Fluorescence Lifetime Imaging
Lambert Instruments' Fluorescence Lifetime Imaging Attachment is
a system that can be attached to almost every fluorescence microscope
and enables to make fluorescence lifetime images but can also be
used for normal fluorescence imaging. The price level of the system
is such that it comes in the reach of many researchers.
Why lifetime imaging?
Quantitative fluorescence microscopy uses the intensity of the fluorescence
to extract information about the local concentrations of molecules
that are labeled with fluorescent probes. This technique suffers
from the fact that the fluorescence of the probe is permanently
destructed by light-induced conversion of the probe material to
a non-fluorescent compound. This photochemical process is called
"bleaching" and makes it necessary to regulate the excitation
dose in an economical way. Another physical property of fluorescent
molecules is the fluorescence lifetime. The fluorescence lifetime
is the decay time of the emission after the excitation has been
stopped. The fluorescence lifetime depends on the local concentrations
of certain molecules or ions. Changes in fluorescence efficiency
as caused by bleaching are not accompanied by changes in fluorescence
lifetime. Fluorescence lifetime imaging microscopy (FLIM) merges
the information of the spatial distribution of the probe with the
lifetime to increase the reliability of the concentration measurements.
Additionally, FLIM enables the discrimination of fluorescence coming
from different dyes, including auto-fluorescent materials, that
exhibit similar absorption and emission properties but showing a
difference in fluorescence lifetime.
How does the system work?
In contrary to other systems that work in the time domain our system
works in the frequency domain. This method requires a modulated
light source and a modulated camera. The excitation light is modulated
in a sinusoidal fashion. The fluorescence intensity shows a delay
or phase-shift with respect to the excitation and a decrease of
modulation-depth. The phase-shift and modulation-depth depend on
the decay constants of the fluorescent material and the modulation
frequency. To extract the phase-shift and the modulation-depth of
the fluorescence light an intensified camera is used. The sensitivity
of the intensifier is modulated with the same frequency as the excitation
modulation, but with an adjustable phase-shift. The output signal
level depends on the phase-shift of the fluorescence signal relative
to the modulated sensitivity of the camera. A series of measurements
is made at increasing phase-shifts and a sine is fitted through
the series. The modulation-depth and phase-shift of the sine are
used to extract the decay constants that are present in the fluorescence
emission. To create the fluorescence lifetime image, a series of
images is captured at different camera modulation delays and the
calculations are performed for each pixel of the images.
As a detector an intensified camera is used. The gain of the intensifier
can be modulated. The camera is connected to a computer by means
of a GPIB interface for controlling the settings of the camera and
a framegrabber imports the image data to the computer. As the excitation
light source a modulated LED or Laserdiode is used which is mounted
on the place of the lamp in the standard lamp house of the microscope.
For modulation both the camera and the LED are connected to the
sinewave generator in the FLIM electronics box. This generator has
two outputs. The frequency and amplitude of both outputs and the
phase shift between them is controlled by software via a RS-232
interface. A software package controls all the settings of the camera
and the light source, calculates the lifetime of each individual
pixel and presents the lifetime image in false colors. The lifetime
value of each individual pixel can be read on the status bar by
pointing it with the mouse.