Fluorescence is emitted energy in the form of light when an electron in an excited state relaxes to the ground state. Fluorescence spectroscopy is the measurement, and characterization of the intensity of the fluorescence emission. Fluorescence is a two-step process: Excitation and Emission.
The molecule is excited by light of a specific wavelength. When light from the excitation monochromator is absorbed by the molecule, an electron in the molecule is excited to a higher energy level. After excitation, the molecule remains in the excited state for some time, ranging from hundreds of picoseconds to hundreds of nanoseconds, and then returns back to the ground state. The transition is accompanied by the emission of a photon, a particle of light. The wavelength of the emission is almost always longer than the wavelength of the excitation. The intensity of the emission is measured by a sensitive light detector.
The processes that compete in the transition from the excited state to the ground state include: emission of a photon, a radiative transition producing fluorescence; internal conversion (IC), the non-radiative loss of excitation energy through conversion of an excited state to a highly excited vibrational level of a lower state of the same multiplicity and subsequent dissipation of this energy into heat; intersystem crossing (ISC), the non-radiative transition from a singlet state to a triplet state (and vice versa) that can lead to phosphorescence; and quenching, the non-radiative loss of excitation energy mediated by a collision with a second molecule.
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It is possible to describe the de-excitation of a simple fluorescent molecule by a first order kinetic scheme:
PROCESS RATE CONSTANT DESCRIPTION
1M* Â® M kf radiative emission
1M* Â® M kic internal conversion
1M* Â® 3M* kisc intersystem crossing
1M* Â® A + B kd dissociation
1M* + Q Â® M kq quenching
1 and 3 designate a singlet and triplet state, respectively. The rate of loss of the excited singlet state may thus be given by
-d[1M*]/dt = [1M*] (kf + kic + kisc + kd + kq [Q])
This equation may be solved to yield
[1M*] = [1M*0] exp(-t/t),
where [1M*0] is the initial concentration of excited molecules and the lifetime t is given by:
t = 1/ (kf + kic + kisc + kd + kq [Q])
Fluorescence intensity can be quantified by measuring the fluorescence quantum yield F:
F = Kf / (Kf + kic + Kisc + kd + kq[Q])
The lifetime (t) and the fluorescence quantum yield (F) are the two basic parameters that characterize fluorescence.
Fluorescence is many orders of magnitude more sensitive than absorption spectroscopy. The sensitivity of a given fluorophore is dependent on both the molecular absorptivity and the fluorescence quantum yield.
Proteins contain three aromatic amino acid residues (tryptophan (W), tyrosine (T), phenylalanine (P)) which may contribute to their intrinsic fluorescence. Changes in intrinsic fluorescence can be used to monitor structural changes in a protein.
The fluorescence of a folded protein is a mixture of the fluorescence from individual aromatic residues. Protein fluorescence is generally excited at 280 nm or at longer wavelengths, usually at 295 nm. Most of the emissions are due to excitation of tryptophan residues, with a few emissions due to tyrosine and phenylalanine.
The three residues have distinct absorption and emission wavelengths. They differ greatly in their quantum yields and lifetimes. Due to these differences and to resonance energy transfer from proximal phenylalanine to tyrosine and from tyrosine to tryptophan, the fluorescence spectrum of a protein containing the three residues usually resembles that of tryptophan.
Trans-membrane glycoprotein gp350/220 was an Epstein-Barr Virus Envelope Glycoprotein (EBV) subunit vaccine candidate licensed back to MedImmune from GSK in September 2009 after a phase II clinical trial in GSK. EBV gp350/220 is the major EBV surface antigen and most of the neutralizing activity in EBV-positive serum is directed against gp350/220  . EBV gp350/220 is also a target for anti-body-dependent cellular cytotoxicity  . In preclinical trials in cottontop tamarins, immunization with purified gp350/220 was effective at preventing EBV-mediated lymphoproliferation. The viral receptor allows EBV gp 350/220 to enter human target cells by binding the cellular membrane protein, CD21  . Transcription from a single gene produces the mRNA for gp350/220 which is differentially spliced to allow both products to be made  , one of 350 kDa and the other 220 kDa.
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Figure 1 shows the sequence and secondary structure of EBV gp 350 fragment from residues 4 to 443  . From Figure 1, it shows that there are at least four tryptophan amino acids existing in thei surface glycoprotein, which should provide sufficient intrinsic fluorescent signals to characterize the subunit protein.
Figure 1: Sequence and secondary structure of gp350 fragment (residues 4-443). Arrows represent b-strands (b1-b25); thin lines depict loop regions; the 14 glycosylated asparagine residues are colored green (residue numbers are listed below); the three peptides known to block EBV attachment to CR2-expressing cells are colored orange, red and magenta 29,30; the four cysteines near the ends of these peptides that form disulfide bonds, stabilizing the peptides as surface loops, are colored cyan; red and blue asterisks mark the specific residues (inside or outside the putative interface, respectively) that were mutated for receptor-binding assays.
Possibly, the tertiary structure changes of EBV gp 350 caused by pH or other environmental factors can be detected by the changes of intrinsic fluorescence in different pH buffers. This protocol will establish the procedures to explore the intrinsic fluorescence of EBV gp350 as a preliminary evaluation for the currently available purified materials.
Materials and Equipment
In this protocol, four different samples will be prepared and evaluated:
Nano Water: To verify the proper operation of PTI fluorometer and also the blank control for BSA samples.
0.5 mM bovine serum albumin (BSA) dissolved in Nano water to verify the instrument can detect proteins properly.
Albumin Monomer bovine (Lyophilized powder containing 98% monomer). BSA Sigma Catalog # A1900-1G, Lot # 127K7405, CAS #: 9048-46-8. Received 24sep08. Light sensitive; stored at 2-8 ï‚°C.
PBS buffer as the blank control For EBV gp 350. GIBCO PBS (Phosphate buffered saline 1X, 155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4). pH 7.4. Cat#: 10010-031. Invitrogen. Eugene, Oregon. US.
EBV in GIBCO PBS buffer, pH 7.4. Source (sub) clone: 30B8-4B1. Batch ID: ID082010-C. 0.24 mg/mL. Purity (densiometry): 80-90% (0ne band at ~ 320 KDa). Prepared by the purification group of MedImmune, Santa Clara.
PTI Fluorometer serial No: 2914. Photon Technology International. Inc. Fluorescence Analysis Software: PTI FeliX32TM.
6Q quartz cuvette.
Mettler Toledo Balance. Equipment ID: WI-022.
BIOHIT Proline 50-1200 uL. MetroCalibrations 415-606-6763. Serial #: 8113319. Pipet (PP-04031): Date: 21-May-2010. Due: November 2010. By: D. Leong. Cert#: 00874.
Refrigerator for 2-8 ï‚°C storage. Aviron Equipment ID: 00077 for BSA and REVCO RF-216 for EBV samples.
Methods and Procedures
IV.1. Procedures to Start the Instrument for Accurate Measurement
1. Ignite the arc lamp. Press the power button and the lamp will light automatically. You do NOT have to press the ignite button. For best results, allow the lamp to warm up for 15 minutes then adjust to display 75 watts.
2. Turn on the computer. Start FeliX32 by opening the FeliX32 icon on the Windows Desktop. This will first prompt you to enter your user ID and password for accessing the database.
User name for Computer: Formulation; Password for Computer: Fluorometer
User name for the software FeliX32: Administrator; Password for FeliX32: pti
3. In no particular order, power up the BryteBox, the Motor Driver Box, the Shutter Controller, the Temperature Controller. Ensure the water tank is full and is plugged in if temperature control is needed.
The Photomultiplier Detector(s) will be turned on by turning on the power of the Motor Driver Box or an optional dedicated power supply. The High Voltage control is set to -1100 volts and it should not need further adjustments.
4. Be Sure the Hardware Configuration is Correct!
FeliX32 must be properly configured. It is very important to make the correct settings in the Configure/Hardware section. If the monochromator is not setup correctly, it can be physically damaged! Make sure that FeliX32 knows where the monochromators are. The wavelengths of the excitation and emission monochromators must be entered into FeliX32. Once FeliX32 knows the position of the excitation and emission monochromators, the software can accurately control the wavelength. With the auto-initializing monochromators, the auto-initialize position should have been entered in the Monochromator Setup dialog box in Configure/Hardware by double-clicking on the monochromator icon. It is possible that the monochromators are damaged and the wavelength counter is incorrect.
5. To Check the Calibration of the Emission Monochromator:
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Check the emission monochromator first. Use the mercury lines from a fluorescent light as a standard. There are three strong lines at 365, 436, and 546 nm. Close both emission slits to 1 nm (a half turn) to avoid damaging the PMT. Raise the lid of the sample compartment and manually open the emission shutter by depressing the lever. Manually set the emission monochromator to 365 nm and observe the signal. The line is very sharp, so if a strong signal is not observed, adjust the wavelength from 363-367 nm. Note the wavelength where a strong response is observed. Small errors are acceptable because of the entrance angle of the light into the monochromator. Change the wavelength to 436 nm, and then 546 nm, and verify that a response is observed. It is often useful to run a wavelength scan covering these wavelengths to determine if the spectral peaks are where they are expected. The first run should be done with a very fast integration time, e.g., 0.01 second to avoid hitting the PMT with too much light. If the signals do not saturate then a scan can be run at slower integration times. Note that in the region 330 - 680 nm there is a smooth background due to the phosphor in the fluorescent lights.
6. To Check the Calibration of the Excitation Monochromator:
After the calibration of the emission monochromator has been verified, place a scatterer in a cuvette in the cuvette holder. Use water as a scatterer. Set all slits at 1 nm (a half turn). Make sure the light source is on. Set the excitation monochromator to 300 nm. While observing the intensity, manually scan the emission monochromator from 295 to 305 nm. The intensity should peak at 300 nm. A DYAG reference crystal can be used to check monochromator calibration.
CAUTION: If the intensity exceeds 1,000,000 counts per second, decrease the bandpass to avoid damaging PMT.
7. Manual Check the Calibration of the Monochromators
If the wavelengths of all monochromators match with what shown in the Monochromator Setup dialog box in Configure/Hardware, proceed to acquire data. If the numbers don't match, manually calibrate the configuration. By following the steps described below. .Figure 1 shows the flowchart of the hardware configuration for both "digital" and "digital-temp".
To initiate the auto-calibration, follow the steps described as below:
Click on "Configuration"
Click on "digital" or "digital-temp" from "Hardware Configuration". The hardware configuration will appear on the screen as shown in Figure 1.
Figure 1: Flowchart of the Hardware Configurations of PTI Fluorometer.
Click on "Initiate" then the diagram as shown in Figure 1 will appear on the screen, and the wavelength readings of "Ex Mono", "Mono A" and "Mono B" will appear as (Example):
Ex Mono 254 nm; Mono A 214.7 nm; Mono B 254 nm
After calibration, the system is ready to acquire data. To start collecting data, choose Acquisition/Open Acquisition to open a previously saved acquisition or Acquisition/New Acquisition to create a new acquisition. If selecting a new acquisition, a dialog box will open allowing the user to select the type of acquisition they would like to perform (Emission Scan, Excitation Ratio, etcâ€¦). Figures 2 and 3 show examples for setting up the excitation scan and emission scan parameters.
Figure 2: An Example for the Setup Parameters of Excitation Scan for Samples.
Figure 3: An Example for the Setup Parameters for Emission Scan for Samples.
Note: Emission wavelength should be always higher than the excitation wavelength.
IV.2. Nano Water to Verify the Proper Operation of the Instrument
The fastest way to verify that the instrument is working properly is to run a known sample and ensure to get the correct results. For fluorescence spectroscopy, the most commonly available material with a known response is water. The Raman band of water has a peak in its emission spectra at 397 nm when excited at 350 nm. Set up the instrument as follows:
Acquire Emission Spectra
Excitation 350 nm
Emission Start: 370 nm Stop: 450 nm
Step Size 0.5 nm
Integration 1 second
Bandpass: 5 nm for the entrance and exits slits of both the excitation and emission monochromators. Fill a clean, 1 cm, quartz cuvette with distilled water, tap it to displace any bubbles adhering to the walls, and place it in the sample compartment. Select ACQUIRE. The emission spectra should peak at 397 nm. Depending on the illuminator type, the intensity at the peak should be between 300,000 - 800,000 counts per second (cps) and the data on the baseline should be relatively noise-free. Figure 4 shows the Emission Scan of Nano water in the quartz cuvette after aligning the system.
Figure 4: Verification of Proper System Operation by Water Emission Scan.
Note: To be strictly correct, the peak in the emission spectra of water is not due to fluorescence, which is the emission of photons after excitation. It is Raman scattering that gives rise to the response of water. It simulates fluorescence nicely in that emission occurs at a longer wavelength than excitation.
IV.3. BSA to Verify Proper Operation of PTI Fluorometer for Proteins
Bovine serum albumin (BSA) is often used as the reference standard for other unknown proteins. The literatures suggested that the BSA maximum excitation peak at 282nm  or 285 nm, and the maximum emission peak reside among 340-348 nm  . BSA has the two trp residues (Trp-135 and Trp-214). Trp-214 is located in a hydrophobic fold and Trp-135 is located on the surface of the molecule. To verify the BSA intrinsic fluorescence in the formulation lab as the reference standard, the BSA samples were prepared as 0.5 mM in Nano water. The sample was conducted an emission scan at excitation wavelength of 282 nm, with the emission scan wavelength between 290 nm and 450 nm (bandpasses of both excitation and emission monochromators are set as 3 nms). Figure 5 shows the result from the initial scanning. The maximum emission wavelength is between 340 nm and 348 nm, which is consistent with what described in the literature.
Figure 5: Emission Scan of 0.5 mM BSA in Nano Water.
IV.4. Evaluation of the Maximum Ex and Em Wavelengths of EBV gp 350
EBV gp 350 is an unknown protein, which likely will have the maximum excitation wavelength between 280 nm to 300 nm, since the tryptophan amino acid has the maximum excitation wavelength at ~ 295 nm.
Click on "Acquisition" icon, and choose "New Acquisition", and choose "Excitation Scan". Find out the maximum excitation wavelength for EBV gp 350. See Figure 2 for the reference parameters.
Based on the maximum excitation wavelength for EBV gp 350, set up the emission scan parameter to find out the emission wavelength profile for EBV gp 350. See Figure 3 for the reference parameters.
If the photon intensity is too weak or too strong, adjust the bandpass to get the optimal range for the future pH and buffer screening study, and other formulation screening studies using the intrinsic fluorescence of EBV gp 350.
Once the maximum excitation and emission wavelengths are found, the same wavelengths and bandpass settings can be used for future pH/buffer screening studies.
V. Interpretation of Results / Analysis and Documentations
All operations should be recorded in a laboratory notebook. For comparison among samples and/or different parameters for the same sample, the data can be exported and copied into Excel Spreasheet, and plotted into graghs. Figure 6 shows that the FeliX 32 software can convert the graph shown in Figure 5 into the data points for Excel data export.