Multi-point calorimeter using distributed fiber Bragg gratings for small field dosimetry in radiotherapy.

BACKGROUND: The interest of using fiber Bragg gratings (FBGs) dosimeters in radiotherapy (RT) lies in their (i) microliter detection volume, (ii) customizable spatial resolution, (iii) multi-point dose measurement, (iv) real-time data acquisition and (v) insensitivity to Cherenkov light. These characteristics could prove very useful for characterizing dose distributions of small and nonstandard fields with high spatial resolution. PURPOSE: We developed a multi-point FBGs dosimeter customized for small field RT dosimetry with a spatial resolution of ∼ $\sim$ 1 mm. METHODS: The 3 cm-long multi-point dosimeter is made by embedding a 80 µ m $\umu{\rm {m}}$ silica fiber containing an array of thirty (30) co-located ∼ $\sim$ 1 mm-long fs-written FBGs inside a plastic cylinder with an UV curing optical adhesive. With its higher thermal expansion coefficient, the plastic cylinder increases the sensitivity of the dosimeter by stretching the fiber containing the FBGs when the temperature rises slightly due to radiation energy deposition. Irradiations (2000 MU at 600 MU/min) were performed with a Varian TrueBeam linear accelerator. RESULTS: The dose profile of a 2  × $ \times$ 2 cm 2 $^{2}$ 6 MV beam was measured with a mean relative difference of 1.8% (excluding the penumbra region). The measured output factors for a 6 MV beam are in general agreement with the expected values within the experimental uncertainty (except for the 2  × $\,\times $ 2 cm 2 $^{2}$ field). The detector response to different energy of photon and electron beams is within 5% of the mean response ( 0.068 ± 0.002 $0.068\pm 0.002$  pm/Gy). The calorimeter's post-irradiation thermal decay is in agreement with the theory. CONCLUSIONS: An energy-independent small field calorimeter that allows dose profile and output factor measurements for RT using FBGs was developed, which, to our knowledge, has never been done before. This type of detector could prove really useful for small field dosimetry, but also potentially for MRI-LINAC since FBGs are insensitive to magnetic fields and for FLASH since FBGs have been used to measure doses up to 100 kGy.

A hybrid strategy combining solution NMR spectroscopy and isothermal titration calorimetry to characterize protein-nanodisc interaction.

NMR is a powerful tool for characterizing intermolecular interactions at atomic resolution. However, the nature of the complex interactions of membrane-binding proteins makes it difficult to elucidate the interaction mechanisms. Here, we demonstrated that structural and thermodynamic analyses using solution NMR spectroscopy and isothermal titration calorimetry (ITC) can clearly detect a specific interaction between the pleckstrin homology (PH) domain of ceramide transport protein (CERT) and phosphatidylinositol 4-monophosphate (PI4P) embedded in the lipid nanodisc, and distinguish the specific interaction from nonspecific interactions with the bulk surface of the lipid nanodisc. This NMR-ITC hybrid strategy provides detailed characterization of protein-lipid membrane interactions.

Applications of isothermal titration calorimetry in pure and applied research from 2016 to 2020.

The last 5 years have seen a series of advances in the application of isothermal titration microcalorimetry (ITC) and interpretation of ITC data. ITC has played an invaluable role in understanding multiprotein complex formation including proteolysis-targeting chimeras (PROTACS), and mitochondrial autophagy receptor Nix interaction with LC3 and GABARAP. It has also helped elucidate complex allosteric communication in protein complexes like trp RNA-binding attenuation protein (TRAP) complex. Advances in kinetics analysis have enabled the calculation of kinetic rate constants from pre-existing ITC data sets. Diverse strategies have also been developed to study enzyme kinetics and enzyme-inhibitor interactions. ITC has also been applied to study small molecule solvent and solute interactions involved in extraction, separation, and purification applications including liquid-liquid separation and extractive distillation. Diverse applications of ITC have been developed from the analysis of protein instability at different temperatures, determination of enzyme kinetics in suspensions of living cells to the adsorption of uremic toxins from aqueous streams.

Isothermal Titration Calorimetry.

Calorimetry is a classical biophysical method that by definition measures heat. In isothermal titration calorimetry (ITC), the heat is the result of titrating interacting components together and allows direct determination of the thermodynamics for this process. The measured heat reflects the enthalpy change (ΔH), and the prospect of determining this in biological systems where high-resolution structural information is available has led to the possibility of rational thermodynamics-guided design of ligands. Although there are limitations to this approach due to the participation of solvent in the thermodynamics, ITC has become an established technique in many labs providing a valuable tool with which to quantify protein-protein interactions. With careful use, ITC can also provide additional insights into the binding process or be used in increasingly complex systems and where interaction is coupled to other molecular events.

Helium radiography with a digital tracking calorimeter-a Monte Carlo study for secondary track rejection.

Radiation therapy using protons and heavier ions is a fast-growing therapeutic option for cancer patients. A clinical system for particle imaging in particle therapy would enable online patient position verification, estimation of the dose deposition through range monitoring and a reduction of uncertainties in the calculation of the relative stopping power of the patient. Several prototype imaging modalities offer radiography and computed tomography using protons and heavy ions. A Digital Tracking Calorimeter (DTC), currently under development, has been proposed as one such detector. In the DTC 43 longitudinal layers of laterally stacked ALPIDE CMOS monolithic active pixel sensor chips are able to reconstruct a large number of simultaneously recorded proton tracks. In this study, we explored the capability of the DTC for helium imaging which offers favorable spatial resolution over proton imaging. Helium ions exhibit a larger cross section for inelastic nuclear interactions, increasing the number of produced secondaries in the imaged object and in the detector itself. To that end, a filtering process able to remove a large fraction of the secondaries was identified, and the track reconstruction process was adapted for helium ions. By filtering on the energy loss along the tracks, on the incoming angle and on the particle ranges, 97.5% of the secondaries were removed. After passing through 16 cm water, 50.0% of the primary helium ions survived; after the proposed filtering 42.4% of the primaries remained; finally after subsequent image reconstruction 31% of the primaries remained. Helium track reconstruction leads to more track matching errors compared to protons due to the increased available focus strength of the helium beam. In a head phantom radiograph, the Water Equivalent Path Length error envelope was 1.0 mm for helium and 1.1 mm for protons. This accuracy is expected to be sufficient for helium imaging for pre-treatment verification purposes.

Operating a graphite calorimeter in quasi-isothermal mode under high-energy x-ray beams.

In this study, we developed a semi-active method to run a graphite calorimeter in the quasi-isothermal mode under high-energy x-ray beams. The rate of energy imparted by the beam during irradiation was compensated mainly by removing the electrical heating power based on the pre-calculation and in part by an active automated algorithm, as well, while the temperature of the calorimeter core was kept constant. Irradiations were performed under the linear electron accelerator x-ray beams at 6, 8, 10, 15, and 18 MV. A simple model was applied to analyze the results. The energy imparted to the core was determined with an uncertainty level of 0.2%-0.3%, and the results were reaffirmed by comparing it with that obtained by the quasi-adiabatic mode. The normalized root-mean-square deviation to the mean from the quasi-adiabatic mode was 0.11%, and the associated uncertainty was 0.16% taking into account the correlation of the uncertainty components. This level of agreement showed that the present method is practical for the high-energy x-ray dosimetry.

A Method to Determine Human Skin Heat Capacity Using a Non-Invasive Calorimetric Sensor.

A calorimetric sensor has been designed to measure the heat flow dissipated by a 2 x 2 cm2 skin surface. In this work, a non-invasive method is proposed to determine the heat capacity and thermal conductance of the area of skin where the measurement is made. The method consists of programming a linear variation of the temperature of the sensor thermostat during its application to the skin. The sensor is modelled as a two-inputs and two-outputs system. The inputs are 1) the power dissipated by the skin and transmitted by conduction to the sensor, and 2) the power dissipated in the sensor thermostat to maintain the programmed temperature. The outputs are 1) the calorimetric signal and 2) the thermostat temperature. The proposed method consists of a sensor modelling that allows the heat capacity of the element where dissipation takes place (the skin) to be identified, and the transfer functions (TF) that link the inputs and outputs are constructed from its value. These TFs allow the determination of the heat flow dissipated by the surface of the human body as a function of the temperature of the sensor thermostat. Furthermore, as this variation in heat flow is linear, we define and determine an equivalent thermal resistance of the skin in the measured area. The method is validated with a simulation and with experimental measurements on the surface of the human body.

Using a small-core graphite calorimeter for dosimetry and scintillator quenching corrections in a therapeutic proton beam.

Organic plastic scintillation detectors (PSDs) are known to produce less light per absorbed dose in highly dense radiations in comparison with e.g. 60Co gamma beams. This so-called ionization density quenching can be experimentally determined by comparison of the scintillator output with the absorbed dose established with a reference detector. The hypothesis of this work was that a newly developed small-core graphite calorimeter (core size: ø5mm × 7mm) can be used as reference for such measurements. The potential benefit of a calorimetric reference would be to have a robust and accurate reference with well-understood dosimetry properties even in high-intensity FLASH beams. As a first step, the hypothesis was tested by comparing previously established quenching parameter estimates for the BCF-60 scintillating material with data obtained with the new instrument at different depths along the central axis of a 170 MeV scanned proton beam. After the calorimetric measurements, scintillator measurements were acquired under equivalent conditions by positioning the PSD in a replica graphite core nominally identical to the core used for calorimetry. To experimentally document details of the irradiations, the spot width was mapped along the central beam axis using a new technique based on a PSD and a time-to-distance conversion procedure. Analysing the proton data in the framework of the Birks model, the graphite calorimeter gave a [Formula: see text] quenching parameter for BCF-60 in agreement with literature values. The consistency between the calorimetric results and the other sources of information supports the validity of the new method, and we therefore aim to apply it for characterization of other detectors in more intense beams where ionometry cannot serve as reference.

Sub-nanowatt microfluidic single-cell calorimetry.

Non-invasive and label-free calorimetry could become a disruptive technique to study single cell metabolic heat production without altering the cell behavior, but it is currently limited by insufficient sensitivity. Here, we demonstrate microfluidic single-cell calorimetry with 0.2-nW sensitivity, representing more than ten-fold enhancement over previous record, which is enabled by (i) a low-noise thermometry platform with ultralow long-term (10-h) temperature noise (80 µK) and (ii) a microfluidic channel-in-vacuum design allowing cell flow and nutrient delivery while maintaining a low thermal conductance of 2.5 µW K-1. Using Tetrahymena thermophila as an example, we demonstrate on-chip single-cell calorimetry measurement with metabolic heat rates ranging from 1 to 4 nW, which are found to correlate well with the cell size. Finally, we perform real-time monitoring of metabolic rate stimulation by introducing a mitochondrial uncoupling agent to the microchannel, enabling determination of the spare respiratory capacity of the cells.

Sub-nanowatt resolution direct calorimetry for probing real-time metabolic activity of individual C. elegans worms.

Calorimetry has been widely used in metabolic studies, but direct measurements from individual small biological model organisms such as C. elegans or isolated single cells have been limited by poor sensitivity of existing techniques and difficulties in resolving very small heat outputs. Here, by careful thermal engineering, we developed a robust, highly sensitive and bio-compatible calorimetric platform that features a resolution of ~270 pW-more than a 500-fold improvement over the most sensitive calorimeter previously used for measuring the metabolic heat output of C. elegans. Using this calorimeter, we demonstrate time-resolved metabolic measurements of single C. elegans worms from larval to adult stages. Further, we show that the metabolic output is significantly lower in long-lived C. elegans daf-2 mutants. These demonstrations clearly highlight the broad potential of this tool for studying the role of metabolism in disease, development and aging of small model organisms and single cells.

Absolute dosimetry of a 1.5 T MR-guided accelerator-based high-energy photon beam in water and solid phantoms using Aerrow.

PURPOSE: In this work, the fabrication, operation, and evaluation of a probe-format graphite calorimeter - herein referred to as Aerrow - as an absolute clinical dosimeter of high-energy photon beams while in the presence of a B = 1.5 T magnetic field is described. Comparable to a cylindrical ionization chamber (IC) in terms of utility and usability, Aerrow has been developed for the purpose of accurately measuring absorbed dose to water in the clinic with a minimum disruption to the existing clinical workflow. To our knowledge, this is the first reported application of graphite calorimetry to magnetic resonance imaging (MRI)-guided radiotherapy. METHODS: Based on a previously numerically optimized and experimentally validated design, an Aerrow prototype capable of isothermal operation was constructed in-house. Graphite-to-water dose conversions as well as magnetic field perturbation factors were calculated using Monte Carlo, while heat transfer and mass impurity corrections and uncertainties were assessed analytically. Reference dose measurements were performed in the absence and presence of a B = 1.5 T magnetic field using Aerrow in the 7 MV FFF photon beam of an Elekta MRI-linac and were directly compared to the results obtained using two calibrated reference-class IC types. The feasibility of performing solid phantom-based dosimetry with Aerrow and the possible influence of clearance gaps is also investigated by performing reference-type dosimetry measurements for multiple rotational positions of the detector and comparing the results to those obtained in water. RESULTS: In the absence of the B-field, as well as in the parallel orientation while in the presence of the B-field, the absorbed dose to water measured using Aerrow was found to agree within combined uncertainties with those derived from TG-51 using calibrated reference-class ICs. Statistically significant differences on the order of (2-4)%, however, were observed when measuring absorbed dose to water using the ICs in the perpendicular orientation in the presence of the B-field. Aerrow had a peak-to-peak response of about 0.5% when rotated within the solid phantom regardless of whether the B-field was present or not. CONCLUSIONS: This work describes the successful use of Aerrow as a straightforward means of measuring absolute dose to water for large high-energy photon fields in the presence of a 1.5 T B-field to a greater accuracy than currently achievable with ICs. The detector-phantom air gap does not appear to significantly influence the response of Aerrow in absolute terms, nor does it contribute to its rotational dependence. This work suggests that the accurate use of solid phantoms for absolute point dose measurement is possible with Aerrow.

Accuracy of the Cosmed K5 portable calorimeter.

PURPOSE: The purpose of this study was to assess the accuracy of the Cosmed K5 portable metabolic system dynamic mixing chamber (MC) and breath-by-breath (BxB) modes against the criterion Douglas bag (DB) method. METHODS: Fifteen participants (mean age±SD, 30.6±7.4 yrs) had their metabolic variables measured at rest and during cycling at 50, 100, 150, 200, and 250W. During each stage, participants were connected to the first respiratory gas collection method (randomized) for the first four minutes to reach steady state, followed by 3-min (or 5-min for DB) collection periods for the resting condition, and 2-min collection periods for all cycling intensities. Collection periods for the second and third methods were preceded by a washout of 1-3 min. Repeated measures ANOVAs were used to compare metabolic variables measured by each method, for seated rest and each cycling work rate. RESULTS: For ventilation (VE) and oxygen uptake (VO2), the K5 MC and BxB modes were within 2.1 l/min (VE) and 0.08 l/min (VO2) of the DB (p≥0.05). Compared to DB values, carbon dioxide production (VCO2) was significantly underestimated by the K5 BxB mode at work rates ≥150W by 0.12-0.31 l/min (p<0.05). K5 MC and BxB respiratory exchange ratio values were significantly lower than DB at cycling work rates ≥100W by 0.03-0.08 (p<0.05). CONCLUSION: Compared to the DB method, the K5 MC and BxB modes are acceptable for measuring VE and VO2 across a wide range of cycling intensities. Both K5 modes provided comparable values to each other.

Isothermal titration calorimetry in a 3D-printed microdevice.

Isothermal titration calorimetry (ITC) can benefit from operating in miniaturized devices as they enable quantitative, low-cost measurements with reduced analysis time and reagents consumption. However, most of the existing devices that offer ITC capabilities either do not yet allow proper control of reaction conditions or are limited by issues such as evaporation or surface adsorption caused inaccurate solution concentration information and unintended changes in biomolecular properties because of aggregation. In this paper, we present a microdevice that combines 3D-printed microfluidic structures with a polymer-based MEMS thermoelectric sensor to enable quantitative ITC measurements of biomolecular interactions. Benefitting from the geometric flexibility of 3D-printing, the microfluidic design features calorimetric chambers in a differential cantilever configuration that improves the thermal insulation and reduces the thermal mass of the implementing device. Also, 3D-printing microfluidic structures use non-permeable materials to avoid potential adsorption. Finally, the robustness of the polymeric MEMS sensor chip allows the device to be assembled reversibly and leak-free, and hence reusable. We demonstrate the utility of the device by quantitative ITC characterization of a biomolecular binding system, ribonuclease A (RNase A) bind with cytidine 2'-monophosphate (2'CMP) down to a practically useful sample concentration of 0.2 mM. The thermodynamic parameters of the binding system, including the stoichiometry, equilibrium binding constant, and enthalpy change are obtained and found to agree with values previously reported in the literature.

A paper-based length of stain analytical device for naked eye (readout-free) detection of cystic fibrosis.

The test of sweat chloride is routinely performed as a worldwide newborn screening (NBS) to the diagnosis of cystic fibrosis (CF) in infants. However, the available methods for measurement of chloride in sweat suffer from such limitations as either low selectivity and/or requiring relatively large sample size. In this work, we have designed an analytical ruler that can measure chloride ion in sweat and hence can be used for the diagnosis of cystic fibrosis. This micro-pad (µ-PAD) device is fabricated by making hydrophilic micro-channel on a filter paper impregnated with silver dichromate. After addition of chloride ion-containing sweat sample, it moves through the channel, leading to the formation of an AgCl sediment, which deposits as a white color stain, the length of which in the channel being proportional to the amount of chloride ion in sweat. A well-defined linear relation was observed between the length of white color stain and the concentration of chloride ion in the sample solutions with a relative standard deviation of 3.6% (n = 3) for an artificial sweat sample containing 100 mM chloride ion. The possible interfering effects of several different cations and anions on the detection of chloride ion were investigated and the results well-confirmed the selectivity of the proposed method. With the use of only 2.0 µL of the sample solution, the µPAD was able to measure the chloride content of sweat over a concentration range of 20.0-100.0 mM, which covers both the healthy range (˂ 40 mM) and the risky range (˃60 mM) of chloride ion. Analysis of chloride content of sweat samples by the µPAD agreed well with those obtained by a standard electrochemical method (with relative errors of lower than 10%).

Microfabricated calorimeters for thermometric enzyme linked immunosorbent assay in one-Nanoliter droplets.

Advances in microfabrication allow for highly sensitive calorimeters with dramatically reduced volume, decreased response time and increased energy resolution. These calorimeters hold the potential for designs of ELISA platforms competitive with fluorescent and chemiluminescent technologies. We have developed a new assay platform using conventional ELISA reagents to produce a thermal signal quantifiable using calorimetry. Our optimized micromachined calorimeters have nL reaction volumes and a minimum detectable power of 375 pW/Hz1/2. We demonstrate rapid quantification in a model system of trastuzumab, a humanized monoclonal antibody used in the treatment of HER2 overexpressing breast cancers, in human serum using a HER2 peptide mimetic. Trastuzumab concentration and reaction time constant correlated well (R2 = 0.954) and can be used to determine trastuzumab concentrations. The limit of detection for the ThermometricELISA (TELISA) was 10 µg/ml trastuzumab in human serum. TELISA allows for a simple readout, reduction in assay time, sample and reagent volumes and has the potential to become a point of care multiplexed platform technology.

Mechanistic Studies of Proton-Coupled Electron Transfer in a Calorimetry Cell.

Mechanistic studies of proton-coupled electron-transfer (PCET) reactions in proteins are complicated by the challenge of following proton transfer (PT) in these large molecules. Herein we describe the use of isothermal titration calorimetry (ITC) to establish proton involvement in protein redox reactions and the identity of PT sites. We validate this approach with three variants of a heme protein cytochrome c (cyt c) and show that the method yields a wealth of thermodynamic information that is important for characterizing PCET reactions, including reduction potentials, redox-dependent p Ka values, and reaction enthalpies for both electron-transfer (ET) and PT steps. We anticipate that this facile and label-free ITC approach will find widespread applications in studies of other redox proteins and enhance our knowledge of PCET reaction mechanisms.

Factors Affecting Performance on an Army Urban Operation Casualty Evacuation for Male and Female Soldiers.

INTRODUCTION: This study was conducted to determine what physical and physiological characteristics contribute to the performance of an urban operation casualty evacuation (UO) and its predictive test, FORCE combat (FC) and describe the metabolic demand of the UO in female soldiers. METHODS: Seventeen military members (9 M and 8 F) completed a loaded walking maximal aerobic test, the UO and FC. Heart rate reserve (HRR) and completion time were used as efficiency/performance measures. Oxygen consumption (VO2) was directly measured for UO on five female participants with a portable indirect calorimetry system, and analyzed using descriptive statistics. Stepwise multiple regression analysis was used to determine the contribution of the non-modifiable (age, sex, height) and modifiable characteristics (lean body mass to dead mass ratio (LBM:DM), VO2max corrected for load (L.VO2max), peak force (PF) measured on an isometric mid-thigh pull (IMTP) and medicine ball chest throw distance (Dist) on to the performance of each exercise. RESULTS: LBM:DM and PF were the only factors included in the stepwise regression model for UO, predicting 70% of UO performance (p < 0.01). For FC, L.VO2max only was included in the stepwise regression model predicting 54% of FC performance (p < 0.01). Sex, age and height were not included in the regression model. The average metabolic cost of UO was 21.4 mL of O2*kg-1*min-1 in female soldiers while wearing PPE. CONCLUSION: This study showed that modifiable factors such as body composition, PF on IMTP and L.VO2max are key contributors to performance on UO and FC performance.

Measurement of human body surface heat flux using a calorimetric sensor.

We have developed a calorimetric sensor that can perform local measurements of the heat flux transmitted by conduction between a human body and thermostat located inside the sensor. The sensor has a detection area of 2 × 2 cm2 and, in its current configuration, facilitates measurement with a resolution of 10 mW. In this paper, measurements of two healthy male subjects of different ages (24 and 60 years) are presented. We study the variation in the power dissipated by the human body surface as a function of time for a thermostat temperature of 28 °C. We also study the same power with thermostat temperatures varying from 24° to 36°C. Measurements are performed on three different surface areas of the human body: the sternum, abdomen, and hand. The ambient room temperature during all measurements was in the range of 22-24 °C, and the subjects were seated and resting. The results show that the heat flux in the trunk is much more stable than that in the hand and that the heat flux in the sternum is greater than that in other areas. Additionally, this flux is higher in the younger subject (42 mW/cm2) than in the older subject (35 mW/cm2). We also defined a thermal parameter that represents the thermal resistance between the sensor thermostat and the skin. The mean value of this parameter varies between 51 and 71 K/W depending on the subject and measurement area.

Density effects of silica aerogel insulation on the performance of a graphite probe calorimeter.

PURPOSE: With the introduction of a novel graphite probe calorimeter, called the Aerrow, various thermal insulating materials are being explored to further improve the device. Silica-based aerogels are proving to be an optimal material due to their low densities, small thermal conductivities, rigidity, and machinability. The aim of this work is to determine how various silica aerogel densities affect the Aerrow's performance. METHODS: Performance concerns three areas: heat transfer from the core, the Aerrow's beam quality dependence, and the effects of an applied magnetic field on its measurement of absorbed dose to water. A numerical heat transfer study was done to determine heat transfer time constants. The EGSnrc radiation transport toolkit was used to determine absorbed dose conversion factors which are used to quantify the Aerrow's beam quality dependence. Dose conversion factors for Cobalt-60 and two clinical photon beams (6 and 10 MV) were determined. Magnetic field perturbation factors are used to characterize the Aerrow's performance under an applied magnetic field. EGSnrc with the magnetic field transport algorithm was used to determine these perturbations for a 1.5 T MR-linac. Several aerogel densities (0.01-0.55 g  cm - 3 ) were examined for each performance area. RESULTS: Heat transfer time constants were found to vary from 52 ± 2 to 117.4 ± 0.4 s. The time constants decreased with increasing aerogel density. The Aerrow's beam quality dependence varied between 0.5% and 1%, decreasing with increasing aerogel density. Beam quality dependence was determined in the range of 60 Co to 10 MV (58.4%  ≤  % d d ( 10 ) x  ≤ 73.5%). Under an applied magnetic field, perturbations were smallest when the Aerrow was parallel to the field. Perturbations varied more so when the Aerrow was perpendicular to the magnetic field and increased with increasing aerogel density. In all cases, perturbations were less than 0.6% from unity with a relative uncertainty of 0.1%. CONCLUSION: Silica-based aerogels demonstrate an improved performance over thermal insulation used in previous iterations of the Aerrow. With it, the Aerrow has shown to be robust in several areas. If heat transfer can be properly corrected for in the dose determination and the parallel orientation is used under a magnetic field, then the high density aerogel is possibly more preferable.

SNAREpin Assembly: Kinetic and Thermodynamic Approaches.

Proteins constantly interact and often form molecular complexes. The dynamics of most biological processes strongly rely on the kinetics and thermodynamics of assembly and disassembly of these complexes. Consequently an accurate characterization of these kinetics and thermodynamics that underlie them provides key information to better understand these processes. Here, we present two efficient techniques to quantify the assembly and disassembly of protein complexes: isothermal titration calorimetry and fluorescence anisotropy. As an example we focus on the formation of SNAREpins and also present how to prepare SNARE proteins to use in these experimental setups. We then show how to use these techniques to determine the critical factors that activate assembly kinetics.