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phospholipid molecules can be calculated. Similarly, membrane proteins such as receptors

or even proteins in a cell can be conjugated to fluorescence labels and their diffusion

coefficients can be determined.

Fluorescence polarisation

A light source usually consists of a collection of randomly oriented emitters, and the

emitted light is a collection of waves with all possible orientations of theEvectors

(non-polarised light). Linearly polarised light is obtained by passing light through a

polariser that transmits light with only a single plane ofpolarisation; i.e. it passes

only those components of theEvector that are parallel to the axis of the polariser

(Fig. 12.14). The intensity of transmitted light depends on the orientation of the

polariser. Maximum transmission is achieved when the plane of polarisation is

parallel to the axis of the polariser; the transmission is zero when the orientation is

perpendicular. The polarisationPis defined as

P¼

IlI$

IlþI$

ð 12 : 7 Þ

IlandI$are the intensities observed parallel and perpendicular to an arbitrary axis.

The polarisation can vary between1 andþ1; it is zero when the light is unpolarised.

Light with 0<|P|<0.5 is called partially polarised.

(b)

O 2 N

CH 3

O

O

O

O

O

O O–

O

P

O

N

N

N

H+

Observation

area under

microscope

(a)

Initial situation

restored

Bleach dye with

focussed

laser beam

More and more

labelled PLs diffuse

into observation area

Dye irreversibly

damaged, no

fluorescence

emission

Determination of diffusion coefficients

between 10–6and 10–12cm^2 s–1

I

t 0 t1/2 t

Fluorescence

emission

monitored with

‘weak’ incident

beam

Fig. 12.13(a) Schematic of a FRAP experiment. Time-based monitoring of fluorescence emission intensity

enables determination of diffusion coefficients in membranes. (b) A commonly used fluorescence label in membrane

FRAP experiments: chemical structure of phosphatidylethanolamine conjugated to the fluorophore NBD.

504 Spectroscopic techniques: I Photometric techniques