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. Commercially-available fiber fixtures can be used as the tube fixture.</li>
	<li>Securely anchor a 650 nm laser pointer, i.e., probe light source, to illuminate the capillary tube. The probe beam should be larger than the capillary tube. Place a screen (e.g., white paper) behind the capillary tube to observe an angular periodic pattern.</li>
	<li>For the detection part, remove any lenses in a webcam to directly capture the scattering pattern. Position the webcam behind the capillary tube at an angle of 25-35&#176; relative to the probe beam direction. Ensure that the angular periodic pattern produced by the capillary tube can be measured with the detector (Figure 1). Observe the angular periodic pattern in the middle of the image sensor when the image sensor is properly positioned.</li>
	<li>Position a 532-nm PT excitation light source to illuminate the capillary tube. Position the PT light source at any angle, as long as the PT excitation light overlaps with probe beam on the capillary tube and does not reach the detector directly. PT excitation of blood samples using high optical power typically improves the PT-AS sensitivity, as it leads to a larger change in the RI.
	<ol>
		<li>Use the highest optical power of the employed PT excitation light source. In addition, ensure that the PT excitation light overlaps the probe light on the capillary tube. Use a beam size of the PT excitation light at least twice that of the probe light to heat the entire probe volume.</li>
	</ol>
	</li>
	<li>Place a long-pass filter in front of the detector to block the 532-nm light and measure only the 650-nm probe light.</li>
	<li>Install an optical chopper in the path of the PT excitation light before illuminating the capillary tube. The optical chopper is employed to modulate the PT excitation light intensity.</li>
</ol>
2. Blood Sample Preparation
<ol>
	<li>Draw 6 ml of fresh whole blood in anemic condition into ethylenediaminetetraacetic acid blood sampling tubes, and mix the samples well. No other processing is required.</li>
	<li>Measure the blood samples using the PT-AS sensor within 24 hr of extraction to prevent coagulation.</li>
</ol>
3. PT-AS Measurement Protocols
<ol>
	<li>Load a micro-capillary tube with a blood sample to measure. Fill the capillary tube with the blood through capillary action by placing the tube into the blood sample. The minimum sample volume required for measurement is determined by the inner diameter of the capillary tube and the probe beam size.
	<ol>
		<li>Employ a tube with an inner diameter of 200 &#956;m. The probe beam size was 2 mm in the representative results, suggesting that the measurement can be performed with a sample volume of &#62;63 nl.</li>
	</ol>
	</li>
	<li>Mount the capillary tube at the designated position in the fixture.</li>
	<li>Turn on the 650 nm probe laser to illuminate the blood-loaded micro-capillary tube. The angular periodic pattern should be observed with the webcam.</li>
	<li>Turn on the 532-nm PT excitation laser to illuminate the tube.</li>
	<li>Run the optical chopper to modulate the intensity of the PT excitation light at 2 Hz. 
	&#8203;NOTE: The rationale for the selection of this operating condition is described in Discussion and Kim et al.12.
	<ol>
		<li>Mount a chopper wheel in the motor head assembly of the optical chopper system.</li>
		<li>Turn on the chopper control box, and use the control knob in the console to set the modulation frequency.</li>
		<li>Run the chopper using the control knob.</li>
	</ol>
	</li>
	<li>Record the fluctuating scattering pattern via the webcam for 5 sec in MPEG-4 (mp4) format.</li>
</ol>
4. Signal Processing
NOTE: PT-AS signal processing was performed using a lab-developed MATLAB code.
<ol>
	<li>Load the video file to extract the images. For each image [see Figure 2(a) for a representative image], obtain the averaged scattering pattern by computing the mean of the pixel values along the vertical direction [Figure 2(b, c)].</li>
	<li>Evaluate the Fourier transform of the averaged scattering pattern, and compute the phase at the peak spatial frequency. Perform these operations for all the frames of all the recorded images.</li>
	<li>Using the phase values obtained from all the images, plot the temporal phase fluctuation [Figure 2(d)]. Note that the phase fluctuates at the PT modulation frequency. Take the Fourier transform of the phase fluctuation in the time domain, and obtain the magnitude at the modulation frequency. This signal is referred to as the PT-AS signal [Figure 2(e)].</li>
	<li>Measure the [Hb] of a blood sample by converting its PT-AS signal into the corresponding [Hb] using the calibration curve that is obtained in Protocol 5.</li>
</ol>
5. PT-AS Calibration
<ol>
	<li>Prepare blood samples, having [Hb] values that are uniformly distributed in the detection range of the PT-AS sensor (e.g., 0 - 18 g/dl).</li>
	<li>Before calibration, quantify the [Hb] values of the samples using a reference hematology analyzer. Measure the PT-AS signals of the samples.</li>
	<li>Derive a calibration curve relating [Hb] to the PT-AS signal by performing a linear least squares fit, [Hb] = A[PT-AS Signal] + B, of the experimental results. For the operating conditions specified in Table 1, the relationship between [Hb] and the PT-AS signal was found to be [Hb] = 5.13 [PT-AS signal] - 0.09. Use MATLAB code to perform the linear fit.</li>
</ol>

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No conflict of interest is declared.

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] measurement with a detection limit of 0.12 g/dl over the range of 0.35-17.9 g/dl, which is comparable to that of commercial [Hb] sensors on the market.
A notable feature of the PT-AS sensor is that it does not require any preconditioning of blood samples or chemicals. Hence, the sensor enables direct, rapid (&#60;5 sec), and environment-friendly measurement. The use of glass-based micro-capillary tubes as a sample container enabled [Hb] assay at a low operating cost. The minimum sample volume in the PT-AS sensor is determined by the inner diameter of the capillary tube and the measurement beam size on the capillary tube. It is estimated to be ~63 nl in the representative results. In comparison to the sample volumes required in the commercial instruments (e.g., 50-200 &#956;l for the reference hematology analyzer), the PT-AS sensor enables [Hb] measurement with a significantly reduced sample volume. Several rapid and low-cost [Hb] detection techniques have been reported11,17,18 but still require the sample volumes of 2-10 &#181;l for operation.
Several features of the PT-AS sensor implementation should be noted. One should ensure that the size of the PT excitation light beam is at least twice that of the probe light beam on the capillary tube. The two light beams should overlap on the capillary tube, as no or partial overlap of the two light beams on the tube will result in either no or a smaller PT-AS response. One should also make sure that the angular scattering pattern is not saturated on the detector. Adjustment of the scattering pattern orientation along the horizontal or vertical direction may be necessary; otherwise, the acquired image should be rotated in the signal processing stage. Note that scattering of 532-nm PT excitation light by the tube also generates an angular scattering pattern on the detector. Thus, a long-pass filter is required to block the 532-nm light. Larger image sensor captures more angular periodic patterns. Fourier transform of the angular pattern would thus produce higher signal at the corresponding spatial frequency, which allows phase measurement with higher precision. Moreover, a higher frame rate would typically result in a PT-AS measurement with an improved SNR, as it enables more sampling of the temporal phase fluctuation. Therefore, the use of a large, high-speed image sensor with a high pixel density is advantageous.
Some comments should also be made on the measurement time and PT modulation frequency. As described in Kim et al.12, the PT-AS signal refers to the magnitude of the Fourier transform of the phase fluctuations of the angular scattering pattern measured at the PT modulation frequency. The noise is defined as the peak amplitude of the Fourier transform of the phase measurement before PT excitation12. The SNR of the PT-AS signal is evaluated by dividing the magnitude of the PT-AS signal by the noise. A longer measurement time typically yields measurements with a higher SNR, but increases the total [Hb] assay time. The measurement time was set to be 5 sec to achieve an SNR greater than 3 even for blood samples of [Hb] &#60; 1 g/dl. The optimal PT modulation frequency can be found by examining the SNR of the PT-AS sensor as a function of the PT modulation frequency. The optimal modulation frequency for the representative results was found to be 2 Hz. Operation with a PT modulation frequency less than 2 Hz did not produce a high SNR owing to low-frequency noise such as excessive motion of the optical chopper and vibration.
In this demonstration, the PT-AS sensor was demonstrated in a benchtop configuration using a commercial laser pointer and webcam. The optical setup is straightforward, and, because no chemicals are involved, the measurement procedures are simple. On the other hand, it should be emphasized that the sensor can potentially be packaged in a compact handheld device. The light sources for probe and PT excitation can be replaced by low-cost laser diodes or light-emitting diodes. A miniaturized complementary metal-oxide-semiconductor image sensor with built-in computational power can also be utilized as a detector. Integrating these components in a small form factor would generate a new portable, chemical-free, and inexpensive platform for [Hb] assay. In addition to [Hb] assay, the detection principle of the PT-AS sensor may be extended to sensing various biomarkers and chemicals that exhibit PT responses. For example, PT assay of organophosphates and pesticides has also been demonstrated19, and can be readily realized with the PT-AS scheme.

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. For measurement, anemic blood samples with three different Hb concentrations were employed. Before the PT-AS measurements, the [Hb] values of the samples were first measured as 5.3, 7.5, and 9.9 g/dl by the hematology analyzer.
Figure 3(a) shows representative time-lapse phase fluctuations of the angular scattering patterns under the modulated PT light illumination. This information was obtained by taking the Fourier transform of the angular scattering pattern and measuring the temporal phase fluctuations at the peak spatial frequency. Note that blood samples with a higher [Hb] exhibit larger phase shifts. The corresponding PT-AS signals were evaluated and converted into [Hb] values. Eleven measurements were performed for each sample, and the mean [Hb] values were found to be 5.46, 7.23, and 9.85 g/dl, respectively. The results agreed well to those obtained using the hematology analyzer [Figure 3(b)]. The [Hb] measurement precision of the PT-AS sensor was found to be &#60;0.89 g/dl. This fluctuation may be in part accounted for by the number fluctuation of erythrocytes in the probe volume and the intensity fluctuations of the employed light sources. Table 2 presents a detailed comparison of the PT-AS measurements against those from the hematology analyzer.
<img alt="Figure 1" src="/files/ftp_upload/55006/55006fig1.jpg" /> 
Figure 1: Schematic of PT-AS sensor. 650-nm probe light from a laser pointer is directed to a blood-loaded capillary tube. The light is then scattered by the blood-containing tube, generating a periodic pattern on a webcam. Upon illumination with 532-nm light, at which Hb molecules exhibit high absorption, Hb molecules absorb the light energy and convert it into heat. The resultant temperature rise changes the RI of the blood. Because the angular periodic pattern varies with the RI and the physical size of the tube, [Hb] in the blood is quantified by measuring this PT shift in the angular periodic pattern. An optical chopper is employed to achieve [Hb] measurement with a high signal-to-noise ratio. A low-cost plastic long-pass filter is located directly in front of the webcam to detect only the probe light. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55006/55006fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55006/55006fig2.jpg" /> 
Figure 2: PT-AS signal processing procedures. (a) Representative webcam images with PT excitation light on and off. The angular scattering pattern shifts because of the PT response of Hb molecules. (b) Each image is averaged along the vertical (y) direction to obtain the averaged pattern. (c) Representative averaged periodic patterns with PT excitation on and off. (d) The averaged periodic pattern is then Fourier transformed, and the phase at the peak spatial frequency is examined as a function of time. Under the modulated PT light illumination, the phase of the periodic pattern fluctuates at the modulation frequency. (e) The measured phase fluctuation is Fourier-transformed, and its magnitude evaluated at the modulation frequency, referred to as the PT-AS signal, is converted into [Hb]. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55006/55006fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55006/55006fig3.jpg" /> 
Figure 3: PT-AS measurement of anemic blood samples. (a) Representative phase fluctuations of the angular scattering patterns measured for three blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl). The blood samples with higher [Hb] values produce larger phase variations. (b) Comparison of [Hb] values measured using the PT-AS sensor with those from the reference hematology analyzer. Eleven PT-AS measurements were performed for each sample. The error bar denotes the standard deviation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55006/55006fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Experimental conditions</td>
		</tr>
		<tr>
			<td>PT modulation frequency</td>
			<td>2 Hz</td>
		</tr>
		<tr>
			<td>PT light intensity</td>
			<td>1.4 W/cm&#178;</td>
		</tr>
		<tr>
			<td>PT beam size</td>
			<td>5 mm</td>
		</tr>
		<tr>
			<td>Probe beam size</td>
			<td>2 mm</td>
		</tr>
		<tr>
			<td>Measurement time</td>
			<td>5 sec</td>
		</tr>
		<tr>
			<td>Frame acquisition rate</td>
			<td>30 fps</td>
		</tr>
	</tbody>
</table>
Table 1: Experimental conditions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Hematology analyzer (g/dl)</td>
			<td colspan="2">PT-AS Sensor</td>
		</tr>
		<tr>
			<td>Mean (g/dl)</td>
			<td>SD (g/dl)</td>
		</tr>
		<tr>
			<td>5.3</td>
			<td>5.46</td>
			<td>0.72</td>
		</tr>
		<tr>
			<td>7.5</td>
			<td>7.23</td>
			<td>0.89</td>
		</tr>
		<tr>
			<td>9.9</td>
			<td>9.85</td>
			<td>0.84</td>
		</tr>
	</tbody>
</table>
Table 2: Comparison of [Hb] measurements by the PT-AS sensor with those by the hematology analyzer.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.6K Views.  Yonsei University. 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.

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

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

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.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

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.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

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.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

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.

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

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 ...

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...