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--- fluorimeter_startup_procedure [2014/06/23 16:20] – stephsam
+++ fluorimeter_startup_procedure [2022/08/26 12:05] (current) – external edit 127.0.0.1
@@ Line 1: / Line 1: @@
-===== Fluorescence in UV/Vis =====
-==== Standard Emission and Excitation Spectra ====
-Instructions that must be followed to maintain the integrity of the fluorimeter components are indicated in red.
-=== INITIATE INSTRUMENT ===
-**1)** Acquaint yourself with the instrument layout and the light path to the detectors using the labeled pictures.
-**2)** Turn on components in following order.
-  * a. Main fluorimeter.  Large black I/O switch on side of box facing the wall that contains the Xenon lamp. WARNING: TURN MAIN SWITCH ON BEFORE TURNING ON LAMP
-  * b. Lamp.  Located directly above Main switch.
-  * c. Dell computer.
-  * d. iHR 320 monochromator.  Black I/O switch on side facing keyboard/monitor.
-  * e. SpectraAco computer.  Switch on the power supply on the top of the back.
-  * f. Symphony power supply. Press button on lower right hand corner of its front face.
-**3)** Allow at least 20 minutes for the lamp to warm up. (For best results; measurements can be taken prior but the lamp spectrum may fluctuate)
-**4)** The Operation Manual and the Test Spectra, which will be needed for calibration, are located in the top left drawer beneath the printer.
-=== CHECK CALIBRATION OF MONOCHROMATORS ===
-**5)** At computer, open the FluorEssenceV3 program on the desktop.
-**6)** Click on M icon (Experiment Menu) (Figure 1).  The various devices will be checked for their connection to the computer.   This takes a few minutes.  If something doesn’t connect, it has likely not been turned on.
-**Figure 1**
-{{:wiki:pl1.png?direct&600|}}
-=== EXCITATION ===
-(See pages 3-4 to 3-9 of Operation Manual.)
-**7)** From the Main Experiment Menu, choose Spectra (Figure 2).
-**Figure 2**
-{{:wiki:pl2.png?direct&400|}}
-**8)** Select Experiment Type: Excitation.
-**9)** The Experiment Setup window opens with the “Monos” menu (see left) open (Figure 3).  The IHR320 is not needed for these measurements, as it is only needed for CCD camera and IR measurements, so uncheck the “Activate” box.
-**Figure 3**
-{{:wiki:pl3.png?direct&600|}}
-**10)** Click on the “Detectors” menu on left.  The Experimental Setup window should look like Figure 4.  The R1 detector (which is a reference detector that does not have any monochromators in front of it) and the S1 detector (which is the PMT on the far side of the table and is for these basic measurements) should be Enabled. Verify the shutter on the PMT detector is open (fully extended) before running scan.
-**Figure 4**
-{{:wiki:pl4.png?direct&600|}}
-**11)** Click Run.   The data is collected and should look like that in Figure 5.  The data from the R1 detector should look like the lamp spectrum in the “Test Spectra” booklet.
-**12)** Click on the Data Reader icon and click on the plot from the R1 detector. Move the crosshairs to the maximum.  It should occur (see numbers in yellow with black background to the bottom left) at 467 nm.   If it does not, follow the directions in the manual from section e on page 3-7 to section m on page 3-9 to calibrate the emission monochromator.
-**Figure 5**
-{{:wiki:pl5.png?direct&600|}}
-=== EMISSION ===
-(See pages 3-10 to 3-12 of Operation Manual.)
-**13)** You use the water Raman spectrum to calibrate the emission monochromator.  Follow the directions in the manual, keeping in mind that you don’t need the iHR monochromator and that you need to activate the R1 and S1 detectors.  Compare the spectrum from the S1 detector to that in the “Test Spectra” booklet.
-**14)** The calibration occurs in the Emission 2 tab of the Monos section of the Real Time Control panel.  The Real Time Control window should look like Figure 6.
-**Figure 6**
-{{:wiki:pl6.png?direct&600|}}
-=== MAKE MEASUREMENTS ===
-**15)** With the excitation and emission monochromators calibrated, you can now take measurements.  If you have a solid sample, you may find front face detection more amenable for measurement.  To do this, make sure the knob next to the sample holder is pointed to “FF” as opposed to “RA”, for right angle detection.
-**16)** There are other adjustments that can be made to optimize data collection (see Chapter 5: Optimizing Data of Operation Manual).  These include
-  * a. Using a grating with a blaze wavelength (the second number in the choices in the grating menu) suitable for your sample
-  * b. Integration time (shorter for quick measurements to see what’s going on/bright samples and longer (0.5 s and longer) for publication quality measurements/dim samples)
-  * c. Saving the Experiment file to make future measurements with same settings easier
-  * d. Measurements can be performed in sequence using the Batch Experiment Button (three buttons to the right of the Experiment Menu button).
-=== DATA ANALYSIS AND SHUTTING DOWN ===
-**17)** Once you’re done, you can save the day’s measurements as a project to open up again in Origin.  Or you can export the data as ascii files to use a program (i.e. Matlab) of your choice.
-  * **!!Shut down the FluorEssenceV3 Software before shutting off any hardware!! Turn off all machines in the reverse order of startup EXCEPT FOR THE MAIN FLUORIMETER.  Wait 30 min for the lamp to cool before switching this off.**
-**18)** Clean off the top of the desk.  Put these instructions and the Operation Manual and Test Spectra in the top left drawer beneath the printer.  Remove your sample and any debris you may have generated from the experiment!  No one wants to clean up after you!