Cornelius DPC 230 Specifications download pdf (Page 31)

Fluorescence Correlation 25

A typical optical setup is shown in Fig. 34. A CW laser beam is focused into the sample

through a microscope objective lens. The fluorescence light from the sample is collected by

the same lens, separated from the laser by a dichroic mirror, and fed through a pinhole in the

upper image plane of the microscope lens. Light from above or below the focal plane is not

focused into the pinhole and therefore gets substantially suppressed. With a high-aperture ob-

jective lens the effective sample volume is of the order of a femtoliter, with a depth of about

1.5 µm and a width of about 400 nm. A similarly small sample volume can be obtained by

two-photon excitation. Typical FCS instruments use several detectors in different wavelength

intervals or under different polarisation. Due to the similarities in the optical system, FCS is

possible with most confocal laser scanning microscopes [2].

Polarising

beamsplitter

Dichroic

Microscope

Lens

Laser

Detectors

Pinhole

Fig. 34: Optical setup for FCS measurements

For good FCS results high detection efficiency is essential. The reason is the unusual signal-

to-noise behaviour of correlation experiments. In a fluorescence lifetime or FLIM measure-

ment a 50% loss in efficiency can be compensated by increasing the acquisition time by a fac-

tor of two. In an FCS measurement a 50% loss in detection efficiency requires the acquisition

time to be increased by a factor of four to obtain the same signal-to-noise ratio. The detectors

of FCS instruments are therefore single-photon avalanche photodiodes (SPADs) or high-

efficiency PMTs with GaAsP cathodes [2].

The electronic setup of an FCS system is shown in Fig. 35. SPADs are connected via a cable

adapter (see Fig. 20) to the LVTTL Inputs of the DPC. Any SPAD module with TTL or

LVTTL output can be used. PMTs are connected to the CFD inputs, see Fig. 35, right.

DPC-230 module

LVTTL

Inputs

Extension cables

SPADs

id Quantique, MPD,

Perkin Elemer, Sensl

Detectors

DPC-230 module

H7422 Power Supply and

DCC-100 Detector Controller

Overload Shutdown

HFAC-26-2

Preamplifier

H7422P-40

Fig. 35: FCS setup with DPC-230. Left: SPAD detectors. Right: GaAsP PMT modules

The measurement control part of the System parameters is shown in Fig. 36. ‘Operation

Mode’ is set to ‘Absolute Time’, ‘Correlation’. The settings shown on the left calculate and

display FCS curves, the accumulated number of counts, and an MCS (intensity) trace online.

The time-tag data are not saved. The settings shown on the right calculate the same curves, but

simultaneously save the time-tag data into a file ‘fluorescein-a.spc’. The maximum file size is

1000 Mb. The measurement stops when this file size is reached. The measurement can also be

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Cornelius DPC 230 Specifications Page 31

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