Q: Is the NanoDrop 3300 an upgrade to the NanoDrop 1000?
A: No, the NanoDrop 3300 is used to measure fluorescence whereas the NanoDrop 1000 is used to measure absorbance.

Q: What is a Blank measurement on the NanoDrop 3300 Fluorospectrometer?
A: It is a stored snapshot of the non-fluorescent signal reaching the detector. It is best generated using water or a non-fluorescent buffer.

Q: How should the Blank be confirmed?
A: Use a fresh aliquot of water or buffer and take a measurement of the fluorescent signal (RFU) as if a sample. The spectral profile should be relatively flat and the RFU signal should be near zero (0). If the fluorescent value isn't < ± 10 RFU, clean the optical surfaces and repeat the blanking and the fresh aliquot measuring sequence until the proper baseline is established.

Q: What is the significance of Blanking in measuring fluorescence?
A: Since the Signal-to-Noise (S/N) ratio is the smallest at the lower detection (or sensitivity) limit, removing all non-fluorescent signal reaching the detector is the most important for those measurements. At higher fluorescence signal (RFU), removal of the blank signal becomes insignificant.

Q: Is the Initialize function required with the white LED equivalent to Blanking?
A: Essentially, yes. Since the white LED is unfiltered, Initialization generates a 'Blank-equivalent' snapshot which includes the LED spectral profile, as well as any other sources of non-fluorescent signal reaching the detector. The stored Initialization signal is removed from all sample, standard, and reference measurements.

Q: How should an appropriate Initialize measurement be confirmed?
A: In the same manner as the Blank measurement.

Q: Are there any general considerations to remember for fluorescence measurements?
A: Yes, protect fluorophores from ambient light and use only polypropylene or glass containers (*check manufacturers' recommendations for each reagent).

Q: Are there any special handling considerations for small volume fluorescence measurements?
A: Yes, be careful not to introduce bubbles while aspirating or pipetting samples. Keep the pedestal clean of all lint and debris as such particles may interfere with readings. Use of low lint labwipes to clean between samples will help to avoid erroneous measurements due to particulates.

Q: What sample size should I use?
A: The NanoDrop 3300 is relatively insensitive to sample size, however it is important to use a consistent sample size to ensure the best reproducibility. It is recommended that 1-2 ul samples may be used. We recommend using calibrated 2ul pipettes and low retention tips to obtain the best results.

Q: Why is it important to blot away the sample and not wipe as recommended for the NanoDrop 1000?
A: Lint and dust can cause erroneous measurements due to fluorescent particulates. Use low lint labwipes to blot and remove samples from the optical surfaces after each measurement to ensure reproducible measurements. Note: Filtration of reagents and use of clean labware is recommended to avoid possible unknown fluorescent contamination.

Q: Is simply blotting the surface enough to prevent carryover?
A: Yes, both surfaces are actually polished ends of a fiber optic cable. There are no holes or crevices for sample to stay in.

Q: Will air bubbles in the sample mixture cause any problems?
A: Yes, air bubbles in a sample will cause scattering of light leading to inaccurate measurements. Although thorough mixing is important for reproducible results, take extreme care when mixing samples and pipetting to avoid introducing air bubbles into your sample.

Q: How does the term "gain" apply to the NanoDrop 3300?
A: Gain as it applies to a fluorometer classically refers to a means of controlling the excitation source voltage to increase the excitation of fluorphores. The NanoDrop NanoDrop 3300 fluorospectrometer applies a constant current for each measurement while using time as a means of maximizing the excitation of the fluorophore. NanoDrop uses the term "gain" to refer to the amount of time the sample is excited. Time is therefore used to maximize the signal to the CCD spectrometer detectors while preventing detector saturation.

Q: What does "Virtual filtering" mean?
A: UV (365 nm) and blue (470 nm) LEDs are used in conjunction with two filters (UV glass and 495 sharp cut interference, respectively) while virtual filtering is used in conjunction with a white LED (500-700 nm). Virtual filtering takes advantage of both the extreme blocking of the off axis excitation by the collection optical fiber, the close coupling of the sample with the collection fiber and the stability of the emission spectrum of LEDs. Based on the inherent "off-angle" light rejection properties of the optical fiber and the reduction in scattering from directly wetted optical surfaces, it is possible to extract the sample fluorescence by subtracting a scaled representation of white LED source.

Q: Can samples that are in high vapor pressure solvents (such as acetone) be analyzed?
A: Solvents such as acetone will likely evaporate during the measurement cycle. Less volatile solvents may be compatible by using larger sample volumes. Note: The emission spectra may shift in different solvents or the solvent itself may have inherent fluorescence. Make sure to match your sample to the correct solvent and blank with the same solvent.

Q: Are there any special concerns when analyzing proteins?
A: Yes. Due to the lower surface tension of protein, a 2 ul sample is required. Also, proteins are harder to wipe off of the sampling surface and require a more vigorous cleaning with a dry lab wipe.

Q: Does the NanoDrop 3300 produce results for a continuous spectrum or just selected wavelengths?
A: Continuous spectrum from 400-750 nm- although most fluorophore methods are written so that narrow wavelength ranges are displayed.

Q: What sort of accuracy should I expect with the NanoDrop 3300?
A: As the NanoDrop 3300 requires that the user generate a standard curve to calculate concentrations, the accuracy of the data is dependent on the accuracy of the standards.

Q: What sort of reproducibility should I expect with the NanoDrop 3300?
A: May vary with fluorophore. The actual relative fluorescent units for any sample will vary from instrument to instrument.

Q: There is not a calibration service plan for the NanoDrop 3300. Why not?
A: Using Beers law, the NanoDrop 1000 software calculates the concentration of samples based on absorbance, pathlength and the molar extinction co-efficient. Therefore accurate pathlength calibration is essential to reporting accurate concentrations. Unlike absorbance spectroscopy, measurement of fluorescence is always reported in RFU (relative fluorescent units) and the actual pathlength-dependence for the fluorospectrometer is much less. NIST SRM reagents (fluorescein and quinine sulfate) routinely used for assessing fluorescence performance are commercially available.

Q: What kind of light sources are being used on NanoDrop 3300?
A: UV (365 nm), blue (470 nm) and white (500-650 nm) light emitting diodes (LEDs)

Q: How long before I need to replace an LED?
A: Each LED should last for a minimum of 150000 measurements. It is expected that the LEDs will last the lifetime of the instrument.

Q: Are the LEDs on continuously, or only when performing a measurement?
A: The LEDs are on only during measurements.

Q: Does NanoDrop 3300 need warm-up time?
A: No. The instrument is ready for measurements as soon as the software is started.

Q: How does temperature affect fluorescence measurements?
A: Measuring standards, blanks, and samples at the same temperature is advised since variations in temperature will have effects on the fluorescent measurements. Samples at higher temperatures will have lower fluorescence in general.

Q: How does pH of the buffer affect fluorescence measurements?
A: Some fluorophores are very sensitive to differences in pH. Make sure that the sample to be measured is at the proper pH for the fluorophore being measured.

Q: How can dried sample be removed from the optical surfaces?
A: Common laboratory reagents including bleach (5.25% sodium hypochlorite), alcohol, and dilute acids or bases can all be used to remove residue from the quartz / stainless steel optical surfaces. Select the one which is most effective for the sample types handled within your laboratory. Follow the cleaning & buffing process by using a water-wetted wipe on the upper and lower optical surfaces and then blot dry.