1. Instrument Setup
<ol>
 <li>Turn on the spectrofluorometer and allow to warm up for 1 hour.</li>
 <li>Using Excel create a file with the Standards and Sample data, as shown in Figure 1 and save it in comma separated values "csv" format. 
 <img src="/files/ftp_upload/2465/2465fig1.jpg" alt="Figure 1" /> 
 Figure 1. Standards and samples data. 
 
 </li>
 <li>Set up fluorometer as follows:
 <ol>
 <li>Click on the <img src="/files/ftp_upload/2465/2465icon1.jpg" alt="Icon 1" /> button, this will open the Analysis Method window, shown in Fig 2. 
 <img src="/files/ftp_upload/2465/2465fig2.jpg" alt="Figure 2" /> 
 Figure 2. Analysis Method Window, General tab. 
 
 Make the following selections:
 <ul>
 <li>In the "Measurement" field, select Photometry from the pull down menu.</li>
 <li>In "Operator" field, enter the appropriate operator's name.</li>
 <li>There is no need to enter information in the "Instrument" field. This is selected automatically by the software.</li>
 <li>The "Sampling" field is inactive when using the microplate reader.</li>
 <li>Enter any comments which you may want included in the report in the 
 "Comments" field.</li>
 <li>In the "Accessory" field, enter information related to the accessories used for this test.</li>
 <li>Note the buttons on the right side of the window.</li>
 <li>The "Load" button allows loading previously saved Analysis Methods.</li>
 <li>The "Save" button allows updating a set of test parameters or Analysis Method previously saved and loaded, using the same name.</li>
 <li>The "Save As" button allows saving a new set of test parameters or Analysis Method under a desired name.</li>
 </ul>
 Now click on the "Quantitation" tab, see Figure 3 and make the following selections. 
 <img src="/files/ftp_upload/2465/2465fig3.jpg" alt="Figure 3" /> 
 Figure 3. Analysis Method Window, Quantitation tab. 
 <ul>
 <li>In the "Quantitation type" field, select Wavelength. This indicates the fluorescence reading will be made using the selected wavelength.</li>
 <li>In the "Calibration type" field, select 1st order.</li>
 <li>In the "Number of wavelengths" field, select 1.</li>
 <li>In the "Concentration unit" field, enter ng/ mL.</li>
 <li>Leave unselected the boxes for "Manual Calibration" and "Force curve through zero".</li>
 <li>In the "Digit after decimal point" field, select 2.</li>
 <li>In the "Lower concentration limit" field, enter 0.</li>
 <li>In the "Upper concentration limit field, enter 10000.</li>
 </ul>
 
 Now click on the "Instrument" tab and make the following selections. 
 <img src="/files/ftp_upload/2465/2465fig4.jpg" alt="Figure 4" /> 
 Figure 4. Analysis Method window, Instrument tab 
 <ul>
 <li>In the "Data mode:" field, select Fluorescence.</li>
 <li>The "Chopping speed" field is inactive at this time, it is used only for phosphorescence measurements.</li>
 <li>In the "Wavelength mode" field, select Both WL fixed.</li>
 <li>In the "EX / WL1" field enter 480nm.</li>
 <li>In the "EM / WL1" field enter 520nm.</li>
 <li>All the other fields under EX and EM are inactive at this time.</li>
 <li>In the "EX slit" field select 5.0nm from the pull down menu.</li>
 <li>In the "EM slit" field select 5.0nm from the pull down menu.</li>
 <li>Leave unselected the box in front of the "PMT Voltage 1-1000V" field.</li>
 <li>In the "PMT Voltage" field, select 700V from the pull down menu.</li>
 <li>In the "Auto statistic calc. number" field select 2.</li>
 <li>In the "Replicates" field select 1.</li>
 <li>In the "Integration time" field select 0.1s.</li>
 <li>In the "Delay" field enter or select 0s.</li>
 </ul>
 Click on the "Monitor" tab. 
 <img src="/files/ftp_upload/2465/2465fig5.jpg" alt="Figure 5" /> 
 Figure 5. Analysis Method window, Monitor tab. 
 <ul>
 <li>In the "Y axis", "Max:" field, select 10000</li>
 <li>In the "Y axis", "Min:" field, select 0.</li>
 <li>Select the box to the left of the "Open data processing after data acquisition".</li>
 <li>Leave unselected the box to the left of the "Print report after data acquisition", unless you want a report to be printed automatically after the readings are completed.</li>
 </ul>
 Click on the "Report" tab, see Figure 6. 
 <img src="/files/ftp_upload/2465/2465fig6.jpg" alt="Figure 6" /> 
 Figure 6. Analysis Method window, Report Tab. 
 <ul>
 <li>In the "Output" field, select Print Report from the pull down menu. You could also select "Use Microsoft Excel" if you want the data exported to Excel, or "Use Print Generator Sheet" if using the optional add on program Report Generator which will allow generating custom made reports.</li>
 <li>Click the boxes for the items you want to be included in the report.</li>
 <li>To save all the selected parameters in the different tabs of the "Analysis Method" window, click again on the "General" tab and save them under a file name and in a location where you could find them for future use, and click the "OK" button to close this window.</li>
 <li>This completes setting up the instrument test parameters.</li>
 <li>Now we will set up the microplate reader.</li>
 <li>Click on the <img src="/files/ftp_upload/2465/2465icon2.jpg" alt="Icon 2" /> button, which will bring the MPR Setup Window on top.</li>
 </ul>
 Select wells for the Standards and Samples in the MPR Setup Window/Plate tab, as shown in Figure 7. 
 <img src="/files/ftp_upload/2465/2465fig7.jpg" alt="Figure 7" /> 
 Figure 7. MPR Setup window, Plate tab. 
 <ul>
 <li>Click on the A1 position and drag the mouse to E1, then click on the <img src="/files/ftp_upload/2465/2465icon3.jpg" alt="Icon 3" /> button. This will select the positions to be used for the standards. Those positions will be highlighted in green color.</li>
 <li>Now we need to select positions F1 to H3 for the samples. Click and drag the mouse starting in position F1, to position H3 and then click on the <img src="/files/ftp_upload/2465/2465icon4.jpg" alt="Icon 4" /> button, this will highlight those positions in yellow color.</li>
 <li>Then repeat the same operation for positions A2 to E3, to select those positions for sample measurements.</li>
 <li>Now, let's load the file Comma Separated Variable (csv) file prepared in advance with the Standards and Sample information.</li>
 </ul>
 Click on the "Well information" tab of the "MPR Setup Window", as shown in Figure 8. 
 <img src="/files/ftp_upload/2465/2465fig8.jpg" alt="Figure 8" /> 
 Figure 8. MPR Setup window, Well Information tab. 
 <ul>
 <li>Then click on the <img src="/files/ftp_upload/2465/2465icon5.jpg" alt="Icon 5" /> button. A Windows explorer window will open which as shown in Figure 9, which will allow locating the "Well information.csv" file and loading it. 
 <img src="/files/ftp_upload/2465/2465fig9.jpg" alt="Figure 9" /> 
 Figure 9. Loading well sample information. 
 After loading the "Well information" file, the "Well information" will look as shown in Figure 10 
 <img src="/files/ftp_upload/2465/2465fig10.jpg" alt="Figure 10" /> 
 Figure 10. Loaded sample well information. 
 </li>
 </ul>
 </li>
 </ol>
 </li>
</ol>
2. Picogreen Assay Sample Preparation
<ol>
 <li>Prepare 1X TE Buffer (10 mM Tris-HCl, 1 mM EDTA) by adding 1 mL of 20 X TE Buffer to 19 mL dI H2O.</li>
 <li>Prepare PicoGreen Reagent Solution by adding 50 &mu;L 200 X PicoGreen Reagent to 9.95 mL 1 X TE Buffer.</li>
 <li>Prepare standards (Lambda dsDNA) by making serial dilutions of stock dsDNA (100 &mu;g/mL). First prepare a solution of 50 &mu;g/mL dsDNA by adding 25 &mu;L of stock dsDNA (100 &mu;g/mL) and 25 &mu;L 1 x TE Buffer. Then prepare the standard solutions by adding the indicated volumes of dsDNA and buffer as listed in the table below:
 <table border="1">
 <tbody>
 <tr>
 <td>Concentration of dsDNA</td>
 <td>Volume of dsDNA</td>
 <td>Volume of Buffer</td>
 </tr>
 <tr>
 <td>1 &mu;g/mL</td>
 <td>20 &mu;L of 50 &mu;g/mL dsDNA</td>
 <td>980 &mu;L 1 X TE Buffer</td>
 </tr>
 <tr>
 <td>0.1 &mu;g/mL</td>
 <td>100 &mu;L of 1 &mu;g/mL dsDNA</td>
 <td>900 &mu;L 1 X TE Buffer</td>
 </tr>
 <tr>
 <td>0.01 &mu;g/mL</td>
 <td>100 &mu;L of 0.1 &mu;g/mL dsDNA</td>
 <td>900 &mu;L 1 X TE Buffer</td>
 </tr>
 <tr>
 <td>0.001 &mu;g/mL</td>
 <td>100 &mu;L of 0.01 &mu;g/mL dsDNA</td>
 <td>900 &mu;L 1 X TE Buffer</td>
 </tr>
 </tbody>
 </table>
 </li>
</ol>
3. Measurement of Standards and Sample Fluorescence
<ol>
 <li>Pipette 150 &mu;L of each standard into wells A1-E1 of a 96 well plate, per Figure 7, and according to the following table:
 <table border="1">
 <tbody>
 <tr>
 <td>Well</td>
 <td>Standard</td>
 </tr>
 <tr>
 <td>A1</td>
 <td>1 &mu;g/mL</td>
 </tr>
 <tr>
 <td>B1</td>
 <td>0.1 &mu;g/mL</td>
 </tr>
 <tr>
 <td>C1</td>
 <td>0.01 &mu;g/mL</td>
 </tr>
 <tr>
 <td>D1</td>
 <td>0.001 &mu;g/mL</td>
 </tr>
 <tr>
 <td>E1</td>
 <td>1 X TE Buffer</td>
 </tr>
 </tbody>
 </table>
 </li>
 <li>Pipette 150 &mu;L unknown samples into wells F1 to H3, shown in Figure 7</li>
 <li>Pour PicoGreen solution prepared in step 1.2 into a trough designed for multi-channel pipette use. Use a multi-channel pipette to deliver 150 &mu;L PicoGreen solution to wells A1-H3. Mix solution and standards gently with pipette.</li>
 <li>Place plate in dark area and wait 2-5 minutes for reaction to develop.</li>
</ol>
4. Representative Results
A good calibration curve is evaluated using the different parameters provided by the F-7000 software upon measurement of the standards and calculation of the calibration curve by the software.
The determination coefficient indicates the goodness of fit of the measured standards and the calculated calibration curve. The closer this value is to "1", the better the fit of the measured value and calibration curve.
If the value is far from "1", the standards must be re-measured, prepared again, or the fitness of the calibration curve changed.
Figure 11 shows an example of a calibration curve with a calculated determination coefficient= 0.99993, obtained for quantification of dsDNA using PicoGreen dye.
<img src="/files/ftp_upload/2465/2465fig11.jpg" alt="Figure 11" /> 
 Figure 11. Example of dsDNA calibration curve.

<ol>
	<li>Singer, V. L., Jones, L. J., Yue, S. T., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+PicoGreen+reagent+and+development+of+a+fluorescence-based+solution+assay+for+double-stranded+DNA+quantitation.">Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.</a> Analytical Biochemistry. 249 (2), 228-238 (1997).</li></ol>

Luis A. Moreno and Kendra L. Cox are employed by Hitachi High Technologies America that produces the instrument used in this article.

Careful pipetting during sample preparation, as well as using a stable and sensitive fluorescence spectrophotometer is essential for obtaining good and reproducible results.
In the case of the fluorescence spectrophotometer used for this test, it is suggested using 300 &mu;L final sample volume in the microplate reader. Lower volume decreases the sensitivity, and a larger volume may cause cross contamination between wells.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Distilled H2O</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Quant-iT Picogreen dsDNA Assay Kit from Invitrogen</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>P7589</td>
<td>&nbsp;</td>
<tr>
<td>Nalge Nunc Fluorescence Microplates p/n 237108</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>237108</td>
<td>&nbsp;</td>
<tr>
<td>12 Channel Eppendorf Micropipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>3516677</td>
<td>&nbsp;</td>
<tr>
<td>1000 &mu;L Eppendorf Pipette</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>200 &mu;L Eppendorf Pipette </td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 mL serological pipet</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Aluminum foil</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette tips</td>
<td>Supply</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hitachi F-7000 Fluorescence Spectrophotometer with FL Solutions 2.1</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>5J1-0003</td>
<td>&nbsp;</td>
<tr>
<td>Microplate Reader Accessory for F-7000</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>5J0-0139</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

quantification of dsdna using the hitachi f-7000 fluorescence spectrophotometer and picogreen dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Watch this Scientific Journal Video about Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye at JoVE.com

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

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24.4K Views.  Hitachi High Technologies America. Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Video: Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...