Composite polymer membranes for laserinduced fluorescence thermometry

: We demonstrate a modified version of laser-induced fluorescence thermometry (LIFT) for mapping temperature gradients in the vicinity of small photothermal devices. Our approach is based on temperature sensitive fluorescent membranes fabricated with rhodamine B and polydimethylsiloxane (PDMS). Relevant membrane features for LIFT, such as temperature sensitivity, thermal quenching and photobleaching are presented for a range of 25 °C to 90 °C, and their performance is evaluated upon obtaining the temperature gradients produced in the proximity of optical fiber micro-heaters. Our results show that temperature measurements in regions as small as 750 µm x 650 µm, with a temperature resolution of 1 °C, can be readily obtained.


Introduction
The advent of new microanalysis platforms, such as lab on a chip and microfluidic systems, has triggered extensive research aimed at studying thermal phenomena at the micron scale [1]. Given the size of these systems, phenomena such as heat transfer and heat loss to the surrounding media become relevant for most practical applications (e.g., DNA replication, electronic cooling, thermal generation of pH gradients [2]) and are of paramount interest for characterization and optimization purposes. In addition to temperature measurements, it is often desirable to obtain a temperature map, which could provide useful information related to thermal phenomena occurring in the system and its vicinity [1,2]. In general, a 'microthermometer' should be able to provide high spatial resolution, high acquisition rate for realtime sampling, low thermal inertia for rapid response and adequate temperature resolution. Among these features, measurements with high spatial resolution are perhaps the most challenging to obtain.
Several methods have shown to provide high spatial resolution (<1 µm) temperature measurements achieving thermal resolutions of less than <0.1 °C [2]. Contact techniques, involving the use of devices such as scanning thermal probes or micro-and nanothermocouples, provide an effective means to obtain local temperature measurements with spatial resolutions of up to 0.01 µm [2]. Non-contact techniques, albeit more elaborated and expensive, can yield a two-dimensional temperature map with high spatial resolution (e.g., 0.1 µm with infrared thermometry) and accuracy (e.g., 1 μK with laser interferometry) [2]. Among the different non-intrusive techniques available, fluorescent thermometry is one of the most widely used owing to its relative simplicity using commercially available cameras (CCD or CMOS); it can further be adapted to a microscope to obtain high spatial resolution measurements [3,4]. Temperature measurements rely on a fluorophore, typically a dye whose fluorescent intensity decreases with temperature, which can be either dissolved in a fluid or deposited on a surface of interest [4][5][6]. Excitation of the fluorophore is customarily done with laser light; hence, the technique is termed laser-induced fluorescence thermometry (LIFT). The spatial resolution depends on the imaging optics whereas the temporal resolution is defined by the framing rate of the camera. Successful applications of LIFT include temperature mapping of microfluidic channels [7-9], characterization of electrical microheaters [10] and thermal mapping of an optical trap [11], to name a few.
Rhodamine B (RhB) is the most widely used dye for LIFT, and its fluorescence intensity strongly depends on the solvent [3-10]. Water and ethanol based solutions are the most common choices for temperature measurements in liquid samples, although other solvents such as sodium carbonate buffer solutions have also shown to perform well [5,6,11,12]. For temperature mapping of dry surfaces, RhB can also be incorporated into polymer matrices to fabricate temperature sensitive films [9,10,[13][14][15]. In particular, integration of RhB into polydimethylsiloxane (PDMS) is of interest because of its dominant role in microfluidic and lab-on a-chip platforms [16]. In this work, we show a simple method for fabricating temperature sensitive PDMS membranes with RhB for LIFT. The PDMS-RhB composite is obtained by simple mixing and is subsequently molded and cured to yield large membranes that can be c contact with t through fluor temperature demonstrate t standard singl

Fabricati
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Laser-induced fluorescence thermometry setup
The setup for LIFT measurements comprised a CW diode-pumped solid-state green laser (532 nm, BWTEK, model BWI532-50-E, beam diameter < 1 mm) with ± 3% power stability. The illumination area is increased by a lens (10x, NA 0.25) yielding a spot size of approximately 2 cm. Images are acquired using a CMOS camera (Thorlabs, DCC1645C) with a 10x microscope objective (see Fig. 1). A notch filter (533 nm, 17 nm FWHM, Thorlabs, NF533-17) was further used to eliminate the green radiation in the images recorded by the camera. As seen in Fig. 1, a 50% beam splitter was used to simultaneously excite with the laser the fluorescence of the PDMS-RhB membranes and for monitoring power fluctuations. The latter was done by means of an optical power meter and a thermal power sensor (Thorlabs, S302C). Data from the sensor allowed us to account for spurious effects of laser power fluctuations that typically require using two dyes (one of them temperature insensitive) or two cameras [7,8]. Our modified version for LIFT allowed for minimizing these effects simply by taking the intensity ratio of the images to that registered by the sensor. More details of this normalization process are provided below.
For temperature calibration, the membranes were placed on the ceramic heater and the temperature was set to a desired value. Fluorescence images were then acquired and postprocessed to yield the calibration curves (i.e., normalized intensity vs. temperature) for the membranes. For all measurements, the camera gain was adjusted manually and kept constant. The resulting curves were then used to obtain the temperature maps across the membranes; with this setup, the spatial resolution of the imaging system was 2.7 µm/pixel.

Temperature calibration and image processing
The calibration curves were obtained from fluorescence images of the PDMS-RhB membrane acquired for a temperature range from 20 to 90 °C with intervals of 10 °C. Each image was acquired after achieving a steady temperature with the controller. The average time required to obtain an adequate thermal equilibrium for calibration purposes was approximately 10 minutes. Notice that this time constraint is due to the heater control system, and is not inherent to the thermal and fluorescence response of the membranes.
A temperature of 25 °C was selected as the baseline reading for all measurements. The PDMS-RhB membranes were exposed to laser excitation for 1 minute and during this time, 10 images along with their corresponding laser power were registered. In order to minimize possible quenching or bleaching effects, the incident laser light was blocked between measurements. The fluorescence intensity was analyzed considering an average intensity value calculated for each image using a gray scale conversion. This allowed us to obtain a ratio of the average fluorescence intensity (I) to the laser power (P) for each temperature; this ratio was then normalized using the ratio of the initial intensity to the initial laser power (I 0 / P 0 ), i.e.: Notice that this normalization procedure accounts for average fluctuations in the laser power. Finally, the calibration curve was obtained plotting the normalized fluorescence intensity as a function of temperature.

PDMS-RhB membranes: calibration and performance
The fabrication process mentioned above allowed us to obtain large PDMS-RhB membranes whose dimensions are limited by the size of the mold. For our experiments, we fabricated membranes with a maximum area of 20 mm by 70 mm and a thickness of 300 µm ± 30 µm. In contrast to other methods, the process to fabricate the membranes allowed us to embed the dye without procedures [9 could be subs square sample An optica the nearly ho magnification during the m Nonetheless, the laser. The while the emi obtained with Before tes their stability the RhB and/ The laser inte 10 minutes du and the proc mW/cm 2 ). As period of tim considerably measurements We also fluorescence i  Fig. 2(a) Fig. 3(b) 3(a), the similar to DMS-RhB obtained n, the plot ller. The temperature obtained from the fluorescence of the membranes showed a similar trend as the data from the thermistor, albeit providing a lower reading. Hence, the fluorescence can readily track the temperature changes, however, an accurate temperature reading can only be obtained after the emission has reached a steady state value. From Fig. 3(b), a stable temperature reading in the thermistor occurs at approximately 200 s, while the fluorescence reading reaches a steady value at around 350 s, yielding a settling time of 2.5 min to obtain an accurate temperature reading. We attribute this limitation to the low thermal conductivity of PDMS host matrix (0.15 Wm −1 K −1 ), which is known to impose some constraints when using PDMS in applications involving heat transfer phenomena [9,18]. Similar response times have been reported when using PDMS functionalized with RhB [9]; in contrast, to previous reports, our fabrication method involves only simple mixing without requiring any chemical process to incorporate the RhB in the host matrix.

Application to micro-thermometry: characterization of optical fiber micro-heaters
Optical fiber micro-heaters (OFMHs) are devices fabricated with layers of absorbing materials deposited on the tip of optical fibers; these photothermal devices are capable to increase the temperature in a highly localized manner [17,19]. Because they are made with standard optical fibers (125 μm diameter), the cross-sectional area of these heating elements is within a few hundred microns. The absorbing layer is deposited on the output end of the fiber and laser light (typically a laser diode) is then launched at the opposite end. Heat is subsequently produced and dissipated in the vicinity of the tip of the device. From previous reports, it is known that the increase in temperature in the vicinity of OFMHs is proportional to the optical power launched into the fiber [19]. However, information of the temperature distribution within the small area heated by these devices becomes relevant for applications involving biological tissue [17]. We therefore explored the feasibility to obtain the temperature maps in the surroundings of an OFMH using the PDMS-RhB membranes and LIFT.
For our experiments, we used OFMHs based on gold nanolayers, which have been previously reported to induce thermal damage in soft tissue [17]. The device was placed in physical contact with one edge of a PDMS-RhB membrane (see Fig. 4(a)). We first acquired 10 reference images at 25 °C, which were averaged and used to obtain the intensity reference ratio for each pixel. Subsequently, for each of the laser diode powers used in the test, 10 images were acquired and averaged and these were then used to estimate the intensity ratio for each pixel. The normalized intensity was then obtained upon dividing the intensity ratio by the reference ratio for the same pixels [3]. Finally, this normalized intensity was converted to temperature using the calibration curve for the membrane, thus yielding an image of the temperature distribution around the OFMH. A graphic depiction of this process is shown in Fig. 4(b). The images shown in Fig. 4(c) were obtained directly from the PDMS-RhB membranes for different powers of the laser diode; a decrease in fluorescence intensity is apparent in the vicinity of the OFMH, located at the bottom of the membrane as depicted in the images. Furthermore, as suggested by the dark areas in the images, the fluorescence intensity seems to be lower for higher powers of the laser diode.
The temperature maps obtained after processing the fluorescence images (Fig. 4(c)) are shown in Fig. 4(d). Clearly, the heat produced by the OFMH is dissipated around the OFMH tip; notice also that the registered temperature increases with the optical power, as previously reported for these devices [17,19]. Interestingly, the heated region seems to be smaller than one millimeter. For this particular case, the OFMH reached a maximum temperature of 78 °C and the total heated area was 750 µm x 650 µm. Using additional image processing, we can further estimate the temperature within a defined region of interest within the image. As an example, Fig. 4(e) shows the average temperature in a region close to the OFMH tip (80 µm X 80 µm) delimited by the red square in the temperature map shown in Fig. 4 Fig. 2(d)). urements. described e used for will also xample, is Fig. 2(a)). ures such mperature re able to mera. The the range olution of mperature embranes estructive med with H devices, the heat d into the than one f LIFT in ution.