Fluorescence lifetime shows temperatures of flowing liquids
Understanding temperature distribution can help optimize chemical processes on microchips
Lynn M. Savage
Many biological analytical
processes would benefit from miniaturization, but some —such as DNA amplification
through polymerase chain reaction (PCR) — depend on homogeneous temperature
gradients to optimize the chemical or molecular reactions. Putting PCR on a microfluidic
chip, for example, should improve its efficiency, response and cost; however, achieving
consistent temperatures through a liquid sample in a chip requires a finely tuned
system for determining what the temperatures are at any given point in space and
Existing temperature-monitoring technology —
including thermocouples and various spectroscopic techniques — do not have
sufficient sensitivity for the task. The time-integrated fluorescence intensity
of fluorophores within a liquid can be measured, but this technique suffers from
significant problems relating to calibration and artifacts resulting from excitation
and detection efficiencies, said Andrew J. deMello, chairman of chemical nanosciences
at Imperial College London.
A team led by deMello and Paul M.W.
French, chairman of the college’s Photonics Group, has used fluorescence
lifetime imaging to create three-dimensional thermal maps of fluorophore-bearing
fluids moving through microchannels on a chip. According to deMello, using fluorescence
lifetime measurements means that the technique is independent of the concentration
of the fluorescent dye within the liquid sample and of the efficiency of the detection
Data in the flow
The investigators report in the March 4 ASAP edition
of Analytical Chemistry that they made 1-mm-thick glass microfluidic chips
etched with channels 139 μm wide and ~38 μm deep. The chips were
injected with solutions of rhodamine B in methanol. They chose rhodamine B because
its fluorescence would not appreciably change as a result of the pH or the viscosity
of the solution.
They used two-photon excitation to
create optical sections of the liquid, which flowed through the channels at rates
between 0.1 and 10 μl/min. Varying the rate enabled them to alter how the liquid
was warmed by the heating elements attached to the chip substrate.
Using 840-nm, 100-fs pulses from a
Ti:sapphire laser made by Spectra-Physics of Mountain View, Calif., that was coupled
with a microscope from LaVision BioTec GmbH of Bielefeld, Germany, the scientists
scanned the flowing liquid. Short pulses of infrared radiation helped control the
amount of heat added to the fluid as it was being scanned.
Two-photon excitation is less efficient
than single-photon, deMello said, but because their system scans up to 64 beams
in parallel, they achieved wide-field detection and good optical sectioning of the
fluid flow. Furthermore, no laser-induced excitation or heating of the fluid occurred
outside of the femtoliter-size excitation volume in the microchannel.
They used the photocathode of a time-gated
intensifier from Kentech Instruments Ltd. of Didcot, UK, to detect the rhodamine
B emission. The fluorescence images were relayed to a CCD camera from LaVision GmbH
of Göttingen, Germany, providing them with a series of 20 fluorescence intensity
images after each excitation pulse. Acquisition times typically were 10 to 30 s
per image, which they noted was compatible with the steady-state flow and heating
conditions of their setup.
The average of each image series provided
a fluorescence decay profile for each pixel in the microscope’s field of view.
The scientists converted the decay data into temperature readings by comparing it
with calibration data from the literature and from measurements of bulk solutions
of rhodamine maintained at various temperatures. Within the three-dimensional map
of fluorescence decay that was created, they achieved a spatial resolution of 1
to 5 μm between pixels in each image section about 100 μm long and a precision
of ±1 °C.
Testing temperature zones
They found that the fluorescence lifetime decreased
by a factor of about two when the temperature of the fluid was increased from 66
to 93 °C — temperature zones commonly used during PCR. Multiple temperatures
are required to perform the basic steps of amplifying DNA with PCR techniques, and
miniaturizing a PCR system would require careful control of the temperature in each
zone to promote the efficiency of the amplification steps.
Researchers used fluorescence lifetime imaging to measure the temperature fluctuations inside
a liquid flowing through a microchannel. Two-photon excitation of rhodamine B in
solution with methanol enabled a 130 x 40 x 100-μm optical section showing
the decay rates of the rhodamine after excitation (top). The data can be mathematically
converted to temperature and smoothed (bottom). Courtesy of Andrew J. deMello.
In PCR, deMello said, if the temperature
at a specific location varies more than 1 °C, efficiencies are drastically
reduced. “Our imaging approach will allow us to detect and correct for these
To test whether the technique would
work in PCR and other applications that require multiple temperature zones, the
investigators acquired time-integrated fluorescence intensity images and fluorescence
lifetime images at points along an S-shaped microchannel to which a temperature
gradient was applied.
They found that the intensity data
was insufficient for recording temperature, but that the lifetime data was very
much suitable. They also found that optimal fluid temperatures were achieved only
within 5 mm of each heating element attached to the chip. The researchers believe
that this knowledge will be important for designers of microchannels and other aspects
of chips for PCR use.
They are looking to improve the sensitivity
of the system by using a fluorophore that exhibits a larger decrease in fluorescence
lifetime as a function of temperature than the rhodamine B does.
They also hope to test a chip substrate
less than 1 mm thick that would permit them to work with lower working distances
and to use objectives with a higher numerical aperture, which they anticipate would
“The ability to image fluid mixing
and flow should allow us to separate out these effects from observed chemical kinetics
— for example, in continuous-flow experiments,” deMello said.
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