature will greatly facilitate its adoption by researchers from diverse backgrounds and disciplines within the broad single-cell metabolism research community.

The general workflow for single-cell microbial metabolic analysis with genetic identification by OPTIR-FISH is summarized in Figure 2. The representative results demonstrating the single-cell metabolic imaging capability of OPTIR are shown in Figure 3. This example used E. coli cells incubated with 12C- or 13C-glucose for 24 h. The incorporation of 13C into proteins has been shown to cause a significant red shift of protein amide...

Read this Scientific Article Protocol about Optical Photothermal Infrared - Fluorescence In Situ Hybridization OPTIR-FISH at JoVE.com

Author Spotlight: Integrated OPTIR-FISH for Single-Cell Metabolic and Identity Analysis in Complex Environments

Here, we present a protocol using optical photothermal infrared-fluorescence in situ hybridization (OPTIR-FISH), also known as mid-infrared photothermal-FISH (MIP-FISH), to identify individual cells and understand their metabolism. This methodology can be applied broadly for diverse applications, including mapping cellular metabolism with single-cell resolution.

Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)

Understanding the metabolic activities of individual cells within complex communities is critical for unraveling their role in human disease. Here, we ...

optical-photothermal-infrared-fluorescence-situ-hybridization-optir

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JoVE Journal

Bioengineering

1.1K Views.  Boston University. Here, we present a protocol using optical photothermal infrared-fluorescence in situ hybridization (OPTIR-FISH), also known as mid-infrared photothermal-FISH (MIP-FISH), to identify individual cells and understand their metabolism. This methodology can be applied broadly for diverse applications, including mapping cellular metabolism with single-cell resolution. 

Video: Author Spotlight: Integrated OPTIR-FISH for Single-Cell Metabolic and Identity Analysis in Complex Environments

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 &deg;C to 62 &deg;C rapidly upon one minute exposure of 750 mW (1.18 W/cm2), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.

We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...