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horiba Fluorolog-3, Operation Manual

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Fluorolog-3 v. 2.2 (10 Sep 2002) 

Optimizing Data Acquisition 

 

4-13

Improving the signal-to-noise ratio 

Because of various hardware or software conditions, occasionally it is necessary to optimize 
the results of an experiment. 
 
The quality of acquired data is determined largely by the signal-to-noise (

S

/

N

) ratio. This is 

true especially for weakly fluorescing samples with low quantum yields. The signal-to-noise 
ratio can be improved by: 

• 

Using the appropriate integration time, 

• 

Scanning a region several times and averaging the results, 

• 

Changing the bandpass by adjusting the slit widths, and 

• 

Mathematically smoothing the data. 

 
The sections that follow discuss the alternatives for improving the 

S

/

N

 ratio and the advan-

tages and disadvantages of each. 

Artisan Technology Group - Quality Instrumentation ... Guaranteed | (888) 88-SOURCE | www.artisantg.com

Summary of Contents for Fluorolog-3

Page 1: ...service in house repair center WE BUY USED EQUIPMENT Sell your excess underutilized and idle used equipment We also offer credit for buy backs and trade ins www artisantg com WeBuyEquipment REMOTE IN...

Page 2: ...n 81 3 5823 0140 Tel 1 732 494 8660 16 18 rue du Canal China 86 10 6836 6542 Fax 1 732 549 5157 91165 Longjumeau cedex Germany 49 89 46 23 17 0 E Mail fluorescence jyhoriba com Tel 33 1 64 54 13 00 It...

Page 3: ...electronic or mechanical photocopying and recording without prior written permission from Jobin Yvon Inc Requests for permision should be requested in writing Information in this manual is subject to...

Page 4: ...signal detector 5 14 Reference signal detector 5 15 Gratings 5 16 Mirrors 5 20 Automated 4 position turret 5 21 6 Components Accessories 6 1 Itemized list 6 2 Model 1940 absorption transmission access...

Page 5: ...sub picomolar concentrations of fluorescein 10 3 Reduced volume samples 10 3 Fluorescence detection of highly scattering samples 10 4 Quantum yield calculations 10 4 Characterizing complex mixtures vi...

Page 6: ...320 Specifications 14 38 Troubleshooting 14 39 15 Technical Specifications 15 1 Spectrofluorometer system 15 2 Minimum computer requirements 15 4 Software 15 4 16 Glossary 16 1 17 Bibliography 17 1 18...

Page 7: ...ost limitless power provided by DataMax refer to the DataMax Data Collection Handbook contains data acquisition information and the Grams 32 User s Guide contains post processing instructions for data...

Page 8: ...le for the Fluorolog 3 7 Troubleshooting Potential sources of problems their most probable causes and possible solutions 8 Introduction to Lifetime Measurements Methods of determining the lifetime of...

Page 9: ...og 3 system including with a CCD detector 15 Technical Specifications Instrument specifications and computer requirements 16 Glossary A list of some useful technical terms related to fluorescence spec...

Page 10: ...hen operating the instruments A hazardous condition exists or danger exists that could damage the equipment Jobin Yvon Inc is not responsible for damage arising out of improper use of the equipment Ge...

Page 11: ...table or bench top Table size varies according to the system configuration an average size of 38 60 96 5 cm 152 4 cm is usually sufficient Table size for standard systems FL3 11 FL3 12 FL3 21 FL3 22...

Page 12: ...with the Fluorolog 3 is ozone free The lamp housing contains an electrically powered fan that removes the heat Environmental requirements Temperature 72 5 F 22 3 C Humidity level 70 No special ventil...

Page 13: ...he lamp never will interfere with the computer or system Make sure enough AC outlets are available for the Computer Printer optional Monitor Xenon lamp System controller SAC Use three prong plugs for...

Page 14: ...ers outside the United States should contact a local representative For up to the minute information about products services upgrades frequently asked questions etc visit our web site http www jyhorib...

Page 15: ...d The resulting radiation is filtered by an emission spectrometer that feeds the signal to a photomultiplier detector e By stepping either or both spectrometers through a wavelength region and record...

Page 16: ...which provides efficient throughput with a choice of standard right angle emission collection or optional front face emission collection The sample compartment module comes equipped with a silicon pho...

Page 17: ...rolog 3 systems include a single or double grating monochromator in the excitation and emission paths in an L configuration Standard systems available Model Source Excitation Spectrometer Sample Compa...

Page 18: ...Single grating Excitation Spectrometer T box Sample Compartment Module Single grating Emission Spectrometer The standard model FL3 11 comes equipped with 450 W light source single grating excitation s...

Page 19: ...grating Emission Spectrometer Like the Model FL3 11 the Fluorolog 3 Model FL3 12 has a single grating excitation spectrometer but the optimum performance of the Model FL3 12 is as a result of a doubl...

Page 20: ...rce Double grating Excitation Spectrometer Single grating Emission Spectrometer T box Sample Compartment Module Features of the Model FL3 21 are 450 W light source double grating excitation spectromet...

Page 21: ...ble grating Excitation Spectrometer T box Sample Compartment Module Double grating Emission Spectrometer Incorporating double grating spectrometers in both the excitation and emission positions places...

Page 22: ...sted below For additional information or for a list of the most recently developed products contact a Spex Fluorescence Sales Representative Sources Pulsed lamp HgXe Your laser Detectors Cooled PMT IR...

Page 23: ...at the emission port of the system Using single grating double grating or the TRIAX 320M imaging spectrometer you can create a system for almost any application Details on using a TRIAX are given in C...

Page 24: ...ion Spectrometer Emission Spectrometer Dectector Dectector Fluorolog 3 model FL3 122 The T configuration allows the Xe source and the signal detector to be positioned at right angles to the sample for...

Page 25: ...citation Spectrometer T box Sample Compartment Module Double grating Emission Spectrometer 270M Imaging Spectrometer Emission Custom optical layout By switching the TRIAX 320M and double grating spect...

Page 26: ...itional information or for a list of the newest options contact a Spex Fluorescence Product Specialist Sources Pulsed lamp HgXe Your laser Detectors Cooled PMT IR CCD Refer to Chapter 6 Components and...

Page 27: ...e system 1 Start the lamp The lamp must be turned on prior to the Fluorolog 3 accessories and peripheral equipment a On the back of the lamp housing turn on the switch marked power b Just above the po...

Page 28: ...rument Control Center The Layout Selection window appears c In the Layout Selection window select the layout to use The Fluorolog 3 spends 1 min initializing then the Instrument Control Center appears...

Page 29: ...ion of the each drive and assigns a wavelength value to this position from a calibration file While the system usually maintains calibration by this method it is wise to check the calibration prior to...

Page 30: ...put and should be the first check performed 1 Secure the lid of the sample chamber in place 2 On the Run Experiment toolbar select the Experiment button This opens the Emission Acquisition dialog box...

Page 31: ...p scan a In the Excitation Acquisition dialog box Name the experimental parameters lamp exp Start the scan at 220 nm Step the wavelength by 0 5 nm Set the emission to 650 nm Perform 1 scan Use a filen...

Page 32: ...on entrance and exit slits to 0 5 mm Set the emission entrance and exit slits to 0 mm closed Click OK to close the window If the slits units are not in millimeters mm then open Visual Instrument Setup...

Page 33: ...ems On a double monochromator system the 467 nm line is no longer the most intense peak within the spectrum Take care to isolate the 467 nm calibration line from other peaks Xenon lamp scan for a doub...

Page 34: ...of this spectrum should be noted for reference although it is not used for instrument specifications Is the peak at 467 0 5 nm Instrument is within specification Go to emission cali bration check Ins...

Page 35: ...at which this peak should be The software will save this change in position to the calibration file 1 Note the wavelength where the 467 nm peak was observed 2 Open the Real Time Display 3 Set the exci...

Page 36: ...tion monochromator The Grating Turret dialog box appears 7 Click on the Calibrate button This opens the Enter Correct Position dialog box 8 Enter the actual xenon lamp peak 467 nm 9 Click OK The excit...

Page 37: ...11 12 Confirm that the excitation monochromator is calibrated by running another lamp scan in Run Experiment This time the peak should occur at 467 0 5 nm Artisan Technology Group Quality Instrumenta...

Page 38: ...water sample should be research quality triple distilled or de ionized water HPLC grade 18 M spec or equivalent water is suggested for the Raman scan Impure samples of water will cause elevated backgr...

Page 39: ...m Set the excitation to 350 nm Perform 1 scan Use a filename water spc End the scan at 450 nm Use an Integration Time of 0 5 s Describe the file as a water Raman emission spectrum c Click on the Signa...

Page 40: ...Click OK to close the box If your slits units are not in bandpass units nm then open Visual Instrument Setup select Options then Units and set the slits units to nm If you have a Front Face Right Ang...

Page 41: ...ion band which is about 10 times the intensity of the Raman band occurs at 350 nm the excitation wavelength The scan begins at 365 in order to avoid detecting the Rayleigh band 4 Find the peak of the...

Page 42: ...correct position for this peak The software will save this change in position to the calibration file 1 Note where the 397 nm peak was observed 2 Open the Real Time Display 3 Reset the emission monoc...

Page 43: ...ono chromator The Grating Turret dialog box appears 7 Click on the Calibrate button This opens the Enter Correct Position dialog box 8 Enter 397 nm the actual peak position using 350 nm excitation 9 C...

Page 44: ...Instrument Setup 12 Confirm that the emission monochromator is calibrated by running another water Raman scan in Run Experiment This time the peak should occur at 397 0 5 nm Artisan Technology Group...

Page 45: ...the Fluorolog 3 Jobin Yvon Inc recommends that the number of hours of xenon lamp use be recorded in a log see sample sheet in Chapter 9 Xenon Lamp Information Record of Use Form Additionally you may...

Page 46: ...glycogen 9005 79 2 Light scattering standard 0 LDS 750 or Styryl 7 114720 33 1 Single exponential life time standard 680 700 0 248 in MeOH exc 568 nm LUDOX or colloidal silica 7631 86 9 Light scatter...

Page 47: ...Spectra can be enhanced by optimization of data acquisition This chapter lists some meth ods of optimizing sample preparation spectrofluorometer setup and data correction to get higher quality data Cu...

Page 48: ...he holder directly Minerals crystals vitamins paint chips and similar samples usually are ground into a homogeneous powder The powder is packed into the depression of the Solid Sample Holder For very...

Page 49: ...right angle detection The optional front face accessory for cuvettes is required for solutions For solid samples use the Model 1933 Solid Sample Holder Determining optimal wavelengths The optimum exc...

Page 50: ...tem components are on 2 Place the sample in the sample compartment 3 Enter the Real Time Display 4 Turn on the high voltage to the signal detector 5 Set the excitation and emission slits to a bandpass...

Page 51: ...eriment button This opens the Emission Acquisition dialog box 8 Click on Exp Type This opens the Select Experiment Type dialog box 9 Choose Emission Acquisition Click OK to close the Select Experiment...

Page 52: ...its to open the Slits dialog box If unsure try 300 nm where many organic samples absorb light Add 15 nm to the exci tation wavelength Try 550 nm Try 2 nm for a rapid scan Try 0 1 second for a rapid sc...

Page 53: ...n scan 11 Enter the data file name and experiment file name and any comments desired to distinguish this experiment from others 12 Click Run to execute the experiment 13 With the preliminary emission...

Page 54: ...itation wavelength for the sample The procedure is very similar to that outlined above 1 On the Run Experiment toolbar choose the Experiment button As before this opens the Emission Acquisition dialog...

Page 55: ...lays intensity versus wavelength and shows the maximum excitation wavelength Optimized excitation and emission spectra of a 1 10 8 M anthracene solution are shown below Because the acquisition modes w...

Page 56: ...erimental parameters are constant In other cases the system can measure the G factor automatically before an ex perimental run Constant Wavelength Analysis Use Constant Wavelength Analysis to determin...

Page 57: ...experimental use of polarizers use Run Experiment Be sure that a layout in cluding polarizers is loaded When selecting the type of experiment the Polarization checkbox must be active A typical Emissio...

Page 58: ...ed information on the G factor see the Polarizers Operation Manual Faster scans with polarizers can be taken with Polar Scan Here the G factor is entered in the Quick Polarization window Artisan Techn...

Page 59: ...e especially for weakly fluorescing samples with low quantum yields The signal to noise ratio can be improved by Using the appropriate integration time Scanning a region several times and averaging th...

Page 60: ...f 1 second S N t 1 2 1 2 1 1 For an integration time of 2 seconds S N t 1 2 1 2 2 1414 or approximately 42 Because S N determines the noise level in a spectrum use of the ap propriate integration time...

Page 61: ...atio improves by n1 2 where n is the number of scans To scan a sample multiple times in an experiment dialog box specify the number of scans in the Number of Scans field in Run Experiment Refer to the...

Page 62: ...mount of fluorescence recorded by the signal detector Bandpass can be calculated using the following formula bandpass nm slit width mm dispersion nm mm The dispersion of the Fluorolog 3 spectrofluorom...

Page 63: ...hing data An additional option Zap for removing outliers also appears under the Arithmetic menu The GRAMS 32 User s Guide covers this option thoroughly In general start with a 9 or 11 point smooth for...

Page 64: ...nd emission correction factors to eliminate response characteristics These files1 xcorrect and mcorrect are included with the software copy them to your hard disk Items that may be convoluted into a s...

Page 65: ...ing acquisition Data can be acquired either as raw data or as corrected data A spectrum composed of raw data exhibits the effects of system parameters while a corrected spectrum displays only the prop...

Page 66: ...is active in the Run Experiment window 2 Select Arithmetic from the toolbar 3 Select Functions This opens the Math Functions dialog box 4 Next to Function select Multiply 5 Next to Operand choose Term...

Page 67: ...box The name of the file appears in the Term File area 9 Click Apply The trace that appears on the screen is a result of the mathematical operation giving a cor rected spectrum Artisan Technology Gro...

Page 68: ...s significantly indicating that the lamp should be replaced Replacing the lamp within the specified time may prevent system failure Jobin Yvon Inc advises to keep a laboratory notebook near the Fluoro...

Page 69: ...housing and when handling the lamp The lamp power supply should not be connected to an AC power line while handling lamp leads Lethal high voltages may exist The lamp will remain extremely hot for ap...

Page 70: ...the 10 Phillips head screws on top of the lamp housing 2 Lift off the cover This exposes the lamp assembly Top view of the lamp housing with cover removed Lamp Mirror Lower bracket Upper bracket Artis...

Page 71: ...Remove the 7 64 cap screw from the upper bracket 3 Remove the upper bracket 4 Remove the lamp 5 Place the protective cover around the xenon lamp 6 Lock the cover into place Dispose of the spent xenon...

Page 72: ...ay attention to the polarity of the xenon lamp Diagram of lamp emphasizing polarity 3 Seat the xenon lamp in the bottom bracket Make sure that the lamp is inserted all the way into the aluminum bracke...

Page 73: ...wer lamp lead and lower power cable to the lower bracket using a 5 32 cap screw 8 Tighten these two 5 32 cap screws on the upper and lower brackets 9 Pull the excess power cables away from the lamp as...

Page 74: ...After installation the lamp should burn in for 24 hours After the burn in the lamp s position may be adjusted to optimize the signal intensity 11 Plug the power cord from the lamp power supply into an...

Page 75: ...uvette in the sample compartment 2 Take a water Raman scan with the following parameters Excitation wavelength 350 nm Scan range 365 nm to 450 nm Increment 0 5 nm Integration time 1 0 s 1 25 mm single...

Page 76: ...and set to 950 V 6 Open the programmable excitation shutter 7 View the incoming data in acquisition mode S signal detector Watch the signal intensity as you adjust the vertical position of the lamp T...

Page 77: ...with the Allen wrench 8 Insert a 5 64 Allen wrench inside the middle port on the top of the lamp housing The Allen wrench is ready to adjust the vertical position of the lamp 9 View the trace and make...

Page 78: ...d the date that the new lamp was installed as well as its maximum intensity 13 Save the protective cover of the new bulb for when the bulb must be replaced in the future Artisan Technology Group Quali...

Page 79: ...equently if needed 1 Remove 10 Phillips head screws on the lamp housing s cover 2 Lift off the cover of the lamp housing 3 Remove the 2 screws that hold the baffle in place inside the housing 4 Carefu...

Page 80: ...p screws were removed replace them 8 Replace the baffle and re install the 2 screws holding it in place 9 Re install the cover and secure it with the 10 Phillips head screws Artisan Technology Group Q...

Page 81: ...ltiplier is replaced with a different detector the emission correction factors must be updated see Chapter 11 Producing Correction Factors To install a new R928P photomultiplier 1 Be sure that the ins...

Page 82: ...aintenance 5 15 Reference signal detector The reference signal detector is a state of the art silicon diode that requires no routine main tenance Artisan Technology Group Quality Instrumentation Guara...

Page 83: ...open position Replacement Gratings require no routine maintenance To use a grating with different specifications than those installed in the system replace the existing ones Jobin Yvon Inc recommends...

Page 84: ...all instrument power 3 Remove the Phillips head screws holding the lid on the monochromator 4 Lift the lid slowly until you feel resistance This is the maximum length of the cables attached to the lid...

Page 85: ...e the grating from the stand a Loosen the thumbwheel screw securing the grating to the grating stand Back view of grating assembly b Pull the grating away from the stand Artisan Technology Group Quali...

Page 86: ...grating stand 2 Tighten the thumbscrew on the back of the grating stand 3 Lift the lid of the spectrometer off of the lid mount 4 Remove the lid mount 5 Place the lid on the spectrometer 6 Secure the...

Page 87: ...ealignment Only the Model 1692M Selection Mirror an option can be adjusted easily to optimize the fluorescence sig nal from the sample This mirror should be adjusted periodically if low signal is caus...

Page 88: ...in the temperature bath or the water can be come hard Automated 4 position turret If a circulating bath is used to regulate the turret s temperature periodically replace the flu ids in the bath Artis...

Page 89: ...he following list represents all the accessories and components including those men tioned above in alphabetical order available for the Fluorolog 3 spectrofluorometers A brief description of each is...

Page 90: ...2D Fiber Optic Mount F 3000 6 14 Filter Cut On 1 2 1938 6 15 Filter Cut On 2 2 1939 6 15 Front Face Viewing Option FL 1001 6 16 Gratings various 6 17 Holder Filter FL 1010 6 18 Holder Four Position Va...

Page 91: ...ly Operation of the Model 1940 Accessory requires two steps a Installation b Optimization of the emission and reference signals Installation 1 Remove the sample holder currently in place 2 Position th...

Page 92: ...p and excitation spectrome ter Note The linear range of the R928P detector operated in the photon counting mode is 2 million cps 2 Place the cuvette containing the blank in the sample holder 3 Set the...

Page 93: ...blank c Acquire and excitation scan of the sample Or When the spectrometers are synchronously scanned using an offset of 0 nm the emission slits should be opened five times as wide as the slits of the...

Page 94: ...e Dewar flask The Teflon cover on the top of the Dewar flask holds any excess liquid nitrogen that bubbled out of the assembly A pedestal holds the Dewar flask in the sampling module A special stove p...

Page 95: ...bly includes an NIST traceable calibrated 200 W quartz tungsten halogen lamp with irradiance values Regulated 6 5 A power supply with lamp holder 1908MOD Scatter Block Assembly see above Emission corr...

Page 96: ...24A Adapter This non fluorescing fused silica cylindrical cell holds 250 L A magnetic stirrer can not be used with this cell The 1924A Adapter is required to enable mounting in the standard 10 mm 10 m...

Page 97: ...Assembly monitors the xenon lamp up to 1 m This acces sory is standard in the Fluorolog 3 package but a photomultiplier can be substituted instead necessary for phosphorescence measurements Model 1967...

Page 98: ...eds Both air cooled and liquid nitrogen cooled CCDs can be in serted into the Fluorolog 3 Available options include extended UV and near IR de tection various pixel sizes and arrays and maximum covera...

Page 99: ...mission signal detector This detector is mounted to the emission spectrometer and operated in the photon counting mode Model 1911F Room Temperature Signal Detector The dark count is specified at 1000...

Page 100: ...o 20 cps the useful wavelength band 190 860 nm remains the same The Thermoelectrically Cooled Signal Detector consists of the housing power supply Model 1630 Field Lens Adapter a black flange and a si...

Page 101: ...he near infrared the FL 1030 Thermoelec trically Cooled PMT is perfect Included in the FL 1030 is the thermoelectrically cooled housing The InGaAs detector has a spectral range from 250 nm all the way...

Page 102: ...he F 3000 couples to the T box light is focused from the ex citation spectrometer onto the fiber optic bundle and then directed to the sample Fluo rescence emission from the sample is directed back th...

Page 103: ...and the appropriate cut on filter in the excitation light path Model 1939 Cut On Filter The Model 1939 Cut On Filter set consists of five 2 2 filters with cut on wave lengths of 350 nm 399 nm 450 nm...

Page 104: ...es a swing away mirror This allows the researcher to change from front face and right angle data collection instantly In the front face col lection mode the viewing angle is 22 5 The FL 1001 is ideal...

Page 105: ...re available 300 grooves mm 600 grooves mm 1200 grooves mm In addition a number of different blazes can be purchased 250 nm 330 nm 500 nm 750 nm 1000 nm All gratings are classically ruled and measure...

Page 106: ...m and T box sampling compartments have three slots that can hold the FL 1010 Fil ter Holder Refer to either Model 1939 Cut On Filter or Model 1938 Cut On Filter for a detailed description of the place...

Page 107: ...ned by an ethylene glycol water mixture pumped through from an external circulating temperature bath not included The holder also includes a magnetic stirrer for mixing turbid or viscous samples Also...

Page 108: ...Art Products Pequannock NJ 2 Place a cuvette in each holder 3 Allow the samples to reach the desired temperature 4 Turn on the magnetic stirrer 5 Select the appropriate mixing speed The speed at whic...

Page 109: ...y an ethylene glycol water mixture pumped through from an external circulating temperature bath not included The holder also includes a magnetic stirrer enabling mixing of turbid or viscous samples Al...

Page 110: ...m cuvette and insert a magnetic stirring bar The stirring bar is available from Bel Art Products Pequannock NJ 2 Place a cuvette in each holder 3 Allow the sample to reach the desired temperature 4 Tu...

Page 111: ...ich a bracket a spring clip and a sample block rest Model 1933 Solid Sample Holder with sample block removed Installation 1 Remove the present holder 2 Position the base on the posts 3 Tighten the two...

Page 112: ...ensures accurate alignment of the Model 1692M Selection Mirror For samples such as thin films microscope slides fibers or other materials 1 Place the material on the block on the side opposite that of...

Page 113: ...Dual Lamp Housing contains both the standard continuous 450 W xenon lamp and a UV xenon flash tube A swing mirror selects which of the two sources ex cites the sample This housing with pulsed lamp is...

Page 114: ...models The F 3005 6 comes with dual syringes for complete control over dispensing and aspirating volumes of liquids into and out of the sample cell A mix function is included With the injector come 18...

Page 115: ...he Microscope Fiber Optic Interface eases the use of the Fluorolog 3 systems for fluorescence microscopy measurements Interfaces are available for Nikon Olympus and Zeiss microscopes Models F 3001 F 3...

Page 116: ...nm to 850 nm for sample excitation The lamp has an approximate life of 2000 hours and is ozone free The lamp is designed to fit into the FL 1039 Xenon Lamp Housing and the FL 1040 Dual Lamp Housing 4...

Page 117: ...fluids the Peltier device heats and cools the sample thermoelectrically and fast The temperature range is 10 C to 120 C To prevent condensation of moisture on chilled cuvettes an injection port for d...

Page 118: ...d This allows the researcher to follow the decay of samples an order of magnitude faster than can be achieved with conventional systems that depend on mechanical choppers To run phosphorescence experi...

Page 119: ...and fluorescence measurements are collected with top reading geometry Thus any titer plates even disposable ones may be used Up to 384 well plates may be inserted into the MicroMax with a scan speed 1...

Page 120: ...be placed at the entrance and the exit of the T box The polarizers are fully automated and are adjustable to within 1 rotation Insertion and removal from the optical path is controlled by the computer...

Page 121: ...he sample compartment With the injec tor in place a lock tight seal is achieved prevented both light and air from reaching the sample The Injector Port is recommended for use with the TRIG 15 25 Trigg...

Page 122: ...th The controller circulates fluids externally with tubes leading to the sample chamber The temperature range is from 25 C to 150 C Sensor and all ca bles are included with the F 1000 1 The Temperatur...

Page 123: ...tored with the associated data file A TTL trigger output also is provided for activating external devices such as a stopped flow unit The front panel has four sets of banana jack inputs for two indepe...

Page 124: ...h Adjust the slits Shutter s is are not com pletely open Open the shutter s Lamp power supply is set to the wrong current rating Call the Spex Fluorescence Service Depart ment 450 W Xe lamp current 25...

Page 125: ...isa_ini dflt set file When system is restarted the system will recreate this file Sample turret is not operating Not turned on Verify that the rocker switch on the front panel of the Model 1976 Acces...

Page 126: ...e following spectra occur with explanations regarding prob lems leading to their appearance Lamp scan Running a lamp scan verifies system integrity and indicates whether the correct pa rameters for th...

Page 127: ...on in the area near the 467 nm peak This lack of spectral resolution appears because the slit widths are set too wide To re solve this problem narrow the slit widths Intensity counts s 1 0 4473 0 3355...

Page 128: ...as contaminated resulting in a high background If a spectrum similar to this is obtained after running a water Raman scan 1 Rotate the cuvette 90 and rerun the scan Clean water Raman scan 0 1 2 3 4 5...

Page 129: ...blem is low intensity of the water signal when compared with the superim posed typical water Raman scan To resolve this problem 1 Make sure the cuvette is filled to the proper level Light should fall...

Page 130: ...and scratches 2 Clean the cuvette or use a new one 3 Verify that the excitation and emission slits are set correctly for a water Raman scan 4 Verify that the excitation spectrometer is at the correct...

Page 131: ...r Raman scan to make sure the system is properly calibrated Print the spectrum for each and note the peak intensities 4 Check this chapter to see if the problem is discussed 5 Visit our web site at ww...

Page 132: ...e a note of the software s and instrument s serial numbers and instrument configuration including all accessories If the problem persists or is unlisted call the Spex Fluorescence Service Department a...

Page 133: ...nd resolving power of the Fluorolog 3 and the high quality Jobin Yvon Inc optics provide the precise collimation to take maximum advantage of the system s CW source thereby ensuring accurate results I...

Page 134: ...domain measurements The figure shown above illustrates a sinusoidal excitation with a circular frequency an amplitude ACx and offset by a DC voltage DCx The sample responds with an identical circular...

Page 135: ...Sep 2002 Introduction to Lifetime Measurements 8 3 tan tan F HG I KJ p p m m AC DC AC DC 1 2 2 1 2 1 2 1 2 1 1 1 m m f f x x c h Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOUR...

Page 136: ...om a protein containing both residues To improve resolution simply measure the lifetime and obtain the spectral characteristics of the in dividual components prior to conducting a lifetime resolved ac...

Page 137: ...n be used to indicate whether the excitation spectrometer is properly calibrated As the xenon lamp ages water Raman spectra have a progressively lower peak intensity During its lifetime the lamp stabi...

Page 138: ...nformation 9 3 Xenon Lamp Record of Use Page _____ of _______ In Service Date Operator Current Date Time On Time Off Total Time Hours Min Total Hours Artisan Technology Group Quality Instrumentation G...

Page 139: ...scence spectra For example fluorescence emission spectra are affected by the response characteristics of the spectrometer and optical components such as gratings and detectors To compen sate for these...

Page 140: ...er plot shows the absorption spec trum of azulene acquired on the same spectrofluorometer system The corrected excita tion spectrum shows strong similarity and fine structure compared with the corresp...

Page 141: ...using an integration of only 1 second a clear demonstration of superior per formance Reduced volume samples Because the samples required to produce spectral data may be expensive or obtained only in l...

Page 142: ...ue to Fluorolog 3 systems is the capacity to measure fluorescence related parameters required to calculate the quantum yield for samples in solution This includes recording excitation source intensity...

Page 143: ...detector is insensitive above 860 nm an IR spectrofluoro meter must be equipped with a red sensitive photomultiplier or a solid state detector whose response is effective far into the IR region With...

Page 144: ...olution of a Fluorolog 3 double grating spectrometer system optimizes measurements under these conditions Monitoring kinetic reactions using time based fluorescence By setting the wavelengths at the e...

Page 145: ...nce polarization the excitation beam is passed through a polarizing prism and the emitted luminescence is analyzed with another polarizer alternately oriented parallel and perpendicular to the excitat...

Page 146: ...gs detectors and other spectrometer components have response characteristics that are functions of wavelength These characteristics are superimposed on spectra and may yield a potentially misleading t...

Page 147: ...can then be applied to the raw fluo rescence data The emission correction factor file mcorrect was acquired in this manner To generate emission correction factors several items are needed a standard...

Page 148: ...08MOD Scatter Block Assembly in the sample chamber so that light is directed toward the right angle Looking down the scatter plate should be toward the left 5 Place the mask over the sample compartmen...

Page 149: ...to the negative black side of the holder c The nipple of the lamp should point upward 7 Fix the lamp holder to the top of the sample compartment with double sided tape Make sure the the filament is ve...

Page 150: ...rn on the constant current power supply Wait until the current ramp function is 6 5 A This may take up to two minutes For valid irradiance values the lamp current must be maintained at 6 5 A 10 Turn o...

Page 151: ...ectrum 5 Name this file stlamp 6 Place the lid over the mask to block the light between the standard lamp and the scatter fixture 7 Run another scan using the same parameters and name this file blank...

Page 152: ...uld appear similar to the one pictured above Its actual appearance however depends on the configuration of your Fluorolog 3 system The lamp scan was acquired with gratings in the emission spectrometer...

Page 153: ...he data will still be off by a factor of c but normalizing the correction factors compensates for this For more information about the theory and application of radiometric correction con sult Accuracy...

Page 154: ...the Select Experiment Type dialog box 4 Enter the following parameters Start the scan at 300 nm End the scan at 850 nm Increment the wavelength by 50 nm each step End the scan at 850 nm Use an integr...

Page 155: ...values will be entered for each 0 point 5 Run the scan with these parameters 6 Name the file CRE After performing the irradiance value multiplication and displaying the file CRE on the screen enter ea...

Page 156: ...radiance values incremented every 50 nm To calculate the actual correction factors the irradiance file needs to have a 5 nm increment 11 Using the instructions presented above create another file Keep...

Page 157: ...s IRR and stdlamp2 These files are required to calculate the emission correction factors for the Fluorolog 3 system IRR file 0 1000 2000 3000 4000 250 350 450 550 650 750 850 Wavelength nm Intensity A...

Page 158: ...intensity 2 Using the Arithmetic menu divide the mcorrect file by this minimum signal intensity 3 Save this new file as mcorrect That is overwrite the existing mcorrect file This normalizes the correc...

Page 159: ...8P photomultiplier detector Once the emission correction factors have been found determination of the excitation correction factors may be necessary The following procedures describe how to obtain exc...

Page 160: ...ill a cuvette with a solution of rhodamine B Use 8 g L 1 of laser grade rhodamine B in 1 2 propanediol 2 Place the cuvette in the sample compartment For single beam sampling modules place the cuvette...

Page 161: ...eriment 10 In Run Experiment choose Collect 11 Under Collect choose Experiment This opens the Emission Acquisition dialog box 12 Click Exp Type This opens the Select Experiment Type dialog box 13 Choo...

Page 162: ...Use the XFER button in the Real Time Display to move the slit widths to Slits Click OK to close the Slits window 18 Click HV This opens the High Voltage dialog box 19 Use the XFER button to move the...

Page 163: ...the scan 22 Save the file as xcorrect 23 If no zeroes are shown invert the data In Run Experiment use the Arithmetic menu to divide the spectrum into 1 24 Normalize the data Start the scan at 240 nm...

Page 164: ...xisting excitation correction factor file To acquire corrected data for an experiment enter the name of this file in the COR RECTION factor file field in the Data Acquisition Parameters dialog box The...

Page 165: ...voltage input to operate The operating voltage determines the sensitivity of the detector To achieve the highest sensitivity possible the plateau voltage determination should be performed when ever a...

Page 166: ...Determining the Plateau Voltage 12 2 High Voltage Volts Signal cps Dark Counts cps 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 Artisan Technology Group Quality Instrumentation Guaranteed 888...

Page 167: ...emission spectrometers to a bandpass of 5 nm 6 Insert a cuvette filled with distilled water in the sample holder 7 Select right angle detection Turn the selection mirror knob located on top of the sam...

Page 168: ...fic high voltage 5 Close the shutter by sliding it to the right This closes the manual shutter on the variable exit slit of the emission spectrometer 6 Record the dark counts at 650 V Write this value...

Page 169: ...0 700 1 28 105 260 750 1 52 105 350 800 1 68 105 480 850 1 74 105 420 900 1 80 105 450 950 1 81 105 490 1000 1 96 105 530 1050 1 96 105 640 1100 2 05 105 560 1150 2 12 105 640 1200 2 25 105 710 Determ...

Page 170: ...at 1000 V there is a plateau both in the signal and dark counts Therefore the point at which there is maximum voltage with minimum dark counts is 1000 V the plateau voltage for this detector Artisan...

Page 171: ...og 3 spectrofluorometer An optional printer may also be included The Fluorolog 3 spectrofluorometer is a combination of discrete modules and components These components include excitation and emission...

Page 172: ...s configuration Each module and the lamp housing has alignment studs alignment receptacles and D connectors on the outside of the housing 1 Assemble the spectrofluorometer system s modules into the pr...

Page 173: ...g on the system s configuration Cable connections These connectors connect the spectrofluorometer components with the System Controller SAC and the computer system The jacks on the rear of the SAC are...

Page 174: ...of each cable During installation refer to the schematic to ensure proper system interconnections Lamp housing Excitation spectrometer Sample compartment module Emission spectrometer Signal detector...

Page 175: ...hell jack on the front of the sample compartment module Plug the BNC plug into the BNC jack next to the D shell jack 3 Plug the remaining D shell end of the cable into the INPUT R connector on the bac...

Page 176: ...77 cable into the INPUT S connector on the back panel of the SAC Sample compartment module 1 Insert the 25 pin 9 pin cable 400108 between the sample compartment and the jack labeled mono drive on the...

Page 177: ...to a properly rated receptacle or power strip Calibrate the system before conducting an experiment Refer to the appropriate chapter for calibration instructions To properly operate the equipment have...

Page 178: ...andard monochromators Four ports generally are available rather than two on a standard monochromator Most TRIAX spectrometers used with the Fluorolog 3 have two entrance ports lateral i e on the side...

Page 179: ...the back of the TRIAX The power supply contains a voltage converter that automatically adjusts to the input AC voltage Internal workings Multiple gratings Generally gratings with different groove den...

Page 180: ...elow is a sketch of the special cable connections required between a TRIAX spec trometer the CCD detector and the system Details of these connections are given on the following pages Other Fluorolog 3...

Page 181: ...e runs from the terminal on the back of the TRIAX to the CCD This controls the CCD shutter The large 37 pin cable bound together with the coaxial cable carries data between the CCD controller and the...

Page 182: ...with dual entrance and exit ports the flip mirrors to choose which entrance and exit ports are controlled in DataMax 1 Click on the mirror icon The Entrance or Exit Mirror dialog box appears Lateral s...

Page 183: ...adio button Allow several seconds for the turret to rotate to the new position 3 Click Close Installing a new grating on the turret 1 Follow instructions in the TRIAX Series User Manual on replacing t...

Page 184: ...the found peak As an example we assume a lamp scan was performed and the 467 nm peak was observed at 480 nm 7 Open the Real Time Display 8 Enter the observed peak position Don t forget to hit the Ente...

Page 185: ...lateral exit while a PMT is mounted on the front exit Adjusting slit widths 1 Click on an icon of a slit The Slits window opens All slits in the TRIAX are adjustable no matter which slit you click 2 E...

Page 186: ...he Acquisition Channel window opens 2 Choose Data Units 3 Choose Gain Level 4 Choose analog digital conversion 5 Click Details The Acquisition Controller Details window opens Details of the CCD contro...

Page 187: ...in the DataMax main window CCD experiments using a TRIAX may be per formed To run a CCD acquisition 1 Choose Collect A drop down menu appears 2 Choose Experiment The CCD Acquisition dialog box opens A...

Page 188: ...CD acquisition type are available such as 1 CCD Areas 2 CCD Regions 3 Image and Scan radio buttons 4 Integration Time 5 Accumulations 6 Mirrors 7 Fast Kinetics Mode These are discussed on the followin...

Page 189: ...nitions and Areas Views These index card tabs give details about which pixels are active mathematically Areas Definitions and graphically Areas Views Comments Unlimited amount of information may be en...

Page 190: ...rmat divides the entire CCD chip equally to within 1 pixel into the value shown in the Number of Areas slider If Number of Areas is 1 then the entire CCD is used as one area In Image mode only one are...

Page 191: ...tral regions which can be attached together to create a single complete spectrum CCD Regions controls how to take and present these snapshots 1 Click CCD Regions The Acquisition Setup dialog box opens...

Page 192: ...h in nm for each region The range shown in nm depends on the chip size the TRIAX model and the grating s groove density 5 Enter the desired integration time in s next to the Int Time field for each re...

Page 193: ...gether several snapshots with automatic baseline matching click Auto Adjust The Nb Cover field changes depending on the grating TRIAX and CCD chip 8 Enter a comment Only the first 80 characters are di...

Page 194: ...nsional detector Image or Binning columns of pixels together to create a one dimensional detector Scan for re cording standard spectra For 2 D detection 1 Click Image For 1 D detection 1 Click Scan Wh...

Page 195: ...is is the amount of time that the CCD chip is exposed to the sample s luminescence 1 Enter the Integration Time in s in the data entry field 2 Click on another data entry field to finalize the value A...

Page 196: ...cence for a particular active CCD area Accumulations are added together if more than one accumulation is specified 1 Enter the desired number of accumulations in the data entry field 2 Click on anothe...

Page 197: ...3 v 2 2 9 Aug 2002 TRIAX operation with the Fluorolog 3 14 20 Mirrors At present this button is inactive in DataMax Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artis...

Page 198: ...photomultiplier tube or other single point detector and NOT to a 2 dimensional CCD detector Therefore corrections to raw data cannot be made in CCD mode Correction The Correction button is useful onl...

Page 199: ...e the Burst or Continuous radio buttons Burst means one rapid exposure With Burst the CCD Acquisition window appears as shown above Burst mode exposes the top edge of the CCD to the luminescence while...

Page 200: ...start of the next exposure d Set the Enable Int Shutter Move checkbox If checked the CCD shut ter opens and closes at each accumulation If unchecked the CCD shut ter opens at the start of the experim...

Page 201: ...n Clear out all charges before an accumulation No Flush Charges are not removed before an accumu lation This is faster but background noise may increase between acquisitions For mul tiple scans the fi...

Page 202: ...velengths are correctly as signed near the center of the CCD but are incorrect near the edges 1 Choose Collect A drop down menu appears 2 Choose Linearization The Linearization Parameters window opens...

Page 203: ...a single wavelength from a source such as the 547 074 nm line from a Hg lamp This takes 5 spectra of the line displaced across the chip and requires a motorized spectrometer a Click Define Experiment...

Page 204: ...i Set the Center to 546 074 nm and press Enter j Calculate the increment i To From 5 to give 5 positions using the maximum amount of the chip in linearization k Set Nb Regions to 5 l Set the first re...

Page 205: ...th fixed grating spectrographs and movable grating systems not attached to the sys tem a Click Define Experiment The CCD Acquisition box appears b Click CCD Areas The Area List window opens c Click Re...

Page 206: ...m Click Run If defined correctly for either technique DataMax collects the spectrum The Select Calibration Peak window appears A dialog box appears requesting the calibration wavelengths for Multi Sc...

Page 207: ...Aug 2002 TRIAX operation with the Fluorolog 3 14 30 Autoscaling the display AutoScale is always on and cannot be switched off Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE...

Page 208: ...me Display is used to optimize the CCD detector and TRIAX settings for the best experimental results To do a CCD acquisition in the Real Time Display 1 In Instrument Control Center click the Run Ex pe...

Page 209: ...CCD de tectors there is no slit at the exit of the TRIAX so any dis played settings are superfluous The Toolbar When a single point detector e g PMT InGaAs IR etc is attached to a TRIAX and activated...

Page 210: ...hip 1 Click CCD 2 The Area List window opens Chip Height and Chip Width This shows the size of the active area using a choice of units Areas Defi nitions and Areas Views These in dex card tabs give de...

Page 211: ...ze lets the user choose an area as the normalization standard DataMax performs a Dark Subtract on all data then divides all areas by the normalization standard Reformat Reformat divides the entire CCD...

Page 212: ...unique file name which needs deciphering Following are rules to guide the correct choice of correction factor files 1 The first two characters show the type of correc tion factor file MC emission corr...

Page 213: ...rrection factor file for an R928P PMT with a grating of 600 grooves mm and a blaze at 300 mm MCCD12x500 spc An emission correction factor file for a CCD array with a grating of 1200 grooves mm and a b...

Page 214: ...ar 3 Choose Functions The Math Functions dialog box opens 4 Next to Function select Multiply 5 Next to Operand choose Term File K 6 Click Select This opens the Select New Term File dialog box 7 Choose...

Page 215: ...grooves mm grating Focal plane 30 mm wide 12 mm high Spectral dispersion 2 64 nm mm nominal with 1200 grooves mm grating Spectral resolution 0 06 nm nominal with 1200 grooves mm grating when scanning...

Page 216: ...ropriate Exit DataMax Check that the software matches the hardware especially device configuration Slit and wavelength drives and accessories do not move Bad electrical connection Test turret as follo...

Page 217: ...o the same ground earth and if possible the same power circuits 4 Add redundant grounds earths to various points in the system Improper grounding Consider a star ground earth of wires radiat ing from...

Page 218: ...11 14 17 14 22 14 26 14 27 14 28 14 29 CCD Areas 14 11 14 12 14 26 14 28 CCD chip 14 4 14 12 14 13 14 16 14 17 14 18 14 33 14 34 CCD controller 14 4 14 9 14 40 CCD detector 14 9 14 12 14 14 14 15 14...

Page 219: ...etector 14 32 14 36 L lamp scan 14 7 laser 14 40 Last Read Out Time 14 23 lateral 14 1 14 3 14 32 Lateral 14 5 layout 14 5 14 32 Linearization 14 12 14 25 14 26 14 28 14 34 Linearization Parameters 14...

Page 220: ...ct Calibration Peak 14 29 Select New Term File 14 37 shutter 14 39 Side 14 32 slits 14 3 14 8 14 28 14 29 14 32 14 39 Slits 14 8 14 32 specifications TRIAX 14 38 SpectrAcq 14 4 Spex Fluorescence Servi...

Page 221: ...lude a printer for hard copy documentation Important The Fluorolog 3 spectrofluorometer system is designed to comply with the require ments of the Low Voltage Directive 73 23 EEC and the EMC Directive...

Page 222: ...gratings Resolution 0 2 nm Accuracy 0 5 nm Speed 150 nm s Range 0 1300 nm Gratings Excitation 330 nm blaze 200 700 nm range Emission 500 nm blaze 300 1000 nm range Other gratings available for ranges...

Page 223: ...microseconds Frequency range 0 2 to 310 MHz Sensitivity Double distilled deionized ICP grade water Raman scan 4000 1 S N at 397 nm 5 nm bandpass 1 s integration time background noise first standard d...

Page 224: ...ded Minimum 4 gigabyte hard drive SVGA display card and SVGA monitor to match display card At least one 3 high density floppy drive CD ROM drive 101 key enhanced keyboard Mouse is strongly recommended...

Page 225: ...a chemical reaction in a living organism Blaze wavelength Wavelength at which a grating is optimized for efficiency Gener ally the gratings are efficient to 2 3 before the blaze wavelength to twice th...

Page 226: ...litude of the excitation voltage and DCx is the offset of the emission voltage Emission scan Shows the spectral distribution of light emitted by the sample Dur ing an emission scan the excitation spec...

Page 227: ...en the modulated emission relative to the excitation Phase modulation method A technique for measuring fluorescence lifetimes where a sample is excited with light whose intensity is modulated sinusoid...

Page 228: ...sam ple It is operated in the photon counting mode of detection to pro vide high sensitivity Different detectors cover different wavelength regions Singlet state The spin paired ground or excited stat...

Page 229: ...l suspensions Xenon lamp Lamp that produces a continuum of light from the ultraviolet to the near infrared for sample excitation Xenon lamp scan A profile of the lamp output as a function of wavelengt...

Page 230: ...David M Jameson and Robert D Hall Multifrequency Phase and Modulation Fluorometry Ann Rev Biophys Bioeng 13 105 124 1984 G G Guilbault Ed Fluorescence Theory Instrumentation and Practice Marcel Dekke...

Page 231: ...luorescence Spectrometry Techniques in Visible and Ultraviolet Spectrometry Vol 2 Chapman and Hall 1981 W G Richards and P R Scott Structure and Spectra of Molecules John Wiley Sons 1985 A Schillen et...

Page 232: ...nce Analysis A Practical Approach Marcel Dekker 1970 J D Winefordner et al Luminescence Spectrometry in Analytical Chemistry Wiley Interscience New York 1972 In addition the following journals may pro...

Page 233: ...liquid nitrogen dewar assembly 6 6 micro cell with Model 1923A adapter 6 8 micro cell with Model 1924A adapter 6 8 microscope interface 6 27 microwell plate reader 6 31 Peltier thermocouple drive 6 29...

Page 234: ...or 14 9 14 12 14 14 15 14 31 CCD icon 14 9 CCD Regions 14 11 14 14 14 27 28 CCD shutter 14 4 14 23 CE marking 15 1 Center 14 15 14 27 center wavelength CCD 14 36 Change 14 5 Change 14 6 Chip Height 14...

Page 235: ...g lamp the 5 3 F F 1000 1 Temperature Bath 6 34 F 3000 Fiber Optic Mount 6 14 F 3004 Sample Heater Cooler Peltier Thermocouple Drive 6 29 F 3005 6 Autotitration Injector 6 26 factors correction 4 18 1...

Page 236: ...IRR 11 11 13 irradiance 11 2 11 5 11 8 11 10 11 isamain ini 7 2 isascan set 7 2 isascan vw 7 2 L lamp adjustment 5 8 exposing 5 3 inserting 5 5 removing 5 4 lamp scan3 5 3 7 3 11 12 5 1 7 3 4 7 8 11 7...

Page 237: ...ulti Scans One Peak 14 26 Multiply 4 20 11 11 14 37 N NADH 3 20 Nb Cover 14 16 Nb region 14 14 Nb Region 14 27 14 29 Nb Regions 14 27 near IR detector 14 35 neutral density filter 11 2 New Polarizatio...

Page 238: ...2 resorufin 10 3 rhodamine B 11 15 ribbon cables 14 39 right angle data collection 4 3 rose Bengal 3 20 RTD CCD 14 31 Run 4 7 4 9 11 18 14 11 14 23 14 28 29 Run Experiment3 2 3 4 3 11 12 3 18 4 5 4 8...

Page 239: ...6 34 Term File 14 37 Term File K 4 20 14 37 thin film 4 2 6 23 24 time based fluorescence 10 6 Time Increment 14 23 time resolved 8 4 time resolved data 10 5 To 14 16 Toolbar 14 32 TRIAX2 9 2 11 14 1...

Page 240: ...service in house repair center WE BUY USED EQUIPMENT Sell your excess underutilized and idle used equipment We also offer credit for buy backs and trade ins www artisantg com WeBuyEquipment REMOTE IN...

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