5. Operating the INFINITE M1000 PRO
54
Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0
2011-09
5.3
Optimize Fluorescence Measurements
Fluorescence measurement results may be improved by optimizing instrument
parameters and selecting the appropriate materials.
5.3.1
FI Scanning (Spectral Intensity Calibration)
Due to wavelength dependence on the intensity of the excitation light and
instrument components (gratings; lenses; PMT) being passed by the excitation
and emission light distortions of measured spectra might be possible.
Excitation spectra are distorted primarily by the wavelength dependence of the
intensity of the excitation light
the
INFINITE M1000 PRO
allows you to correct the
spectra.
To calculate corrected emission spectra, one needs to know the wavelength-
dependent efficiency of the detection system. Therefore a calibration curve is
saved on the
INFINITE M1000 PRO
for correction.
For more details see also‚ Principles of Fluorescence Spectroscopy’, Third
Edition; Joseph R. Lakowicz.
What is the reason for intensity differences between scan
measurement values and fixed wavelength measurement values?
Excitation scan versus fixed wavelength
The
INFINITE M1000 PRO
uses a reference fiber to compensate for fluctuations
of the flash lamp. The sensitivity of the reference fiber needs to be adjusted
(automatically done by the software) before each measurement to make sure that
the fiber is working in an optimal sensitivity range and does not show overflow
values. This reference measurement is performed differently for scan
measurements and fixed wavelength measurements. For fixed wavelength
measurements, the calibration of the reference fiber is performed at the selected
measurement wavelength.
For scan measurements, the same reference method would be possible. But in
the worst case (3D scan over full wavelength range) over 600 reference points
(one per wavelength) have to be measured and saved.
Depending on the number of measurement points, this can take a few seconds to
up to nearly one minute. To improve the measurement speed, we decided to
perform the reference measurement at one wavelength, which is expected to give
the highest light intensity. This procedure has proven successful in avoiding
overflow errors and in providing sufficient sensitivity. The fixed wavelength
measurement and scan measurement performed with the same measurement
parameters (gain, number of flashes, z-position), have one side effect the results
do not show the same RFU values.
Emission scan versus fixed wavelength
The reference measurement is performed at the selected excitation wavelength in
both modes. Fixed wavelength values might deviate from scan wavelength values
±10% due to energy fluctuations of the flash lamp.
