that the energy difference between a blue and red photon is always lost
as heat [1].
There is generally a positive correlation between Chl a and fluorescence. However, Chl a per cell (or other biomass unit) will vary
depending on the light environment. For example, the Chl a content
typically increases in dim light as an acclimation mechanism to lower
photon flux and similarly decreases if the amount of light increases.
When using constant light, the light acclimation will be stable, and the
fluorescent measurement is more closely correlated with biomass than
for algae grown under natural light.
In addition to using single fluorescent measurements as proxy for
biomass, more advanced measurements can be made when making
some assumption about the loss of energy as heat (e.g., that it is
constant over short time periods). One of the key measures is the
variable fluorescence (F v ), which is the difference between the minimum (F 0 ) and maximum (F m ) fluorescence [1]. These variables can be
used to calculate the photochemical efficiency, F v /F m , which can be
used as a measure of stress to the algal cells. The minimum fluorescence
can only be measured when all the reaction centers are open, and this is
achieved by dark acclimation of the culture prior to measurement.
Then the whole photosynthetic machinery will be ready for processing
absorbed photons, and all reaction centers are open. Maximum fluorescence is measured when all the reaction centers are closed; this can
be induced either chemically (typically by adding an herbicide such as
DCMU that blocks the photosynthetic electron transport chain) or by
adding a strong light flash that fills the electron transport chain
completely. There are some different approaches to the latter, the
most common being pulse amplitude modulated (PAM) fluorometer,
in which one strong pulse of light is provided, and fast repetition rate
fluorometry (FRRF) where a sequence of rapid light flashes gradually
fills the electron transport chain [2]. Some fluorometers also provide
the fluorescent transient from F 0 to F m , often termed the OJIP curve
[2, 3]. An OJIP curve is the buildup of fluorescence from the origin
(O) through inflections (J and I) up to a peak (P) (Fig. 1). The
curvature of the OJIP curve is related to the filling of the electron
transport chain, can be divided into photochemical and thermal phases,
and is affected by the reduction of the intermediate electron acceptors
in the electron transport chain, most notably Q A [3].
2 Materials
1. Culture of Chlorella sp. grown in F/2 medium [4].
2. Fluorescence cuvettes (quartz glass, all four sides clear).
3. Fluorescence spectrophotometer.
4. Syringe and 0.2 μm syringe filter.
42
Kristian Spilling and Jukka Sepp€ al€ a
as heat [1].
There is generally a positive correlation between Chl a and fluorescence. However, Chl a per cell (or other biomass unit) will vary
depending on the light environment. For example, the Chl a content
typically increases in dim light as an acclimation mechanism to lower
photon flux and similarly decreases if the amount of light increases.
When using constant light, the light acclimation will be stable, and the
fluorescent measurement is more closely correlated with biomass than
for algae grown under natural light.
In addition to using single fluorescent measurements as proxy for
biomass, more advanced measurements can be made when making
some assumption about the loss of energy as heat (e.g., that it is
constant over short time periods). One of the key measures is the
variable fluorescence (F v ), which is the difference between the minimum (F 0 ) and maximum (F m ) fluorescence [1]. These variables can be
used to calculate the photochemical efficiency, F v /F m , which can be
used as a measure of stress to the algal cells. The minimum fluorescence
can only be measured when all the reaction centers are open, and this is
achieved by dark acclimation of the culture prior to measurement.
Then the whole photosynthetic machinery will be ready for processing
absorbed photons, and all reaction centers are open. Maximum fluorescence is measured when all the reaction centers are closed; this can
be induced either chemically (typically by adding an herbicide such as
DCMU that blocks the photosynthetic electron transport chain) or by
adding a strong light flash that fills the electron transport chain
completely. There are some different approaches to the latter, the
most common being pulse amplitude modulated (PAM) fluorometer,
in which one strong pulse of light is provided, and fast repetition rate
fluorometry (FRRF) where a sequence of rapid light flashes gradually
fills the electron transport chain [2]. Some fluorometers also provide
the fluorescent transient from F 0 to F m , often termed the OJIP curve
[2, 3]. An OJIP curve is the buildup of fluorescence from the origin
(O) through inflections (J and I) up to a peak (P) (Fig. 1). The
curvature of the OJIP curve is related to the filling of the electron
transport chain, can be divided into photochemical and thermal phases,
and is affected by the reduction of the intermediate electron acceptors
in the electron transport chain, most notably Q A [3].
2 Materials
1. Culture of Chlorella sp. grown in F/2 medium [4].
2. Fluorescence cuvettes (quartz glass, all four sides clear).
3. Fluorescence spectrophotometer.
4. Syringe and 0.2 μm syringe filter.
42
Kristian Spilling and Jukka Sepp€ al€ a