We would like to thank Taarna Studemund and Hagen Wendler for taking photographs of the experimental setup as well as preparing them for figure publication. Furthermore, we would like to thank Anne Hildebrandt for the sample preparation and Sreedhar Unnikrishnakurup, Alexander Battig and Felix Fritzsche for proof-reading.

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proc. SPIE 9761. , (2016).</li><li>Thiel, E., Kreutzbruck, M., Ziegler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proc. WCNDT 2016. , 6 (2016).</li><li>Mandelis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diffusion-Wave Fields: mathematical methods and Green functions. , (2001).</li><li>Almond, D., Patel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Photothermal Science and Techniques. 10, (1996).</li><li>Salazar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+propagation+of+thermal+waves.">Energy propagation of thermal waves.</a> Eur. J. Phys. 27 (6), 1349-1355 (2006).</li><li>Bennett, C. A., Patty, R. 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The authors have nothing to disclose.

The presented protocol describes how to locate artificial subsurface defects oriented perpendicular to the surface. The main idea of the method is to create interfering thermal wave fields which interact with the subsurface defect. The most important steps are (i) to combine an SLM with a diode laser in order to create two alternating high-power illumination patterns at the sample surface; these patterns are photothermally converted into coherent thermal wave fields, (ii) to let them destructively interfere whilst intera...

The laser projected photothermal thermography (LPPT) method is used to locate subsurface defects that are embedded in the volume of the test specimen and oriented predominantly perpendicular to its surface.
The method uses the destructive interference of two anti-phased thermal wave fields of the same elongation and frequency as shown in Figure 1b. In isotropic defect-free materials, the thermal waves neutralize destructively (i.e. zero temperature oscillation) at the symmetry plane by coherent superposition. In case of a material with a subsurface defect, the method takes advantage of the interaction of the lateral (i.e. in-plane) components between the transient heat flow and this defect. This interaction can be measured in a recreated oscillating temperature elongation at the symmetry line on the sample surface. Now, the defect containing sample is scanned by the superposed thermal wave field and the level of temperature elongation is measured in relation to the sample position. Due to symmetry, the destructive interference condition is satisfied once again when the defect crosses the symmetry plane; this enables us to locate the defect very sensitively. Moreover, since the level of maximal disturbance of the destructive interference correlates with the depth of the defect, it is possible to determine its depth by analyzing the temperature scan1.
The LPPT can be assigned to the active thermography methodology, a well-established non-destructive method, where transient heating is actively generated and the resulting, also transient, temperature distribution is measured via a thermal IR camera. In general, the sensitivity of this methodology is limited to defects which are oriented essentially perpendicular to the transient heat flow. Moreover, since the governing transient heat conduction equation is a parabolic partial differential equation, the heat flow into the volume is strongly damped. As a consequence, the probing depth of the active thermography methodology is limited to a near surface region, usually in the millimeter-range. Two of the most common active thermography techniques are pulsed and lock-in thermography. They are fast due to planar optical surface illumination2, but lead to a transient heat flow perpendicular to the surface. Therefore, the sensitivity of these techniques is limited to defects predominantly oriented parallel (e.g. delaminations or voids) to the heated sample surface. An empirical rule for pulsed thermography states that &#34;the radius of the smallest detectable defect should be at least one to two times larger than its depth under the surface&#34;3. To increase the effective interaction area between a perpendicularly oriented defect (e.g. a crack) and the heat flow, the direction of the heat flow needs to be changed. Local excitation, by using a focused laser with a linear or circular spot for instance, generates a heat flow with an in-plane component that is able to effectively interact with the perpendicular defect4,5,6,7.
In the presented method, we also use the lateral heat flow components to detect subsurface defects, but we use the fact that thermal waves can be superposed, whereas defects, especially vertically oriented ones, disturb this superposition. In this way, the presented method resembles a reference-free, symmetric and very sensitive method, as it is possible to detect artificial subsurface defects at a width/depth ratio of far below one8,9. Until now, it was difficult to create two anti-phased thermal wave fields supplying sufficient energy. We achieved this by coupling a spatial light modulator (SLM) to a high-power diode laser, which enabled us to merge the high optical power of the laser system with the spatial and temporal resolution of the SLM (see Figure 2) into a high-power projector. The thermal wave fields are now created by photothermal conversion of two anti-phased sinusoidally modulated line patterns via the pixel brightness of the projected image (see Figure 2, Figure 1a). This leads to structured heating of the sample surface and results in well-defined destructively interfering thermal wave fields. In order to find a subsurface defect, the disturbance of the destructive inference is measured as a temperature oscillation at the surface using an IR camera.
The term thermal wave, is controversially discussed because thermal waves do not transport energy due to the diffusive character of the heat propagation. Still, there is wave-like behavior when heating periodically, allowing us to use similarities between real waves and diffusion processes10,11,12. Thus, a thermal wave can be understood as highly damped in the propagation direction but periodic over time (Figure 1b). The characteristic thermal diffusion length <img alt="Equation 1" src="/files/ftp_upload/55733/55733eq1.jpg" /> is hereby described by its material properties (thermal conductivity k, heat capacity cp and density &#961;), and the excitation frequency &#402;. Although the thermal wave is decaying strongly, its wave nature can be applied to gain insight into the properties of the sample. The first application of thermal wave interference was used to determine the thickness of layers. In contrast to our method, the interference effect was used in the depth dimension (i.e. perpendicular to the surface)13. Extending the idea of interference to a second dimension by splitting up a laser beam, thermal wave interference was used to size subsurface defects14. Still this method was applied in transmission configuration, which means that it was limited by the penetration depth of the thermal wave. Furthermore, because only one laser source has been used, this method applies constructive interference, meaning that a defect-free reference is needed. Apart from the idea of using thermal wave interference, the first technical approach to spatially and temporally controlled heating was performed by Holtmann et al. using an unmodified liquid crystal display (LCD) projector with the built-in light source, which was severely limited in its optical output power15. Further approaches by Pribe and Ravichandran aimed at increasing the optical output power by also coupling a laser to a SLM16,17.
The protocol presented herein describes how to apply the LPPT method to locate subsurface defects oriented perpendicularly to the surface of steel samples. The method is at an early stage, yet powerful enough to validate the proposed approach; however, it is still limited in terms of the achievable optical output power of the experimental setup. Since the increase of the optical output power remains a challenge, the presented method is applied to coated steel containing artificial electrically discharge machined notches. Nevertheless, the most important and most critical steps of the protocol, generating a homogeneous structured illumination, meeting prerequisites for destructive thermal wave interference, and locating the defect, still hold for more demanding defects as well. Since the governing quantity is the thermal diffusion length &#956;, the LPPT method can be applied to numerous different materials as well.
<img alt="Figure 1" src="/files/ftp_upload/55733/55733fig1.jpg" /> 
Figure 1: Principle of destructive interference effect. (a) Schematic of the illumination pattern used during experiments. The sample is spatially and temporally heated by two periodically illuminated patterns with a phase shift of &#960;. The dashed line represents the symmetry line between both patterns. This line will be used for evaluation as a &#34;depletion line&#34;. (b) Diagram of the spatially and temporally resolved alternating thermal result as calculated from the analytical solution of the thermal heat conduction equation. It shows the responding thermal waves to the illumination of (a) with an irradiance of the two patterns with Popt1 = 1.5 W sin(2&#960; 0.125 Hz t) + 1.5 W and Popt2 = 1.5 W sin(2&#960; 0.125 Hz t + &#960;) + 1.5 W for constructional steel &#961; = 7,850 kg/m3, cp = 461 J/(kg&#183;K), k = 54 W/(m&#183;K). The temperature profile at the dashed line shows no thermal oscillation for homogeneous, isotropic material. <a href="http://cloudflare.jove.com/files/ftp_upload/55733/55733fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55733/55733fig2.jpg" /> 
Figure 2: Schematic of the measurement principle of structured heating used in active thermography. A Gaussian beam homogenized to a top hat profile is applied to a Spatial Light Modulator (SLM). The SLM resolves the beam spatially by its switchable elements and temporally by its switching speed. Each element represents an SLM pixel. In this experiment, the SLM is a digital micro mirror device (DMD). By modulating the pixel brightness A with a time deterministic control software, the sample surface is heated in a structured way. In case of the presented experiment, we modulate two anti-phased lines (phases: &#966; = 0, &#960;), which are the origin of coherently interfering thermal wave fields at the angular frequency &#969;. The wave fields interact with the sample&#39;s inner structure also influencing the temperature field at the surface. This is measured via its thermal radiation by a mid-wave infrared camera. <a href="http://cloudflare.jove.com/files/ftp_upload/55733/55733fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>500 W diode laser system, 940 nm</td><td>Laserline</td><td>LDM 500 - 20</td><td>Pilot laser class 2 @ 650&amp;nbsp;nm, diode laser is a class 4 laser system --&gt; special laboratory needed</td></tr><tr><td>Laser control box</td><td>Laserline</td><td>Laser control box LDM</td><td>Add on to the laser system, used to switch electronically, laser threshold, shutter, laser on 0 V..5 V TTL</td></tr><tr><td>Control box scanner</td><td>Laserline</td><td></td><td>Add on to the laser system, used to adjust the optical output power via analog signal from 0&amp;nbsp;V..10 V</td></tr><tr><td>Fiber Laser Mount 2&quot;, f = 80 mm</td><td>Laserline</td><td></td><td>Add on to the laser system</td></tr><tr><td>Multifunction Data Aquisition (DAQ) Device + BNC Terminal</td><td>National Instruments</td><td>NI-USB 6251</td><td>The DAQ card is used to trigger the IR camera,&amp;nbsp; the DLP Light Commander 5500, control Laser and diode PDA 36A</td></tr><tr><td>Standard - PC&amp;nbsp;</td><td></td><td></td><td>Control PC - graphic card for two screens, at least 4 x USB, Windows based</td></tr><tr><td>BNC cabel</td><td></td><td></td><td>Standard cable</td></tr><tr><td>HDMI cable</td><td></td><td></td><td>Standard cable</td></tr><tr><td>Micro USB to USB cable</td><td></td><td></td><td>Standard cable</td></tr><tr><td>LabVIEW 2013 SP1 Development System</td><td>National Instruments</td><td></td><td>Development environment for device control</td></tr><tr><td>LPPT control software</td><td>BAM</td><td></td><td>part of the LPPT software package by LabVIEW 2013 SP1</td></tr><tr><td>LPPT intensity&amp;nbsp; software</td><td>BAM</td><td></td><td>part of the LPPT software package by LabVIEW 2013 SP1</td></tr><tr><td>LPPT laser control software</td><td>BAM</td><td></td><td>part of the LPPT software package by LabVIEW 2013 SP1</td></tr><tr><td>Matlab 2016b</td><td>MathWorks</td><td></td><td>Postprocessing of the measurement data</td></tr><tr><td>LPPT postprocessing software</td><td>BAM</td><td></td><td>Postprocessing of the measurement data</td></tr><tr><td>IR camera control PC</td><td>InfraTec</td><td></td><td>Control PC is supplied by camera distributor</td></tr><tr><td>IR camera control software</td><td>InfraTec</td><td>Irbis 3 Professional</td><td></td></tr><tr><td>InfraTec SDK</td><td>InfraTec</td><td></td><td>Dynamic Link Library as interface between the native data aquisition format of Infratec and Matlab</td></tr><tr><td>IR camera</td><td>InfraTec</td><td>Image IR 8300</td><td>640 x 512, cooled InSb detector, wavelength 2&amp;nbsp;&amp;micro;m..5.7 &amp;micro;m, noise = 20&amp;nbsp;mK + accessories (LAN cable, Digital in/out cable, space ring, power supply, case)</td></tr><tr><td>Tripod</td><td>Manfrotto</td><td>161MK2B</td><td></td></tr><tr><td>IR camera mount</td><td>Manfrotto</td><td>405</td><td></td></tr><tr><td>Projector development kit (PDK) for digital light processing (DLP) technology (DLP Light Commander 5500)</td><td>Logic PD</td><td>DLP-LC-DLP5500-10R</td><td>DLP5500 Digital Micromirror Device from Texas Instruments included , light engine and case need to be disassembed</td></tr><tr><td>PDK control software</td><td>Logic PD</td><td></td><td>Included when delivered, DLP Light Commander control software</td></tr><tr><td>Mechanical platform for the PDK</td><td>BAM</td><td></td><td>Self made (140 x 230 x 420) mm&lt;sup&gt;3&lt;/sup&gt;</td></tr><tr><td>Power meter control unit</td><td>Ophir</td><td>Vega</td><td>USB Interface</td></tr><tr><td>30 W power meter head&amp;nbsp;</td><td>Ophir</td><td>30(150)A-LP1-18</td><td>Power meter head to determine Transmission of the projector system</td></tr><tr><td>500 W power meter head</td><td>Ophir</td><td>FL500A</td><td>Power meter for process supervision</td></tr><tr><td>Motion controller</td><td>Newport</td><td>ESP301</td><td>with USB Interface</td></tr><tr><td>Translation stage</td><td>Newport</td><td>M-ILS200CC</td><td>Connected to ESP301</td></tr><tr><td>Photodiode with amplifier</td><td>Thorlabs</td><td>PDA 36A-EC</td><td>1&quot; mount</td></tr><tr><td>Reflective filter ND1</td><td>Thorlabs</td><td>ND10A</td><td>to be mounted to the PDA 36A</td></tr><tr><td>Pinhole 1&quot;</td><td>Thorlabs</td><td>P1000S</td><td>to be mounted to the PDA 36A</td></tr><tr><td>Optical aluminium breadboard&amp;nbsp;</td><td>Thorlabs</td><td>MB60120/M</td><td>(1,200 mm x 900 mm) base</td></tr><tr><td>Plano Convex Lens f = 200 mm</td><td>Thorlabs</td><td>LA1979-B</td><td>Coated for IR, first telescope lens</td></tr><tr><td>Plano Convex Lens f = 75 mm</td><td>Thorlabs</td><td>LA1145-B</td><td>Coated for IR, second telescope lens</td></tr><tr><td>xy-translation stage</td><td>Newport</td><td>M401</td><td>Used for adjusting the telecope</td></tr><tr><td>Beamsampler</td><td>Thorlabs</td><td>BSF20-B&amp;nbsp;</td><td>Splits the optical output, used to reduce the optical input for the projector system</td></tr><tr><td>Mirror</td><td>Thorlabs</td><td>BB2-E03</td><td>Mirror for coupling the beam to the DLP Light Commander</td></tr><tr><td>Heavy duty lab jack</td><td>Thorlabs</td><td>L490</td><td>Used for the fiber mount and on top of the linear stage to position the sample (2x)</td></tr><tr><td>PDK-objective&amp;nbsp;</td><td>Nikon</td><td>Nikon AF Nikkor 50 mm 1:1:8:D&amp;nbsp;</td><td>Objective for DLP Light Commander, 50 mm</td></tr><tr><td>Plano Convex Lens f = 100 mm</td><td>Thorlabs</td><td>LA1050 -B</td><td>Lens is attached to the Nikon Objective</td></tr><tr><td>Bi-Convex Lens f = 60&amp;nbsp;mm</td><td>Thorlabs</td><td>LB1723 -B</td><td>Lens to be attached to the Nikon objective in order to determine the optical transmission with the 30 W measurement head</td></tr><tr><td>Square protected gold mirror</td><td>Thorlabs</td><td>PFSQ20-03-M01</td><td></td></tr><tr><td>High power IR sensor card</td><td>Newport</td><td>F-IRC-HP-M</td><td>Sensor card to check the optical pathway</td></tr><tr><td>2&quot; crosshairs</td><td>BAM</td><td></td><td>Self-made</td></tr><tr><td>1&quot; crosshairs</td><td>BAM</td><td></td><td>Self-made</td></tr><tr><td>Bullseye level</td><td>Thorlabs</td><td>LCL01</td><td></td></tr><tr><td>Translation Stage</td><td>Newport</td><td>M-UMR8.25</td><td>Used for measuring the beam profile</td></tr><tr><td>Micrometer screw</td><td>Newport</td><td>DM17-25</td><td>Used with translation stage M-UMR8.25</td></tr><tr><td>Mounted Zero Aperture Iris</td><td>Thorlabs</td><td>ID75Z/M</td><td>used to check the optical pathway</td></tr><tr><td>Bases and Post Holders Essentials Kit, Metric and Universal Components</td><td>Thorlabs</td><td>ESK01/M</td><td>Basis</td></tr><tr><td>Posts &amp;amp; Accessories Essentials Kit, Metric and Universal Components</td><td>Thorlabs</td><td>ESK03/M</td><td></td></tr><tr><td>M6 Cap Screw and Hardware Kit</td><td>Thorlabs</td><td>HW-KIT2/M</td><td></td></tr><tr><td>Construction Rails</td><td>Thorlabs</td><td>XE25L700/M</td><td></td></tr><tr><td>1&quot; Construction Cube</td><td>Thorlabs</td><td>RM1G</td><td>Used to mount construction rails</td></tr><tr><td>Electrical discharge machining</td><td>Sodick</td><td>AG60L</td><td>www.sodick.de</td></tr><tr><td>St37 block of steel&lt;br /&gt;(100 x 100 x 40) mm&lt;sup&gt;3&lt;/sup&gt;</td><td>BAM</td><td></td><td>self-made, hidden defect with remaining wall thicknesses of 0.25&amp;nbsp;mm, 0.5 mm, 0.70 mm, 1.25&amp;nbsp;mm (shown in &lt;strong&gt;Figure 5&lt;/strong&gt;)</td></tr><tr><td>St37 block of steel&lt;br /&gt;(100 x 100 x 40) mm</td><td>BAM</td><td></td><td>self-made, hidden defect with remaining wall thicknesses of 1&amp;nbsp;mm, 1.5&amp;nbsp;mm, 1.75 mm, 2 mm (shown in &lt;strong&gt;Figure 5&lt;/strong&gt;)</td></tr><tr><td>Graphite spray</td><td>CRC Industries Europe NV</td><td>GRAPHIT 33</td><td>Ref. 20760, 200 mL aerosol (Kontakt-Chemie)</td></tr><tr><td>Protective tape</td><td>Tesa</td><td>tesakrepp 4348</td><td>used to protect the hidden defects while coating</td></tr></tbody></table>

subsurface defect localization by structured heating using laser projected photothermal thermography

The presented method is used to locate subsurface defects oriented perpendicularly to the surface. To achieve this, we create destructively interfering thermal wave fields that are disturbed by the defect. This effect is measured and used to locate the defect. We form the destructively interfering wave fields by using a modified projector. The original light engine of the projector is replaced with a fiber-coupled high-power diode laser. Its beam is shaped and aligned to the projector&#39;s spatial light modulator and optimized for optimal optical throughput and homogeneous projection by first characterizing the beam profile, and, second, correcting it mechanically and numerically. A high-performance infrared (IR) camera is set up according to the tight geometrical situation (including corrections of the geometrical image distortions) and the requirement to detect weak temperature oscillations at the sample surface. Data acquisition can be performed once a synchronization between the individual thermal wave field sources, the scanning stage, and the IR camera is established by using a dedicated experimental setup which needs to be tuned to the specific material being investigated. During data post-processing, the relevant information on the presence of a defect below the surface of the sample is extracted. It is retrieved from the oscillating part of the acquired thermal radiation coming from the so-called depletion line of the sample surface. The exact location of the defect is deduced from the analysis of the spatial-temporal shape of these oscillations in a final step. The method is reference-free and very sensitive to changes within the thermal wave field. So far, the method has been tested with steel samples but is applicable to different materials as well, in particular to temperature sensitive materials.

Following the protocol, side 1 of the steel sample with a subsurface defect at a depth of 0.25 mm was chosen to generate representative results. The defect was initially positioned approximately at the center of the illuminated area. The sample was then moved from -5 mm to 5 mm via the linear stage at a speed of 0.05 mm/s. Using these parameters, Figure 11a shows the scan data after extracting them from the depletion line. At this stage, the success of the experiment can ...

Watch this Scientific Journal Video about Subsurface Defect Localization by Structured Heating Using Laser Projected Photothermal Thermography at JoVE.com

Subsurface Defect Localization by Structured Heating Using Laser Projected Photothermal Thermography

This method aims at locating vertical subsurface defects. Here, we couple a laser with a spatial light modulator and trigger its video input to heat a sample surface deterministically with two anti-phased modulated lines while acquiring highly resolved thermal images. The defect position is retrieved from evaluating thermal wave interference minima.

The presented method is used to locate subsurface defects oriented perpendicularly to the surface. To achieve this, we create destructively interfering ...

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JoVE Journal

Engineering

11.1K Views.  Bundesanstalt für Materialforschung und -prüfung (BAM).  Deze methode streeft naar het lokaliseren van verticale ondergrondse defecten. Hier koppelen we een laser aan met een ruimtelijke lichtmodulator en trekken de video-ingang om deterministisch een monsteroppervlak te verwarmen met twee antifase gemoduleerde lijnen, met het verwerven van sterk opgeloste thermische beelden. De defectpositie wordt opgehaald om de thermische golfinterferentie minima te evalueren. 

Video: Subsurface Defect Localization by Structured Heating Using Laser Projected Photothermal Thermography

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be streamlined by heat treatment (blanching). A blanching apparatus can be designed more precisely if the thermal properties of the leaves are known in detail, i.e., the specific heat capacity and thermal conductivity. The measurement of these properties is time consuming and labor intensive, and usually requires invasive methods that contact the sample directly. This can reduce the product yield and may be incompatible with containment requirements, e.g., in the context of good manufacturing practice. To address these issues, a non-invasive, contact-free method was developed that determines the specific heat capacity and thermal conductivity of an intact plant leaf in about one minute. The method involves the application of a short laser pulse of defined length and intensity to a small area of the leaf sample, causing a temperature increase that is measured using a near infrared sensor. The temperature increase is combined with known leaf properties (thickness and density) to determine the specific heat capacity. The thermal conductivity is then calculated based on the profile of the subsequent temperature decline, taking thermal radiation and convective heat transfer into account. The associated calculations and critical aspects of sample handling are discussed.

A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample.

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be ...

Biochemistry

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

Medicine

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Genetics

High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient optical micro-imaging technique which can be used to map and quantify such behavior. Europium thenoyltrifluoroacetonate (EuTFC) has a 612 nm luminescence line whose activation efficiency drops strongly with increasing temperature, due to T-dependent interactions between the Eu3+ ion and the organic chelating compound. This material may be readily coated on to a sample surface by thermal sublimation in vacuum. When the coating is excited with ultraviolet light (337 nm) an optical micro-image of the 612 nm luminescent response can be converted directly into a map of the sample surface temperature. This technique offers spatial resolution limited only by the microscope optics (about 1 micron) and time resolution limited by the speed of the camera employed. It offers the additional advantages of only requiring comparatively simple and non-specialized equipment, and giving a quantitative probe of sample temperature.

Europium thenoyltrifluoroacetonate (EuTFC) has an optical luminescence line at 612 nm, whose activation efficiency decreases strongly with temperature. If a sample coated with a thin film of this material is micro-imaged, the 612 nm luminescent response intensity may be converted into a direct map of sample surface temperature.

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Bioengineering

Plasmonic Photothermal Cancer Therapy: Nanoparticle-embedded Tumor-tissue-mimicking Phantoms for Visualizing Photothermal Temperature Distribution

Plasmonic photothermal therapy (PPTT), an emerging cancer treatment, involves delivering nanoparticles (NPs) to a tumor, followed by near-infrared (NIR) irradiation to generate localized heat that destroys cancer cells. Before administering PPTT, the therapeutic parameters -- NP concentration, irradiation intensity, and duration -- need to be estimated. For this, numerical simulations are performed. However, to ensure robust computation, these simulations must be validated through photothermal experiments on tumor-tissue-mimicking phantoms replicating the optical properties of tumor tissue. For PPTT, therapeutic parameters are governed by the scattering and absorption of incident radiation by the tissue and NPs. Therefore, validation experiments can be conducted on phantoms mimicking the reduced scattering coefficient (&#181;s&#39;) and absorption coefficient (&#181;a) of the target tumor/tissue.
Specifically, this protocol provides instructions for preparing phantoms mimicking &#181;s&#39; and &#181;a of breast tumor injected with gold nanorods, surrounded by normal breast tissue. The protocol also details NIR irradiation, temperature monitoring, and validation of numerical results by comparing spatiotemporal temperatures with those measured using thermocouples. The protocols presented in this study facilitated the preparation of hydrogel-based cylindrical breast tumor-tissue phantoms with dimensions (&#981;40 x 12 mm) and a central tumor region (&#981;20 x 6 mm), comprising 1% agarose as the base matrix and intralipid as the scattering constituent and tumor region embedded with gold nanorods at 25 &#181;g/mL concentration. Representative results from a case study illustrate the application of fabricated phantoms for validating numerical simulations for PPTT. The study concludes that the demonstrated protocols are valuable for conducting photothermal experiments aimed at optimizing and planning therapeutic parameters prior to in vivo experiments and validating numerical simulations for PPTT.

This article presents the protocol for preparing tumor-tissue phantoms that replicate optical properties for plasmonic photothermal therapy. It details phantom preparation, photothermal evaluations, and validation of the developed numerical model based on photothermal temperature measurements for assessing therapeutic parameters, offering an ethical, cost-effective alternative to in vivo studies for preliminary testing.

Plasmonic photothermal therapy (PPTT), an emerging cancer treatment, involves delivering nanoparticles (NPs) to a tumor, followed by near-infrared (NIR) ...

Cancer Research

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Flapping Soft Fin Deformation Modeling using Planar Laser-Induced Fluorescence Imaging

Propulsive mechanisms inspired by the fins of various fish species have been increasingly researched, given their potential for improved maneuvering and stealth capabilities in unmanned vehicle systems. Soft materials used in the membranes of these fin mechanisms have proven effective at increasing thrust and efficiency compared with more rigid structures, but it is essential to measure and model the deformations in these soft membranes accurately. This study presents a workflow for characterizing the time-dependent shape deformation of flexible underwater flapping fins using planar laser-induced fluorescence (PLIF). Pigmented polydimethylsiloxane fin membranes with varying stiffnesses (0.38 MPa and 0.82 MPa) are fabricated and mounted to an assembly for actuation in two degrees of freedom: pitch and roll. PLIF images are acquired across a range of spanwise planes, processed to obtain fin deformation profiles, and combined to reconstruct time-varying 3D deformed fin shapes. The data are then used to provide high-fidelity validation for fluid-structure interaction simulations and improve the understanding of the performance of these complex propulsion systems.

The present protocol involves the measurement and characterization of 3D shape deformation in underwater flapping fins built with polydimethylsiloxane (PDMS) materials. Accurate reconstruction of these deformations is essential for understanding the propulsive performance of compliant flapping fins.

Propulsive mechanisms inspired by the fins of various fish species have been increasingly researched, given their potential for improved maneuvering and ...

Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 &#181;m is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase.
To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time.
The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.

An original experimental setup for heating cells in a culture dish using 1.94 &#181;m continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser ...

Biology

Laser Induced Thermoplasmonic Puncture for the Study of Plasma Membrane Repair

A Thermoplasmonic Approach for Investigating Plasma Membrane Repair in Living Cells and Model Membranes

The cell membrane is crucial for cell survival, and ensuring its integrity is essential as the cell experiences injuries throughout its entire life cycle. To prevent damage to the membrane, cells have developed efficient plasma membrane repair mechanisms. These&#160;repair mechanisms can be studied by combining confocal microscopy and nanoscale thermoplasmonics to identify and investigate the role of key proteins, such as annexins, involved in surface repair in living cells and membrane model systems.
The puncturing method employs a laser to induce highly localized heating upon nanoparticle irradiation. The use of near-infrared light minimizes phototoxicity in the biological sample, while the majority of the absorption takes place in the near-infrared resonant plasmonic nanoparticle. This thermoplasmonic method has been exploited for potential photothermal and biophysical research to enhance the understanding of intracellular mechanisms and cellular responses through vesicle and cell fusion studies. The approach has shown to be complementary to existing methods for membrane disruption, such as mechanically, chemically, or optically induced injuries, and provides a high level of control by inflicting extremely localized injuries. The extent of the injury is limited to the vicinity of the spherical nanoparticle, and no detrimental damage occurs along the beam path as opposed to pulsed lasers using different wavelengths. Despite certain limitations, such as the formation of nanobubbles, the thermoplasmonic method offers a unique tool for investigating cellular responses in plasma membrane repair in an almost native environment without compromising cell viability.
When integrated with confocal microscopy, the puncturing method can provide a mechanistic understanding of membrane dynamics in model membrane systems as well as quantitative information on protein responses to membrane damage, including protein recruitment and their biophysical function. Overall, the application of this method to reduced model systems can enhance our understanding of the intricate plasma membrane repair machinery in living cells.

Author Spotlight: Exploring Plasma Membrane Repair Mechanisms with Innovative Thermoplasmonic Puncturing 

The thermoplasmonic puncture method integrates confocal microscopy, optical tweezers, and gold nanoparticles to study protein responses during plasma membrane repair in cells and giant unilamellar vesicles. The technique enables rapid and localized membrane puncture, allowing the identification of key proteins and their functional roles in the intricate plasma membrane repair machinery.

The cell membrane is crucial for cell survival, and ensuring its integrity is essential as the cell experiences injuries throughout its entire life cycle. ...

Subsurface Defect Localization by Structured Heating Using Laser Projected Photothermal Thermography

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