I think this could depend on what type of microalgae u are using and if they are naturally adapted to dark and/or ligth conditions. 

I grew them in light intensity of 25 uE for one week. The fact is that I should expect a higher value of QY after dark adaptation, but when I measured it I got a slight lower value compared to light-adapted... For dark adaptation I put the samples in darkness for 20 minutes.

Basically there are two Chl Fluorescence approaches used to monitor photosynthesis efficiency in microalgae culture, the rapid fluorescence induction and the saturation-pulse method. The rapid fluorescence induction method explore photosynthetic activity using dark-adapted samples, the other is a in situ measurement in turbulent cultures. 

In the dark PSII should become fully oxidized and all non-photochemical quenching (npq) mechanisms should get switched off. This results in the lowest possible fluorescence in the dark and the highest possible fluorescence with a saturating pulse and give you the maximal yield. In the light the yield should go down because PSII becomes partially reduced due to excitation and npq can start to play a role at higher light intensities, this is called the active yield. It sounds like your algae are well adapted to the low light you gave them which means that PSII is still basically oxidized and no npq is needed so there is basically no difference in maximal and active yield, although dark yield should be slightly higher in green algae but if the difference is not significant I wouldn't worry.

If you just want to know how your culture 'is doing', happy, stressed out or dying than the yield in the light is most useful because light is the most relevant condition. Determining the dark yield is something of a reference point, so that you know which yield represents 'happy'. PSII yield differ between strongly between species and somewhat between culture conditions.

I agree with Rosa.

Moreover, I am using Aquapen too and I recommend you to compare two measurements: 20 min dark and 5 min dark. If the difference is not relevant, you can use 5 min dark measurements. I am doing this 5 min test and I find enough good correlation between light stress and Qy

(I am growing T-Iso in Spain and I use this test at solar noon (14:00 h) when I appreciate foam due to excess O2 in my PBrs).

Thank you very much for your precious suggestions!

Federico, how do you make the dark adaptation of the sample? I've seen that the best way is leaving the sample inside the AquaPen (inside the cuvette) and than taking the measurement, after mixing simply by inverting the cuvette.

That's right if you have just one sample, Lorenza.

But I usually measure three or more samples simultaneously, thus I cover the cuvettes with alu-foil and change samples very fast in dark conditions.

As you know, the reason to keep the sample a few minutes in darkness is to "open" the reaction centers, so, as just a few photons escape into the cuvettes, one minute between sample and sample will be enough because we will provide the cells enough time to find electron acceptors to relax photosystem II. As someone has exposed above, if you wait 20 min or more, you will get higher Qy values; in contrast, without dark adaptation you will get also the non-photochemical quenching and therefore a lower Qy, and hence you have to consider that you are measuring a steady-state quantum yield strongly dependent on light stress. (Personally I find the steady state Qy more interesting than the maximum Qy -the one with dark-adapted cells- to  immediately follow how light-stressed the algae are).

You can aditionally perform a test by yourself: After 5 min in darkness determine Qy; then, open the lid and expose the cuvette to dim light for one or two seconds; measure again, and check wether the results are different. Moreover, you can perform some consecutive pulses (push repeatedly the measure button) and compare results. Once you have made all this tests (and anymore you can argue) you will determine the most proper working parameters, that in turn will change amongst different algae strains and/or culture conditions.

With this sort of procedures and a handy portable device such as Aqua pen you will not get absolute results (those you can use for publising) but you will find a useful and rapid test for daily personal measurements. If you want to get completely reproducible results, then you better use the dark adapted test (determine maximum Qy).

I have worked in this field for marine macroalgae. It is better to adapt the sample at least for half an hour in dark adaptation and switch on the light when you want to take the reading.

Fv/Fm in dark adapted state gives the information about the potential maximum photocehemical quantum effciency of PS II (i.e. what is the maximum efficiency of your culture) and Fv'/Fm' (Fv/Fm in light adapted state) is the operational quantum efficientcy of PSII (i.e. what is the current top photosynthetic efficiency of the culture). Difference between the two figures roughly corresponds to the degree of short-term stress.

This reading might be of some help for naviagation in the world of Chl fluoerscence:

http://jxb.oxfordjournals.org/content/51/345/659.short

http://link.springer.com/article/10.1007/s11120-014-0024-6

With regard to your first question - it depends on why you are taking the measurements. If you wish to consider the general performance of your culture under your applied conditions then the "operational" or "effective" QY as mentioned by Alexei would be most useful. Performing this measurement at different times of the day may provide some interesting data on diel fluctuations. Analysing NPQ relaxation kinetics would also provide useful data on the photosynthetic functioning of your organism.

With regard to your dark adaptation results:

I am surprised that nobody has mentioned the propensity of chorophytes to exhibit symptoms of chororespiration (such as dark-reduction of the plastoquinone pool). Use of a far-red light prior to measurement can help mitigate this impact.

NEVER just assume that throwing an alga  in the dark with result in achieving peak QY values (Fv/Fm). The protocol required changes from strain to strain little own between species and will also change depending on both light quantity and quality applied to the culture. Preliminary tests should always be done to determine best protocol for achieving Fv/Fm. You may only need to dark adapt for 5min!

Regarding leaving your sample in the cuvette holder:

As Federico mentions, this is fine if you don't have heaps of samples to do. Always check beforehand how your culture behaves in the cuvette. If cells maintain suspension over 20 plus minutes then you don't even need to take the cuvette out to invert it (in a darkened place). With motile cells you will need to be aware of any form of tropism they display.

Best of luck.

It really depends on what you want to evaluate:

If you want to have the maximal PSII quantum yield, you should dark acclimate your sample before your measurement

If you want to have the operational PSII quantum yield, do the measurement under light at a similar light intensity than the growth light. This way you will have an indication of the photosynthetic activity when you have continuous electron flow between photosystems.

Thank you so much for all the explanations, they were very precious!

I have done some work onred algae.pl refer the same