Team:UNIPV-Pavia/Methods Materials/Fluorescence

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Fluorescence and water dispensation

Differently from the measures of absorbance, we find out that the dispensation of water in the well causes an amplification of the fluorescence signal, both for bacterial coltures and simple liquid growing medium (in figure the results for a colture producing GFP).

This amplification seams to be non linear and characterized by a kind of saturation, if analised on a large scale of volumes.

You can find more informations about the experiment at Download Protocol - Test n.9, 07/07/09.

Fluorescence (GFP) and water dispensation, bacterial colture

Fluorescence and dilutions in water

The proportionality between fluorescence measurements (GFP and RFP) and different dilutions of LB was verified in two volumetric dilutions.

This considerations has not to be read together with the results of the non-liner relationship between fluorescence and water dispensation, because even if different amounts of water in the well means different dilution of the colture, in that simulation we didn't work with costant volume in the well, as in this case we do.

The first two measurements must not be taken into account because of the high measure's rumour.

You can find more informations about the experiment at Download Protocol - Test n.18, 06/08/09.

Fluorescence (GFP) and water dilution, LB

Fluorescence and volume

The relationship between fluorescence (both GFP and RFP) and volume of LB in the well present a non-linear characteristic.

This test will be done also using bacterial coltures, because we note that the behaviour of the signal from fluorescent proteins is different from that coming from the autofluorescence.

You can find more informations about the experiment at Download Protocol - Test n.14, 30/07/09.

Fluorescence and volume, LB

Fluorescence and dilutions in liquid growing medium

This experiment was done in two different growing mediums, LB and M9, and in three different total volumes in the well, 100μl, 200μl and 300μl; a black plate was also used to repeat the experiment in LB.

The hypothesis of proportionality between fluorescence measurement and colture's dilutions was not verified.

We observed a non-linear relationship between bacterial quantity in the well and fluorescence (both GFP and RFP) measures, in the two cases, and a non-negligible reliance on the total volume in the well.

With total volumes larger than 200μl the function shows a kind of saturation, suggesting not to work with those volumes.

The situation gets a little bit better using the black-home-made plate.

You can find more informations about the experiments at Download Protocol - Test n.20, 13/08/09, Test n.20 "M9", 28/08/09 and Test n.20 "black", 25/08/09.

Fluorescence and dilutions in LB

Fluorescence and dilutions in M9

Fluorescence and dilutions in LB, black plate

Temporal evolution of fluorescence measurements

We observed a particular descending evolution in fluorescence measurements, with regard to the signal of the growing medium.

This behaviour depends in part on temperature, as it changes its evolution with temperature's changes (see the figure below), and probably in part on evaporation of the volume in the well.

You can find more informations about the experiments at Download Protocol - Test n.14, 30/07/09.

Fluorescence evolution, LB

Bleaching and autofluorescence

A simple experiment was made to understand if the temporal descending evolution of fluorescence measurement in LBis related to the bleaching phenomenon of the fluorescence signal or not. The results (see the figure) let us exclude this hypothesis.

You can find more informations about the experiment at Download Protocol - Test "bleaching", 31/08/09.

Bleaching in fluorescent signal, LB