3.2 Measuring
Variable Fluorescence
Using a Standard
Fluorescence
Spectrophotometer
1. Take a 3 ml subsample from the culture and fill the fluorescence
cuvette, and let the cuvette stay in darkness for 15 min to make
the culture dark acclimated (see Note 3).
2. Filter and measure a blank sample as described above.
3. Measure fluorescence of the sample using excitation wavelength
440 nm and emission wavelength 680 nm, and subtract the
blank reading. This is the F 0 value.
4. After the dark-acclimated measurement, take the cuvette and
add 10 μl DCMU solution (see Note 4).
5. Let the cuvette stay in light for 2 min.
6. Measure fluorescence again using the same procedure as step
4. This is the F m value.
7. Calculate the variable fluorescence: F v ¼ F m – F 0 (see Notes
5 and 6).
8. Calculate the photochemical efficiency index: F v /F m ¼ (F m – F 0 )/
(F m – blank) (see Note 7).
3.3 Measuring
Variable Fluorescence
Using a Variable
Fluorometer
1. Take a 3 ml subsample from the culture and fill the fluorescence
cuvette.
2. Let the cuvette stay in darkness for 15 min to make the culture
dark acclimated.
3. Measure fluorescence using excitation wavelength 450 nm and
emission wavelength 680 nm, using the OJIP option.
4. The F v will automatically be calculated from the measurements
(see Note 8).
4 Notes
1. You might need to adjust your fluorometer to get fluorescence
values that are not too low or over range. In most fluorometers
there are two ways this can be done, either by adjusting the slit
widths of emission and/or excitation filter or by adjusting the
voltage in the photomultiplier tube, i.e., increasing voltage
increases sensitivity.
2. Before starting, it is advisable to test your instrument and the
settings you are using at a wide range of biomass concentrations.
The increase in fluorescence with increasing Chl a is initially
linear but will at very high Chl a concentrations start to level
off, e.g., due to increasing reabsorption of fluorescent light.
Finding the linear range can easily be done by running a dilution
series where a dense culture is diluted with filtered (0.2 μm)
water.
44
Kristian Spilling and Jukka Sepp€ al€ a
Variable Fluorescence
Using a Standard
Fluorescence
Spectrophotometer
1. Take a 3 ml subsample from the culture and fill the fluorescence
cuvette, and let the cuvette stay in darkness for 15 min to make
the culture dark acclimated (see Note 3).
2. Filter and measure a blank sample as described above.
3. Measure fluorescence of the sample using excitation wavelength
440 nm and emission wavelength 680 nm, and subtract the
blank reading. This is the F 0 value.
4. After the dark-acclimated measurement, take the cuvette and
add 10 μl DCMU solution (see Note 4).
5. Let the cuvette stay in light for 2 min.
6. Measure fluorescence again using the same procedure as step
4. This is the F m value.
7. Calculate the variable fluorescence: F v ¼ F m – F 0 (see Notes
5 and 6).
8. Calculate the photochemical efficiency index: F v /F m ¼ (F m – F 0 )/
(F m – blank) (see Note 7).
3.3 Measuring
Variable Fluorescence
Using a Variable
Fluorometer
1. Take a 3 ml subsample from the culture and fill the fluorescence
cuvette.
2. Let the cuvette stay in darkness for 15 min to make the culture
dark acclimated.
3. Measure fluorescence using excitation wavelength 450 nm and
emission wavelength 680 nm, using the OJIP option.
4. The F v will automatically be calculated from the measurements
(see Note 8).
4 Notes
1. You might need to adjust your fluorometer to get fluorescence
values that are not too low or over range. In most fluorometers
there are two ways this can be done, either by adjusting the slit
widths of emission and/or excitation filter or by adjusting the
voltage in the photomultiplier tube, i.e., increasing voltage
increases sensitivity.
2. Before starting, it is advisable to test your instrument and the
settings you are using at a wide range of biomass concentrations.
The increase in fluorescence with increasing Chl a is initially
linear but will at very high Chl a concentrations start to level
off, e.g., due to increasing reabsorption of fluorescent light.
Finding the linear range can easily be done by running a dilution
series where a dense culture is diluted with filtered (0.2 μm)
water.
44
Kristian Spilling and Jukka Sepp€ al€ a