Name: a rapid and chemical-free hemoglobin assay with photothermal angular light scattering
Uploaded: 2016-12-07T15:00:00
Description: Watch this Scientific Journal Video about A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering at JoVE.com

This research was supported by the research programs of the National Research Foundation of Korea (NRF) (NRF-2015R1A1A1A05001548 and NRF-2015R1A5A1037668).

Experiments with blood samples were performed in compliance with the relevant laws and institutional guidelines. The samples were the residual blood samples that had been acquired and processed in clinical tests at the institution.
1. PT-AS Optical Setup
NOTE: One may use an empty micro-capillary tube for an initial PT-AS setup.
<ol>
	<li>Mount an empty micro-capillary tube with inner and outer diameters of 200 and 330 &#956;m, respectively, and a length of greater than ~5 cm on a capillary tube fixture. ...

<ol>
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S., 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=Direct+measurement+of+the+in+vitro+hemoglobin+content+of+erythrocytes+using+the+photo-thermal+effect+of+the+heme+group.">Direct measurement of the in vitro hemoglobin content of erythrocytes using the photo-thermal effect of the heme group.</a> Analyst. 135 (9), 2365-2371 (2010).</li><li>Lapotko, D., Lukianova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+micro-bubbles+in+cells.">Laser-induced micro-bubbles in cells.</a> International Journal of Heat Mass Transfer. 48 (1), 227-234 (2005).</li><li>Lapotko, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+bubbles+in+living+cells.">Laser-induced bubbles in living cells.</a> Lasers in surgery and medicine. 38 (3), 240-248 (2006).</li><li>Lapotko, D. O., Romanovskaya, T. y. R., Shnip, A., Zharov, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothermal+time-resolved+imaging+of+living+cells.">Photothermal time-resolved imaging of living cells.</a> Lasers in surgery and medicine. 31 (1), 53-63 (2002).</li><li>Yim, 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=Photothermal+spectral-domain+optical+coherence+reflectometry+for+direct+measurement+of+hemoglobin+concentration+of+erythrocytes.">Photothermal spectral-domain optical coherence reflectometry for direct measurement of hemoglobin concentration of erythrocytes.</a> Biosens. Bioelectron. 57, 59-64 (2014).</li><li>Kim, U., 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=Capillary-scale+direct+measurement+of+hemoglobin+concentration+of+erythrocytes+using+photothermal+angular+light+scattering.">Capillary-scale direct measurement of hemoglobin concentration of erythrocytes using photothermal angular light scattering.</a> Biosens. Bioelectron. 74, 469-475 (2015).</li><li>S&#0248;rensen, H. S., Larsen, N. B., Latham, J. C., Bornhop, D. J., Andersen, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+biosensing+based+on+interference+from+light+scattering+in+capillary+tubes.">Highly sensitive biosensing based on interference from light scattering in capillary tubes.</a> Appl. Phys. Lett. 89 (15), 151108 (2006).</li><li>Swinney, K., Markov, D., Bornhop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasmall+volume+refractive+index+detection+using+microinterferometry.">Ultrasmall volume refractive index detection using microinterferometry.</a> Rev. Sci. Instrum. 71 (7), 2684-2692 (2000).</li><li>Tarigan, H. J., Neill, P., Kenmore, C. K., Bornhop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary-scale+refractive+index+detection+by+interferometric+backscatter.">Capillary-scale refractive index detection by interferometric backscatter.</a> Anal. Chem. 68 (10), 1762-1770 (1996).</li><li>Bornhop, D. 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=Free-solution,+label-free+molecular+interactions+studied+by+back-scattering+interferometry.">Free-solution, label-free molecular interactions studied by back-scattering interferometry.</a> science. 317 (5845), 1732-1736 (2007).</li><li>Yang, 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=Simple+paper-based+test+for+measuring+blood+hemoglobin+concentration+in+resource-limited+settings.">Simple paper-based test for measuring blood hemoglobin concentration in resource-limited settings.</a> Clin. Chem. 59 (10), 1506-1513 (2013).</li><li>Zhu, H., 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=Cost-effective+and+rapid+blood+analysis+on+a+cell-phone.">Cost-effective and rapid blood analysis on a cell-phone.</a> Lab Chip. 13 (7), 1282-1288 (2013).</li><li>Poga&#269;nik, L., Franko, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+organophosphate+and+carbamate+pesticides+in+vegetable+samples+by+a+photothermal+biosensor.">Detection of organophosphate and carbamate pesticides in vegetable samples by a photothermal biosensor.</a> Biosens. Bioelectron. 18 (1), 1-9 (2003).</li></ol>

The PT-AS sensor represents an all-optical method capable of direct [Hb] measurement of unprocessed blood samples. The method quantifies [Hb] in blood using the intrinsic PT response of hemoglobin molecules in erythrocytes. Under illumination by 532-nm light, Hb molecules absorb the light energy and produce heat. The resultant temperature rise changes the RI of the blood sample. The high RI sensitivity of BSI was exploited to measure this RI change in blood. Previously, we demonstrated that the PT-AS sensor enables [Hb] ...

A blood test is commonly performed to evaluate overall human health and to detect biomarkers related to certain diseases. For example, the cholesterol concentration in blood serves as a criterion for hyperlipidemia, which is closely related to cardiovascular diseases and pancreatitis. The blood glucose contents should be measured frequently, as the glucose level is associated with complications such as diabetic ketoacidosis and hyperglycemic hyperosmolar syndrome. Serious illnesses such as malaria, human immunodeficiency virus and acquired immune deficiency syndrome are diagnosed by blood examinations, and quantification of blood components including erythrocytes, thrombocytes, and leukocytes enables screening of pancreatic and renal diseases.
Hemoglobin (Hb), a critical component of blood, makes up about 96% of erythrocytes, and transports oxygen to human organs. Significant alteration of its mass concentration ([Hb]) may indicate metabolic changes, hepatobiliary disease, and neurological, cardiovascular and endocrinological disorders1. [Hb] is therefore routinely measured in blood tests. In particular, anemic patients, dialysis patients, and pregnant women are strongly recommended to monitor [Hb] as a vital task2.
Various [Hb] detection methods have thus been developed. The hemoglobin cyanide method, one of the most common techniques for [Hb] quantification, employs potassium cyanide (KCN) to destroy the lipid bilayer of erythrocytes3. The cyanide hemoglobin produced by the chemical exhibits high absorption around 540 nm; hence, [Hb] measurements can be made via colorimetric analysis. This method is widely employed owing to its simplicity, but the employed chemicals (e.g., KCN and dimethyllaurylamine oxide) are toxic to humans and the environment. The hematocrit scheme measures the volume ratio of red blood cells compared to the total blood volume through centrifugal separation; however it requires a relatively large blood volume (50-100 &#956;l)4. Spectrophotometry methods measure [Hb] precisely without any chemicals, but measurements at multiple wavelengths and a large blood volume are required5,6. Similarly, several optical methods for measuring [Hb] have been proposed including detection methods based on light-scattering, but their measurement accuracies depend strongly on the accuracy of the theoretical blood model.
To overcome these limitations, [Hb] detection methods based on the photothermal (PT) effect of Hb have recently been proposed7. Hb, which is composed mainly of iron oxides, absorbs light at 532 nm and converts the light energy into heat8-10. This PT temperature increase can be detected optically by measuring a change in the refractive index (RI) of blood samples. Yim et al. employed spectral-domain optical coherence reflectometry to measure the PT optical path-length change in a blood-containing chamber11. Although the method enables chemical-free and direct [Hb] measurement, the use of a spectrometer and an interferometric arrangement may hinder its miniaturization. We recently presented an alternative [Hb] detection method, termed photo-thermal angular light scattering (PT-AS) sensor, which is more suitable for device miniaturization12. The PT-AS sensor exploits the high RI sensitivity of the back-scattering interferometry (BSI) to measure PT changes in the RI of a blood sample inside a capillary tube. BSI have been utilized to measure RI of various solutions13-15 and to monitor biochemical interactions in free solution16. The PT-AS sensor employs similar optical arrangement as in BSI, but combines photothermal excitation setup to measure PT increase of RI in blood samples. Operating principles of the BSI and the PT-AS sensors are described in detail elsewhere12,15. PT-AS sensor demonstrated high-sensitivity [Hb] measurement over a wide detection range (0.35-17.9 g/dl) and is capable of operating with sample volumes of &#60;100 nl. No preconditioning of blood sample is required, and the measurement time is only ~5 sec. Here, the experimental setup and a detailed measurement protocol are described. Representative PT-AS results are provided using blood samples from anemic patients, and the results are compared against those from a hematology analyzer to assess the accuracy of the PT-AS sensor.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>650nm laser pointer</td>
			<td>LASMAC</td>
			<td>LED-1</td>
			<td>Probe light</td>
		</tr>
		<tr>
			<td>Hollow round glass capillaries</td>
			<td>Cm Scientific</td>
			<td>CV2033</td>
			<td>Blood sample container</td>
		</tr>
		<tr>
			<td>Webcam</td>
			<td>Logitech</td>
			<td>C525</td>
			<td>CMOS optical sensor</td>
		</tr>
		<tr>
			<td>532-nm DPSS laser</td>
			<td>CNI Laser</td>
			<td>MGL-&#8546;-532</td>
			<td>Photothermal light source</td>
		</tr>
		<tr>
			<td>Optical chopper system</td>
			<td>Thorlabs</td>
			<td>MC2000-EC</td>
			<td>Optical chopper</td>
		</tr>
		<tr>
			<td>Plastic long-pass filter</td>
			<td>Edmund Optics</td>
			<td>#43-942</td>
			<td>To reject 532-nm PT excitation light</td>
		</tr>
		<tr>
			<td>Fiber clamp</td>
			<td>Thorlabs</td>
			<td>SM1F1-250</td>
			<td>Capillary tube fixture</td>
		</tr>
		<tr>
			<td>EDTA coated blood sampling tube</td>
			<td>Greiner Bio-One</td>
			<td>VACUETTE 454217</td>
			<td>Blood sampling &#38; anticoagulating</td>
		</tr>
		<tr>
			<td>Hematology analyzer</td>
			<td>Siemens AG</td>
			<td>ADVIA 2120i</td>
			<td>Reference hematology analyzer</td>
		</tr>
	</tbody>
</table>

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 hemoglobin assay was performed using the PT-AS sensor, and its measurements were compared with those from a hematology analyzer. The experiment was conducted with a PT excitation light intensity of 1.4 W/cm2, PT modulation frequency of 2 Hz, and measurement time of 5 sec. Table 1 summarizes the experimental conditions. The beam sizes of the probe and PT excitation light were 5.5 and 2 mm, respectively. The webcam recorded the images at a frame rate of 30 fps....

Watch this Scientific Journal Video about A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering at JoVE.com

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

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 ...

The goal of this presentation is to demonstrate a direct and rapid hemoglobin assay of anemic blood samples using a photothermal angular light scattering sensor. The PT-AS sensor is a novel, all optical sensor capable of measuring mass concentration of hemoglobin in unprocessed nanoliter volume blood samples without any chemicals. The sensor can be readily implemented with a low cost consumer electronic devices, such as a laser pointer and webcam.And so, provides ideal platform for point of care testing devices. The sensor can also be utilized to measure various kinds of biological and chemicals species that exhibit strong photothermal response, such as hemoglobin. The blood samples employed in this presentation are the red steel blood samples that had been acquired and processed in the clinical test at the institution.To begin setting up the photothermal angular light scattering sensor. First mount a clean capillary tube upright on a capillary tube fixture. Then, mount a 650 nanometer probe laser directed at the tube.Turn on the laser and check the scattering pattern with a sheet of paper. Next, place a camera without a lens on the other side of the capillary tube at an angle of about 25 to 35 degrees relative to the laser beam. Connect the camera to a computer and open the custom camera recording software.Check that the scattering pattern is in the center of the camera feed. Then, mount a 532 nanometer photothermal excitation laser directed towards the capillary tube. Ensure that the excitation laser light intersects the probe laser at the capillary tube, but does not directly shine into the camera.Turn off both lasers and remove the capillary tube from the mount. Place a long pass filter in front of the camera so only the 650 nanometer light is detected. Then, mount an optical chopper with a chopper wheel in front of the 532 nanometer laser.First derive calibration curve for the PT-AS sensor based on reference samples with known mass hemoglobin concentrations. Then, obtain a fresh whole blood sample drawn into a blood sampling tube containing ethylenediaminetetraacetic acid. Fill a clean capillary tube from the sample.Place the sample filled capillary tube in the mount. Then, turn on the 650 nanometer laser and check that the scattering pattern is centered correctly on the camera. Turn on the 532 nanometer laser at its highest power and turn on the optical chopper control box.Set the modulation frequency to two hertz and then start the chopper wheel. Using the custom designed software, record the scattering pattern from the camera. Then, turn off the lasers.Compute the average scattering pattern for each frame of the recording. Take the Fourier transform and calculate the phase at the peak spatial frequency for each scattering pattern. Plot the phase fluctuation over all of the frames as a function of time.Then, take the Fourier transform in the Time domain to determine the PT-AS signal magnitude at the PT modulation frequency. Convert the PT-AS signal to the hemoglobin mass concentration using the calibration curve. Three different samples were tested with PT-AS and with a hematology analyzer.Larger phase fluctuations are observed in samples with higher mass hemoglobin concentrations. The PT-AS signals averaged over 11 measurements corresponded to mean mass hemoglobin concentration values of 5.46, 7.23, and 9.85 grams per deciliter, which were similar to the values obtained with the hematology analyzer. Once properly aligned and calibrated, the PT-AS sensor enables high accuracy measurement of mass concentration of hemoglobin in unprocessed blood samples.A sensor can be built with low cost consumer electronic devices and requires only a blood loaded microcapillary tube for operation. These unique features of the PT-AS sensor will therefore enable it's wide adoption in clinical laboratories and research limited settings.

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Research

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Bioengineering

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A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

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We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

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We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

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This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

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This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

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This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...