This work was funded by the NSERC Individual Discovery program, the NSERC/CIHR Collaborative Health Research Program (CHRP), and the NSERC CREATE Canadian Astrobiology Training Program (CATP).

1. QD Solubilization
This is a method appropriate for CdTe. Similar methods can be used with other types of QDs such as InP/ZnS20 and CdSe/ZnS 21.
<ol>
 <li>Prepare a solution of CdTe QDs in toluene at 15 &mu;M (optical density = 2.5 at the first exciton peak).</li>
 <li>Transfer 200 &mu;L of this stock into a glass vial. Do not use plastic!</li>
 <li>Add 800 &mu;L toluene, 1 mL of 200 mM borate buffer (pH 9) and 2 &mu;L of 11.5 M mercaptopropionic acid (MPA).</li>
 <li>Cap the vial and shake it vigorously for 30-60 seconds.</li>
 <li>The aqueous and organic-solvent phases will separate spontaneously. The aqueous phase will become the color of the QDs. To avoid the toluene layer, remove it and discard it. Then transfer the aqueous phase into a clean vial.</li>
 <li>Purify the solubilized QDs by washing/filtering four times using a 500 &mu;L, 10000 molecular weight cutoff centrifuge filter. The filters should be spun at 3000 x g for 13 minutes. For at least the last two washes, wash with the desired final buffer.</li>
 <li>After the final wash, suspend the washed QDs in 50 mM borate buffer at the desired pH and store at 4 &deg;C. They will be stable for 1-2 weeks.</li>
 <li>Measure absorbance and emission spectra to estimate concentration based upon published formulas 22.</li>
</ol>
Representative results: Figure 1 shows an image of CdTe QDs under UV lamp illumination, and emission spectra before and after water solubilization, showing negligible change from the cap exchange. The size values are the core diameter measured by electron microscopy.
2. QD Conjugation to Antibiotic
This part of the protocol is applicable to any negatively-charged water-solubilized nanoparticle, including most commercial QDs, metal particles, and more19.
<ol>
 <li>Any molecule will work that either self-assembles to the negatively-charged solubilized QDs, or that has a reactive group that can be conjugated, such as a primary amine. In this example, we are using polymyxin B (PMB), which is positively charged and self-assembles without need for conjugation reagents. Dissolve the PMB in H2O at 60 &mu;M. (If the antibiotic chosen is not soluble in H2O, dissolve in DMSO or ethanol at a concentration of 0.1-1 mM). This will be a 10x solution (in H2O) or a 100x solution (in solvent).</li>
 <li>Calculate how much QD solution you will need for the bacterial toxicity experiments. For a 96-well plate with 0.3 mL per well in quadruplicates, 0.5 mL of 200 nM conjugate is needed. Prepare two tubes containing 100 &mu;L of 1 &mu;M quantum dots in 50 mM borate buffer.</li>
 <li>If coupling to a primary amine, prepare a 19 mg/mL (100 mM) solution of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) in H2O. EDC in solution is not stable; use immediately and throw the rest away.</li>
 <li>Add 50 &mu;L of the 10x antibiotic solution (in H2O) or 5 &mu;L of the 100x solution (in solvent) to the conjugate tube. For PMB:CdTe conjugation at molar ratio of 30:1, add 50 &mu;L of 60 &mu;M PMB. Add H2O or solvent to the QD only control tube.</li>
 <li>If needed, add 1 &mu;L of the EDC stock solution to the conjugate tube.</li>
 <li>Bring up the conjugate volume to 0.5 mL with the appropriate buffer (borate buffer or PBS; no amine-containing buffers such as Tris, as they will inhibit EDC).</li>
 <li>Shield tubes from light with aluminum foil, and place on a nutator or rocker for 1 hr. If aggregation occurs, repeat the conjugation with lower concentrations of antibiotic. Titrate the concentration upwards until the particles do not aggregate.</li>
 <li>Wash the conjugates by passing through an appropriate filter. 10000 molecular weight cutoff works for most antibiotics, but not for proteins or antibodies.</li>
 <li>Depending upon the molecule, different methods can be used to estimate the number of antibiotic molecules per QD: absorbance or fluorescence spectroscopy, gel electrophoresis, or Fourier transform infrared spectroscopy (FTIR).</li>
</ol>
Representative results. In this example, PMB conjugation is characterized by changes in QD emission spectrum. Figure 2 shows the spectra of CdTe QDs with PMB addition.
3. Preparation of Bacteria for 96-well Screen; Determination of Antibiotic IC50
This is applicable to almost any bacterial strain grown in the appropriate medium18. The exact length of time the recordings should continue depends upon the bacterial growth rate. In our example, we use Escherichia coli grown in lysogeny broth (LB) medium.
<ol>
 <li>In order to choose the appropriate QD conjugate concentrations, it is important to know the IC50 of the antibiotic to be conjugated and if the QDs alone are toxic. This should be begun 2 days before the conjugation experiment. In the evening, seed 10 mL of bacterial growth medium from a fresh colony, using correct sterile and biosafety technique.</li>
 <li>The next day, 1-2 hours before the experiment, fill each of the wells of a clear-bottom 96-well plate with a nearly-maximal amount of growth medium. Using a multichannel pipette, seed each well with 1-50 &mu;L of bacterial culture (the concentration will depend upon how fast the particular strain grows and will need to be calibrated by your laboratory).</li>
 <li>Put the plate onto the plate reader and set it to read every 10 minutes for 2 hours at a determined wavelength (usually 600 nm). Monitor the plate so that the bacteria do not grow too dense too quickly.</li>
 <li>When the cells all reach OD=0.1-0.15, remove the plate from the plate reader and stop the recording.</li>
 <li>Add drug alone and QDs alone at different concentrations in at least triplicates. Use one side of the plate as a "dark" control and one half to be illuminated. A suggested layout is given in Figure 3. The concentrations should span a wide enough range to include one concentration that inhibits the bacteria very little, and one that kills them all.</li>
 <li>Shield the "dark" side of the plate with aluminum foil. Expose the other side to a lamp at the desired wavelength. We use a custom 96-well, 440-nm lamp made from 2.4 mW LEDs (Figure 4) and irradiate for 30-60 min. The black plates with clear bottoms help shield the unexposed bacteria from stray light. An empty column may also be used between the exposed and un-exposed sides.</li>
 <li>After light exposure, put the plate into the plate reader and record OD 600 every 10 minutes for 5-12 hours depending upon growth rate. Keep the temperature &lt; 32 &deg;C if possible to avoid drying of the cultures.</li>
 <li>Plot the growth curves at a selected time point vs. log [concentration] and determine the IC50 of the antibiotic using the Hill equation:</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/3969/3969eq1.jpg" /> 
where H is the Hill coefficient, ymax is the highest point of growth (ideally on a plateau), and ymin is the zero-point, also ideally on a plateau. It is unlikely that QDs alone will show much toxicity to the cells at the concentrations used, so a value will not be determined.
Representative results. At the end of the recording period, clear wells will indicate complete cell death, and a gradient of cell density should appear along increasing concentrations of the drug. The bacteria should show S-shaped growth curves (Figure 5 A); the location of the maximum plateau will vary greatly from strain to strain and also depends upon temperature. A given time point can be chosen as representative and the values plotted vs. Log[antibiotic] to give the IC50 (Figure 5 B). To evaluate QD toxicity, survival vs. Log[QD] may also be plotted, but achieving significant bacterial killing with QDs alone is rare (Figure 5 C).
4. Preparation of Bacteria for 96-well Screen with Antibiotic/QDs
<ol>
 <li>The day before the experiment, seed 10 mL of culture from a fresh colony into the appropriate rich medium, using correct sterile and biosafety technique. The QD-antibiotic conjugate should be prepared on this day, unless it is highly unstable.</li>
 <li>1-2 hours before the experiment, fill each of the wells of a clear-bottom 96-well plate with a nearly-maximal amount of growth medium. Using a multichannel pipette, seed each well with 1-50 &mu;L of bacterial culture.</li>
 <li>Put the plate onto the plate reader and set it to read every 10 minutes for 2 hours at the same wavelength as in Part 3.</li>
 <li>When the cells all reach OD=0.1-0.15, remove the plate from the plate reader and stop the recording.</li>
 <li>Add QD conjugates at different concentrations in at least quadruplicate. Use one side of the plate as a "dark" control and one half to be illuminated. A suggested layout is given in Figure 6. A bacteria-only control strip should always be included in case problems with the culture, temperature, etc. affect growth conditions. A single strip of drug only and QD only should also be included to ensure that they match the control plate done previously. The rest of the wells are dedicated to conjugates in order to allow for good statistics.</li>
 <li>Shield the "dark" side of the plate with aluminum foil. Expose the other side to a lamp at the desired wavelength.</li>
 <li>After light exposure, put the plate into the plate reader and record OD 600 every 10 minutes for 5-12 hours. Keep the temperature &lt; 32 &deg;C if possible to avoid drying of the cultures.</li>
</ol>
Representative results. The combination of QDs and antibiotic may be less toxic than antibiotic alone; equally toxic; or more toxic. This can be quantified using the growth curves and IC50 measurements. Figure 7 shows an example of conjugates that equally toxic as antibiotic alone, and an example of conjugates that are more toxic.
5. Plate Count
<ol>
 <li>Choose one or more wells of the treated 96-well plate to be used for serial dilutions and colony counting.</li>
 <li>Make a serial dilution of each of the selected bacterial samples with PBS or saline solution (0.9 % NaCl). Transfer 20 &mu;L of the bacterial solution and dilute it with 180 &mu;L saline solution, change the pipette tip, mix and transfer 20 &mu;L of diluted bacterial solution to 180 &mu;L saline solution. Repeat 6 times.</li>
 <li>Take 10 &mu;L from each dilution and plate on an appropriate solid media plate. All 8 concentrations of one sample may be plated on a 10 cm round Petri dish, though rectangular dishes are preferred as it is easier to line things up.</li>
 <li>Let it dry on the bench for 15 min. then incubate at appropriate temperature for your bacteria for 16 hours.</li>
 <li>Count colonies and calculate colony forming units (CFU) according to (# of colonies x dilution factor)/volume plated= CFU/mL.</li>
</ol>
Figure 8 shows an example CFU plate.
6. Representative Results
<img alt="Figure 1" src="/files/ftp_upload/3969/3969fig1.jpg" /> 
Figure 1. CdTe QDs. (A) Eight preparations of CdTe QDs illuminated with a UV wand (365 nm). (B) Absorbance and emission spectra of five selected sizes before and after water-solubilization. The dashed lines are spectra in toluene, the solid lines are in water.
<img alt="Figure 2" src="/files/ftp_upload/3969/3969fig2.jpg" /> 
Figure 2. Spectral and gel analysis of QD-PMB conjugates. Orange-emitting CdTe QDs were used for this example; the effects on other types of QDs will need to be evaluated for each experiment. (A) Typical absorbance (grey line) and emission spectra (black line) of the QDs before conjugation of PMB and the emission (dashed line) after the addition of 160 molar equivalents of PMB. (B) Relationship between the ratio of PMB and the QD emission intensity (squares) and peak wavelength location (triangles).
<img alt="Figure 3" src="/files/ftp_upload/3969/3969fig3.jpg" /> 
Figure 3. Suggested plate layout for control growth plate. A wide range of PMB and QD concentrations is represented. One half of the plate is irradiated (highlighted blue), and an identical half is protected from light. <a href="https://www.jove.com/files/ftp_upload/3969/3969fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/3969/3969fig4.jpg" /> 
Figure 4. Custom 96-LED lamp for uniform plate irradiation, showing appearance off and on. A typical hand-held UV lamp may also be used, but will not cover the entire plate uniformly.
<img alt="Figure 5" src="/files/ftp_upload/3969/3969fig5.jpg" /> 
Figure 5. Example results for control growth plate. (A) Representative bacterial growth curves with different drug concentrations, from 0 to complete cell death. The open symbols are PMB only with concentrations given; the solid symbols are CdTe-PMB without irradiation; and the half-filled symbols are CdTe-PMB with irradiation. Irradiation had no effect on the PMB-only samples, so these curves were omitted for clarity. All PMB-CdTe conjugates are 30:1 PMB:QD ratios. (B) Plots of growth curve values at 200 min vs. Log[PMB] and fits to Eq. (1). To control for the effects of light, a curve is done with antibiotic only with 30 min of light exposure. (C) Bacterial survival at 200 min vs. QD concentration, using CdTe QDs. Some toxicity is seen with light exposure, but too little to determine an IC50 value. <a href="https://www.jove.com/files/ftp_upload/3969/3969fig5large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 6" src="/files/ftp_upload/3969/3969fig6.jpg" /> 
Figure 6. Suggested layout for conjugate test plate. The blue-highlighted half of the plate should be exposed to light, and the unhighlighted half is protected. <a href="https://www.jove.com/files/ftp_upload/3969/3969fig6large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 7" src="/files/ftp_upload/3969/3969fig7.jpg" /> 
Figure 7. Example results for conjugate test plate. Growth curve values at 200 min were plotted and fit to Eq. (1). (A) CdTe-PMB conjugates show increased toxicity over PMB alone. (B) Gold nanoparticle Au-PMB conjugates show no increased toxicity over PMB alone.
<img alt="Figure 8" src="/files/ftp_upload/3969/3969fig8.jpg" /> 
Figure 8. Example of a CFU plate. E. coli seeded in a 96-well plate was treated with QD-PMB with or without irradiation for 30 min. then incubated at 32 &deg;C for 4 hours. Serial dilutions of each bacterial sample were made with saline solution, and 10 &mu;L of 100 X to 107 X dilutions were plated on agar plates. The plates were incubated at 37 &deg;C and colonies were counted after 16 hours. The plate shows the dilutions along the rows as indicated; the columns are: (A) 0.06 &mu;M PMB + 2 nM CdTe, (B) 0.12 &mu;M PMB + 4 nM CdTe (C) 0.2 &mu;M PMB + 6.7 nM CdTe, (D) 0.06 &mu;M PMB + 2 nM CdTe irradiated, (E) 0.12 &mu;M PMB + 4 nM CdTe irradiated, (F) 0.2 &mu;M PMB + 6.7 nM CdTe irradiated.

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No conflicts of interest declared.

Nanoparticles represent a promising approach to creation of new anti-microbial agents. Growth curve analysis is a way to monitor bacterial cell density that distinguishes actively-growing cells from growth-inhibited cells. When coupled with plate counts, it allows for a thorough analysis of the antibiotic potential of a conjugate. The 96-well format permits relatively high-throughput variations of concentration and other conditions such as light exposure; the latter is crucial for light-activated agents such as quantum d...

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Borate Buffer Component #1</td>
			<td>Fisher</td>
			<td>Boric acid A-74-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Borate Buffer Component #2</td>
			<td>Sigma-Aldrich</td>
			<td>Sodium Tetraborate B9876</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPA</td>
			<td>Sigma-Aldrich</td>
			<td>M5801</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vivaspin 500</td>
			<td>GE Healthcare</td>
			<td>28-9322</td>
			<td>Various MWCO available</td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Fisher</td>
			<td>03-338C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDC</td>
			<td>Sigma-Aldrich</td>
			<td>E6383</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polymyxin B</td>
			<td>Sigma-Aldrich</td>
			<td>P1004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacterial growth medium (LB) Component #1</td>
			<td>Fisher</td>
			<td>NaCl S271</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacterial growth medium (LB) Component #2</td>
			<td>BD</td>
			<td>Tryptone 211705</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bacterial growth medium (LB) Component #3</td>
			<td>BD</td>
			<td>Yeast Extract 211929</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lamp for light exposure</td>
			<td>Custom</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Clear-bottom 96-well plates</td>
			<td>Fisher</td>
			<td>07-200-567 or 07-200-730</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence spectrometer</td>
			<td>Molecular Devices</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Absorbance plate reader</td>
			<td>Molecular Devices</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BactoAgar for solid media</td>
			<td>Bioshop</td>
			<td>AGR001.1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petri dishes round</td>
			<td>Fisher</td>
			<td>08-75-12</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petri dishes rectangular</td>
			<td>Fisher</td>
			<td>08-757-11A</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. QDs have attracted a lot of interest for imaging, diagnostics, and therapy due to their bright, stable fluorescence1,2 3,4,5. QDs can be conjugated to a variety of bio-active molecules for binding to bacteria and mammalian cells6.
QDs are also being widely investigated as cytotoxic agents for targeted killing of bacteria. The emergence of multiply-resistant bacterial strains is rapidly becoming a public health crisis, particularly in the case of Gram negative pathogens 7. Because of the well-known antimicrobial effect of certain nanomaterials, especially Ag, there are hundreds of studies examining the toxicity of nanoparticles to bacteria 8. Bacterial studies have been performed with other types of semiconductor nanoparticles as well, especially TiO2 9,10-11, but also ZnO12 and others including CuO 13. Some comparisons of bacterial strains have been performed in these studies, usually comparing a Gram negative strain with a Gram positive. With all of these particles, mechanisms of toxicity are attributed to oxidation: either the photogeneration of reactive oxygen species (ROS) by the particles or the direct release of metal ions that can cause oxidative toxicity. Even with these materials, results of different studies vary greatly. In some studies the Gram positive test strain is reportedly more sensitive than the Gram negative 10; in others it is the opposite 14. These studies have been well reviewed 15.
In all nanoparticle studies, particle composition, size, surface chemistry, sample aging/breakdown, and wavelength, power, and duration of light exposure can all dramatically affect the results. In addition, synthesis byproducts and solvents must be considered16 17. High-throughput screening techniques are needed to be able to develop effective new nanomedicine agents.
CdTe QDs have anti-microbial effects alone18 or in combination with antibiotics. In a previous study, we showed that coupling of antibiotics to CdTe can increase toxicity to bacteria but decrease toxicity to mammalian cells, due to decreased production of reactive oxygen species from the conjugates19. Although it is unlikely that cadmium-containing compounds will be approved for use in humans, such preparations could be used for disinfection of surfaces or sterilization of water.
In this protocol, we give a straightforward approach to solubilizing CdTe QDs with mercaptopropionic acid (MPA). The QDs are ready to use within an hour. We then demonstrate coupling to an antimicrobial agent.
The second part of the protocol demonstrates a 96-well bacterial inhibition assay using the conjugated and unconjugated QDs. The optical density is read over many hours, permitting the effects of QD addition and light exposure to be evaluated immediately as well as after a recovery period. We also illustrate a colony count for quantifying bacterial survival.

Watch this Scientific Journal Video about Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count at JoVE.com

Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count

Nanoparticles such as semiconductor quantum dots (QDs) can be used to create photoactivatable agents for anti-microbial or anti-cancer applications. This technique shows how to water-solubilize cadmium telluride (CdTe) QDs, conjugate them to an antibiotic, and perform a bacterial inhibition assay based upon growth curves and plate count.

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. ...

solubilization-bio-conjugation-quantum-dots-bacterial-toxicity-assays

Research

JoVE Journal

Bioengineering

14.2K Views.  McGill University, Montreal, QC Canada. Nanoparticles such as semiconductor quantum dots (QDs) can be used to create photoactivatable agents for anti-microbial or anti-cancer applications. This technique shows how to water-solubilize cadmium telluride (CdTe) QDs, conjugate them to an antibiotic, and perform a bacterial inhibition assay based upon growth curves and plate count.

Video: Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with turn-key performance and microjoule-level pulse energy is used to seed the regenerative chirped-pulse amplifier. The amplifier is placed in airtight housing. It operates at room temperature and exhibits stable operation at a 5 kHz repetition rate, with a pulse-to-pulse stability less than 1%. By employing a 1.5 mm-thick beta barium borate crystal, the frequency of the laser output is doubled to 515 nm, with an average power of 70 W, which corresponds to an optical-to-optical efficiency of 70%. This superior performance makes the system an attractive pump source for optical parametric chirped-pulse amplifiers in the near-infrared and mid-infrared spectral range. Combining the turn-key performance and the superior stability of the regenerative amplifier, the system facilitates the generation of a broadband, CEP-stable seed. Providing the seed and pump of the optical parametric chirped-pulse amplification (OPCPA) from one laser source eliminates the demand of active temporal synchronization between these pulses. This work presents a detailed guide to set up and operate a Yb:YAG thin-disk regenerative amplifier, based on chirped-pulse amplification (CPA), as a pump source for an optical parametric chirped-pulse amplifier.

A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here.

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

A Combined Micro-Device System for Promoting and Monitoring Coral Growth

An Integrated Micro-Device System for Coral Growth and Monitoring

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of the coral culture environment is highly in demand for coral conservation and study. Here, we developed a semi-closed coral culture micro-device system as a multi-functional platform, which can provide accurate and programmable temperature control, a sterile initial environment, long-term stable water quality, an adjustable dissolved oxygen concentration, and a customized light spectrum for corals. Owing to the modular design, the coral culture system can be upgraded or modified by installing desirable new modules or removing existing ones. Currently, under appropriate conditions and with proper system maintenance, the sample corals can survive for at least 30 days in a healthy state. Furthermore, due to the controllable and sterile initial environment, this coral culture system can support research into the symbiotic relationship between corals and associated microorganisms. Therefore, this micro-device system can be applied to monitor and investigate sea corals in a relatively quantitative manner.

Author Spotlight: Advancing Coral Culture - Creating a Semi-Quantitatively Controlled Microenvironment System to Counter Current Limitations 

This protocol describes the development of a modular controllable micro-device system that can be applied for the long-term culturing and monitoring of sea corals.

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of ...