Fluorescence Spectroscopy Data Research Articles

Insight into the binding of alpha-linolenic acid (ALA) on human serum albumin using spectroscopic and molecular dynamics (MD) studies

Alpha-linolenic acid (ALA) is one of the necessary fatty acids for the human body. It is a precursor for synthesizing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The consumption of ALA as an anti-inflammatory factor plays a significant role in preventing cardiovascular diseases. The current study was investigated to evaluate the binding affinity and molecular interactions between human serum albumin (HSA) with ALA through spectroscopic and computational studies. In the existence of ALA, the absorbance of HSA is reduced, confirming the development of the HSA-ALA complex. The fluorescence spectroscopic data implied that the quenching mechanism for HSA-ALA interaction was static mode. The negative values of ΔH° and ΔS° indicated that hydrogen bonding and van der Waals are the predominant intermolecular forces in the complex between HSA and ALA. The binding of ALA induces changes in the structure of HSA, which were revealed by far-UV circular dichroism (CD). The reduction of root mean square deviation (RMSD) after binding to ALA proved the stability of the structure of HSA in the presence of ALA. The results of the esterase activity of HSA presented that ALA competitively binds with HSA. This study demonstrated the importance of ALA in medicine and emphasized its main pharmacological significance.

Self‐Assembly and Transport Behaviour of Non‐ionic Fluorinated and Alkylated Amphiphiles for Drug Delivery

AbstractA newer series of AB2 typed non‐ionic amphiphiles have been synthesized using p‐hydroxybenzoate as a central core, which is further functionalized with two hydrophilic units, i. e. D‐lactose/poly(glycerol) dendron [G2.0]/monomethoxy poly(ethyleneglycol) and a hydrophobic alkyl/fluoroalkyl moiety. The aggregation behaviour of the synthesised amphiphiles in aqueous medium was studied using fluorescence spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM) and were found to form nano‐sized aggregates. The potential of these amphiphiles to act as nanocarrier was studied using model hydrophobic probe Nile red and nimodipine by recording their UV‐visible and fluorescence spectroscopic data. For comparison purpose, analogous AB type amphiphiles were also synthesized and their transport ability analysed. A study of cell viability in HeLa cells revealed that all synthesized amphiphiles are well tolerated at the concentrations studied. The immobilized hydrolase Novozym‐435 was used to release the encapsulated dye from the inner hydrophobic core of micellar structures of synthesized amphiphiles in vitro.

Petrogenesis of arsenic and platinum-group minerals from a partially serpentinized dunite in East Dover, Vermont, USA

In the Appalachian Mountains of Vermont, USA, variably serpentinized ultramafic rocks mark the Ordovician Taconic orogenic suture zone. These ultramafic rocks provide evidence for several alteration events that occurred during Appalachian orogenesis. The largest of these ultramafic bodies occurs as a partially serpentinized meta-dunite located in East Dover, Vermont. Whole-rock X-ray fluorescence spectroscopy and electron microprobe data on variably serpentinized meta-dunite samples are interpreted with respect to several processes including fluid/melt-rock interaction in the mantle, serpentinization, and subsequent regional metamorphism. We report the first discovery of nickel arsenide minerals hosted in this meta-dunite, as well as rare occurrences of platinum-group mineral inclusions in chromitite. Although the platinum-group minerals and chromitite are rare, their occurrence and chemistry suggest that they formed by fluid/melt-rock interaction during partial melting events that produced the dunite, likely in a supra-subduction zone setting. Arsenic minerals are rare in un-serpentinized samples but are ubiquitous in highly serpentinized samples, which suggests that most of the arsenic was introduced into the ultramafic rocks during serpentinization. Whole-rock geochemical analyses also indicate that highly serpentinized samples contain the highest concentrations of arsenic. The discovery of arsenic minerals identifies a potential source to explain elevated arsenic in groundwater in Vermont, which is a serious health concern in places where wells have been drilled in serpentinite bedrock.

An innovative fluorometric bioanalysis strategy towards recognition of DNA methylation using opto-active polymer: Anew platform for DNA damage studies by genosensor technology.

Efficient pharmacotherapy of cancer is related to accurate recognition of genetic mutations and epigenetic alterations in the early-stage diagnosis. In the present study, a novel optical genosensor based on toluidine blue as photonic probe was developed to detection of DNA methylation using hybridization of pDNA with cDNA. Biomedical analysis was performed using UV-vis and fluorometric methods. For the first time, this strategy was applied for the distinction of methylated DNA from unmethylated-DNA-based on the interaction of optical probe with methylated-DNA and unmethylated DNA. Fluorescence spectroscopic data showed that poly-toluidine blue could be bind to DNA sequences and lead to different fluorescence patterns and could be used as an efficient geno-platform for the sensitive bioassay of mutation. The excitation and emission wavelengths were 580 and 630 nm, respectively. Non-binding of mismatch sequences with the optical probe was used as negative control. Under optimal conditions, linear range was 1zM to 0.2 pm and the lower limit of quantitation was obtained as target concentrations ranging 1zM. The designed genosensor showed high capability to distinct methylation from un-methylated. Therefore, the designed DNA-based bioassay could detect DNA methylation significantly. Finally, bioanalysis of real samples showed that the designed genosensor could use to detect DNA methylation which is a new platform for point of care analysis.

English

Bacteriophytochromes (BphPs) are light-regulated biliprotein photoreceptors widely occurring in bacteria. BphPs exists in two spectral distinct states: the red-light absorbing state (Pr) and the far-red light-absorbing state (Pfr). BphPs are composed of an N-terminal photosensory core module and a light-controlled C-terminal histidine kinase module. The detailed mechanism on how photoconversion, initiated by a light-triggered isomerization of the biliverdin chromophore, is conducted from the N-terminal to the C-terminal domain and how the histidine kinase is modulated is still not clear. In this work Agp1 from Agrobacterium tumefaciens is used as a model phytochrome. The method of labeling full-length Agp1 mutants with single accessible Cys residue using fluorescence probes is described. Different absorption spectra showed that neither mutagenesis nor fluorescence labeling exerts significant effects on the photoconversion behavior of various Agp1 mutants. The histidine kinase activity assay revealed that the labeled holoproteins exhibit wild-types like autophosphorylation activity that depends on the Pr and the Pfr state. Fluorescence spectroscopic data suggested that fluorescence resonance energy transfer occurs from the donor Atto495 to the acceptor Atto565 in double-labeled Agp1-A362C, K514C, H554C, R603C, I653C, and V674C mutants. Red light irradiation gave rise to small changes on fluorescence emission intensity in some mutants but in some no remarkable inter-subunit distance changes occurred during the photo-conversion from Pr to Pfr in full-length Agp1. In contrast to double-labeled samples, significant changes in fluorescence intensity were observed in single-labeled samples.   Key words: Bacteriophytochromes (BphPs), C-terminal domain, photosensory, phytochrome. 

Detection of protein oxidation products by fluorescence spectroscopy and trilinear data decomposition: Proof of concept

Current analytical methods studying protein oxidation modifications require laborious sample preparation and chromatographic methods. Fluorescence spectroscopy is an alternative, as many protein oxidation products are fluorescent. However, the complexity of the signal causes misinterpretation and quantification errors if single emission spectra are used. Here, we analyzed the entire fluorescence excitation-emission matrix using the trilinear decomposition method parallel factor analysis (PARAFAC). Two sample sets were used: a calibration set based on known mixtures of tryptophan, tyrosine, and four oxidation products, and a second sample set of oxidized protein solutions containing UV-illuminated β-lactoglobulin. The PARAFAC model succeeded in resolving the signals of the model systems into the pure fluorophore components and estimating their concentrations. The estimated concentrations for the illuminated β-lactoglobulin samples were validated by liquid chromatography-mass spectrometry. Our approach is a promising tool for reliable identification and quantification of fluorescent protein oxidation products, even in a complex protein system.

Mechanistic Insights into the Inhibitory Activities of Chemical Constituents from the Fruits of Terminalia boivinii on α-Glucosidase.

Regulation of key digestive enzymes is currently considered an effective remedy for diabetes mellitus. In this study, bioactive constituents were purified from Terminalia boivinii fruits and identified by 1 H-NMR, 13 C-NMR and EI-MS. In vitro and in silico methods were used to evaluate α-glucosidase, α-amylase, and lipase inhibition activities. Compounds 1, 2, and 4-7 with IC50 values between 89 and 445 μM showed stronger α-glucosidase inhibitory activities than the antihyperglycemic drug acarbose (IC50 =1463.0±29.5 μM). However, the compounds showed lower inhibitory effects against α-amylase and lipase with IC50 values above 500 μM than acarbose (IC50 =16.7±3.5 μM) and ursolic acid (IC50 =89.5±5.6 μM), respectively. Lineweaver-Burk plots showed that compounds 1, 2, and 7 were non-competitive inhibitors, compounds 4 and 5 were competitive inhibitors and compound 6 was a mixed-type inhibitor. Fluorescence spectroscopic data showed that the compounds altered the microenvironment and conformation of α-glucosidase. Computer simulations indicated that the compounds and enzyme interacted primarily through hydrogen bonding. The findings indicated that the compounds were inhibitors of α-glucosidase and provided significant structural basis for understanding the binding activity of the compounds with α-glucosidase.

Binding effects of antibiotic drug sulfamethazine on the serum albumins: Multi-spectroscopic and computation approach

Over the past decades, bio-macromolecules-drug binding interaction studies have been of great interest with varied applications in drug modification and development. Such reports help us to recognize the molecular aspects of the bindings correlating with the structural phenomenon. The binding study of an antibiotic drug, Sulfamethazine (SMZ) with plasma proteins, Human Serum Albumin (HSA), and Bovine Serum Albumin (BSA) have been investigated using spectroscopic and computational techniques. The UV–Vis and fluorescence spectroscopic data indicate towards potential binding affinity of the drug to the proteins through the formation of stable ground state complexes. Binding constants obtained were in the order of 104M − 1 for both the cases, the values were comparatively higher for HSA than BSA. Thermodynamic parameters were estimated from temperature-dependent fluorescence analyses; enthalpy driven, exothermic nature of the binding interactions along with the predominance of electrostatic and hydrophobic forces were established in both the cases. Synchronous fluorescence and FT-IR spectra revealed the conformational changes in the protein structure(s). Molecular docking and dynamic studies further supported the complex formation in accordance with the experimental report(s). Molecular docking denoted binding of the drug at pocket-I of the active site in proteins, while molecular dynamic simulation established the stability and compactness of the complex due to binding.

Enhanced luminescence efficiency and thermal stability via introduction of non-rare earth Bi3+ in Gd5Si2BO13:Eu3+

A series of Gd5Si2BO13:Eu3+ and non-rare earth Bi3+ ions doped Gd5Si2BO13:Eu3+ phosphors was successfully synthesized via high-temperature solid-state method, and the as-obtained phosphors were studied on their phase structures, luminescence characteristics, thermal stability and luminescence lifetime. Transient fluorescence spectroscopy data show that the addition of Bi3+ can obviously enhance the emission intensity of Eu3+ in the near-ultraviolet band owing to energy transfer from Bi3+ to Eu3+. Besides, the addition of Bi3+ can improve the thermal stability of a single-doped phosphor Gd5Si2BO13:0.35Eu3+ from 70.33% to 87.45% at 150 °C and the quantum yield from 58.80% to 78.61% at room temperature. Finally, Gd5Si2BO13:Eu3+,Bi3+ was used to encapsulate white light-emitting diodes (WLEDs) with green ((Ba,Sr)2SiO4:Eu2+) and blue (BaMgAl10O17:Eu2+) commercial phosphors. The color rendering index of WLEDs was calculated to be larger than 90, and the color temperature was estimated to be 4300–4500 K, which demonstrate that Gd5Si2BO13:Eu3+,Bi3+ can be regarded as a red phosphor with great potential application. This paper can provide a new insight into design of high-efficiency phosphors by introducing non-rare earth Bi3+ ions via energy transfer from Bi3+ to Eu3+.

Selection of DNA Aptamers for Sensing Uric Acid in Simulated Tears

AbstractUric acid is a biomarker for a range of diseases and hyperuricemia is the cause of many diseases including gout. While most biosensors for detecting uric acid relied on enzymatic reactions, in this work a library‐immobilization method was used to obtain DNA aptamers for uric acid. After 18 rounds of selection, two representative aptamers were obtained with a Kd around 1.2 μM measured by isothermal titration calorimetry (ITC). Based on their difference in binding to xanthine, which differs from uric acid by only one oxygen atom, these two aptamers have different binding orientations to uric acid. ITC also indicated that the UA‐1 aptamer specifically required a high concentration of Na+ for binding, which cannot be replaced by Li+, K+ or Mg2+. Combined ITC and fluorescence spectroscopy data indicated the need of three Na+ ions, which explained the requirement of a high Na+ concentration. The UA‐1 aptamer was engineered into a fluorescent biosensor based on the strand‐displacement reaction, resulting in a limit of detection of 90 nM uric acid. This sensor was also tested in simulated tears with a limit of detection of 350 nM uric acid.

Discrimination of onion subjected to drought and normal watering mode based on fluorescence spectroscopic data

Drought stress can affect the yield and quality of cultivated plants. The deficit of water may result in the physiological and anatomical reactions at organ, tissue and cellular levels of the plant species. The objective of this study was to discriminate different onion samples with the use of innovative models based on fluorescence spectroscopic data using different classifiers. The onion growing under drought and normal watering conditions were compared. Additionally, the five different samples of onion including three varieties (Konkurent bql, Asenovgradska kaba, Trimoncium) and two lines (white, red) subjected to both the drought mode and normal watering mode were differentiated. The results were evaluated based on confusion matrices, average accuracies, and the values of TP (True Positive) Rate, FP (False Positive) Rate, Precision, F-Measure, ROC (Receiver Operating Characteristic) Area and PRC (Precision-Recall) Area. In the case of the discrimination of two classes: drought mode and normal watering mode, an average accuracy reached 100% for white line of onion for a model built using the Naive Bayes, Multilayer Perceptron, JRip and LMT classifiers and for red line of onion for all used classifiers (Naive Bayes, Multilayer Perceptron, IBk, Multi Class Classifier, JRip, LMT). The values of TP Rate, Precision, F-Measure, ROC Area and PRC Area were equal to 1.000, and FP Rate was 0.000. For onion samples subjected to drought, five classes including the Konkurent, Asenovgradska kaba, Trimoncium varieties and the white and red lines were discriminated with an average accuracy of up to 90% for the LMT classifier. The same classes of samples but subjected to normal watering were correctly distinguished in 84% for the Naive Bayes classifier.

Study on the interaction of ethylene glycol with trypsin: Binding ability, activity, and stability

The study of the structural changes and thermal stabilities of proteins in the presence of various co-solutes is a critical element of basic research, drug discovery, and development. Ethylene glycol (EG) is typically known to be poisonous and teratogenic, causing skeletal and external abnormalities as well as other disorders. At pH 8, the influence of EG on the structure and thermal stability of trypsin was investigated using a variety of spectroscopic techniques. Spectroscopic techniques, fluorescence spectroscopy, circular dichroism (CD), and molecular dynamics simulation studies were used to evaluate the binding reaction between ethylene glycol and trypsin in the current study. Circular dichroism, Trp fluorescence, and absorption tests demonstrated that EG has an effect on secondary and tertiary structures of trypsin, and it boosts the protein's thermal stability, which increases with increasing EG concentration. This contact occurs through a static quenching process, according to fluorescence spectroscopy data, and van der Waals contributions and hydrogen bonds were also important in binding ethylene glycol to trypsin. The ethylene glycol binding constant (Kb) and the number of binding sites (n) were calculated using fluorescence quenching data. Kinetic technique analysis displayed a decrease in trypsin activity in the presence of ethylene glycol. Also, circular dichroism results confirmed the conformation changes resulting from the binding of ethylene glycol to trypsin. Molecular dynamics simulation using RMSD was used to test the stability of the trypsin-ethylene glycol complex in this study. Molecular docking research revealed that EG interacts with trypsin amino acid residues via hydrogen bonding and van der Waals interactions.

A Primal-Dual algorithm for nonnegative N-th order CP tensor decomposition: application to fluorescence spectroscopy data analysis

A Primal-Dual algorithm for nonnegative N-th order CP tensor decomposition: application to fluorescence spectroscopy data analysis

Inhibition of Maillard reaction during alkaline thermal hydrolysis of sludge

The Maillard reaction (MR) occurs during the alkaline thermal hydrolysis (ATH) of sludge, which affects the quantity and quality of recovered protein. In this paper, four different sulfites were added to investigate their inhibitory effects on melanoidin production. The results showed that sulfites inhibited melanoidin production during ATH of sludge and the inhibitory rate increased with their concentration. At a concentration of 5.71 g/L, the inhibitory rates of NaHSO3 on melanoidin were 63.27%. Furthermore, the 3D-EEM (Three-Dimension Excitation-Emission-Matrix) fluorescence spectroscopy and protein testing data showed that the inhibition of melanoidin production was accompanied by an increased protein concentration, and protein increased with increasing sulfites concentration. A 2.5-fold increase in protein concentration with Na2S2O4 significantly enhanced the quantity of protein recovered. Therefore, the addition of sulfite during ATH of sludge reduces the amount of non-biodegradable melanoidin, which in turn benefits protein recovery.

A Luminescent Guest@MOF Nanoconfined Composite System for Solid-State Lighting.

A series of rhodamine B (RhB) encapsulated zeolitic imidazolate framework-8 (RhB@ZIF-8) composite nanomaterials with different concentrations of guest loadings have been synthesized and characterized in order to investigate their applicability to solid-state white-light-emitting diodes (WLEDs). The nanoconfinement of the rhodamine B dye (guest) in the sodalite cages of ZIF-8 (host) is supported by fluorescence spectroscopic and photodynamic lifetime data. The quantum yield (QY) of the luminescent RhB@ZIF-8 material approaches unity when the guest loading is controlled at a low level: 1 RhB guest per ~7250 cages. We show that the hybrid (luminescent guest) LG@MOF material, obtained by mechanically mixing a suitably high-QY RhB@ZIF-8 red emitter with a green-emitting fluorescein@ZIF-8 “phosphor” with a comparably high QY, could yield a stable, intensity tunable, near-white light emission under specific test conditions described. Our results demonstrate a novel LG@MOF composite system exhibiting a good combination of photophysical properties and photostability, for potential applications in WLEDs, photoswitches, bioimaging and fluorescent sensors.

Design and synthesis of C3-symmetric molecules containing oxepine and benzofuran moieties via Metathesis

• We demonstrated a simple synthetic strategy to assemble C 3 -symmetric star-shaped phenyl and triazine central core based oxepine and benzofuran derivatives. • The key steps involved are Claisen rearrangement, double-bond isomerization and cyclo-trimerization. • We introduced oxepine and furan ring systems in C 3 -symmetric star-shaped phenyl and triazine core derivatives using olefin metathesis. • We studied photophysical properties of prepared molecules by fluorescence spectroscopic data. We report a new synthetic strategy to C 3 -symmetric star-shaped phenyl and triazine central cores bearing oxepine and benzofuran ring systems. In this regard, we have explored the application of metathetic strategy to construct C 3 -symmetric molecules starting with commercially available p -hydroxy acetophenone and p -hydroxy benzonitrile through cyclotrimerization, double bond isomerization, and ring-closing metathesis as key steps. We also studied photophysical properties of these molecules by fluorescence spectroscopic data. All compounds except 1 and 2 exhibit strong fluorescence.

Detection and Identification of Organic Pollutants in Drinking Water from Fluorescence Spectra Based on Deep Learning Using Convolutional Autoencoder

Three dimensional fluorescence spectroscopy has become increasingly useful in the detection of organic pollutants. However, this approach is limited by decreased accuracy in identifying low concentration pollutants. In this research, a new identification method for organic pollutants in drinking water is accordingly proposed using three-dimensional fluorescence spectroscopy data and a deep learning algorithm. A novel application of a convolutional autoencoder was designed to process high-dimensional fluorescence data and extract multi-scale features from the spectrum of drinking water samples containing organic pollutants. Extreme Gradient Boosting (XGBoost), an implementation of gradient-boosted decision trees, was used to identify the organic pollutants based on the obtained features. Method identification performance was validated on three typical organic pollutants in different concentrations for the scenario of accidental pollution. Results showed that the proposed method achieved increasing accuracy, in the case of both high-(>10 μg/L) and low-(≤10 μg/L) concentration pollutant samples. Compared to traditional spectrum processing techniques, the convolutional autoencoder-based approach enabled obtaining features of enhanced detail from fluorescence spectral data. Moreover, evidence indicated that the proposed method maintained the detection ability in conditions whereby the background water changes. It can effectively reduce the rate of misjudgments associated with the fluctuation of drinking water quality. This study demonstrates the possibility of using deep learning algorithms for spectral processing and contamination detection in drinking water.

Elucidation of Interaction between Whey Proteins and Proanthocyanidins and Its Protective Effects on Proanthocyanidins during In-Vitro Digestion and Storage.

Whey proteins and oligomeric proanthocyanidins have nutritional value and are widely used in combination as food supplements. However, the effect of the interactions between proanthocyanidins and whey proteins on their stability has not been studied in depth. In this work, we aimed to characterize the interactions between β-Lactoglobulin (β-LG) and α-lactalbumin (α-LA) and oligomeric proanthocyanidins, including A1, A2, B1, B2, B3, and C1, using multi-spectroscopic and molecular docking methods. Fluorescence spectroscopic data revealed that all of the oligomeric proanthocyanidins quenched the intrinsic fluorescence of β-LG or α-LA by binding-related fluorescence quenching. Among the six oligomeric proanthocyanidins, A1 showed the strongest affinity for β-LG (Ka = 2.951 (±0.447) × 104 L∙mol−1) and α-LA (Ka = 1.472 (±0.236) × 105 L∙mol−1) at 297 K. β-LG/α-LA and proanthocyanidins can spontaneously form complexes, which are mainly induced by hydrophobic interactions, hydrogen bonds, and van der Waals forces. Fourier-transform infrared spectroscopy (FTIR) and circular dichroism spectroscopy showed that the secondary structures of the proteins were rearranged after binding to oligomeric proanthocyanidins. During in vitro gastrointestinal digestion, the recovery rate of A1 and A2 increased with the addition of WPI by 11.90% and 38.43%, respectively. The addition of WPI (molar ratio of 1:1) increased the retention rate of proanthocyanidins A1, A2, B1, B2, B3, and C1 during storage at room temperature by 14.01%, 23.14%, 30.09%, 62.67%, 47.92%, and 60.56%, respectively. These results are helpful for the promotion of protein–proanthocyanidin complexes as functional food ingredients in the food industry.

Molecular collisions or resonance energy transfer in lipid vesicles? A methodology to tackle this question

In this work, molecular interactions in a lipid membrane are discussed through fluorescence spectroscopy data, both experimentally and theoretically. In particular, the fluorescence quenching mechanisms between the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) and the potential drug 2-nitrobenzaldehyde-thiosemicarbazone (2-TSC) were studied, both inserted in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DMPC) model membrane. The fluorescence intensity and the lifetime of Laurdan decrease dramatically in the presence of 2-TSC, in both gel and fluid phases of the DMPC bilayer. It is shown here how to identify the correct quenching mechanism, by conducting a careful analysis of the fluorescence data. The analysis of the bimolecular constant values obtained through the Stern-Volmer equation, considering the collisional mechanism, made clear the incompatibility of the obtained values with estimated diffusion coefficients for Laurdan and 2-TSC inserted into lipid bilayers. On the other hand, using the Förster’s theory of resonance energy transfer (FRET) we obtained results in good agreement with the already known dynamic characteristics of a DMPC bilayer, at its both gel and fluid phases. Through spectroscopy data and computational calculation, Förster distance, energy transfer efficiency and distance distribution were obtained for the donor/acceptor pair Laurdan/2-TSC, at both gel and fluid phases of the bilayer. The distance distribution reflects the occurrence of FRET involving donor/acceptor pairs in the same leaflet of the lipid bilayer and pairs in opposite leaflet, and these results are in good agreement with our previous proposal about the lateral organization and position of Laurdan and 2-TSC molecules in a DMPC bilayer. All these results lead us to conclude that FRET between the donor Laurdan and the acceptor 2-TSC is the mechanism responsible for non-radiative deexcitation of Laurdan. The methodology used here could be extended to other pairs of donor/acceptor molecules, to contribute to the knowledge about their localizations in lipid membranes.

Fast and robust two-dimensional inverse Laplace transformation of single-molecule fluorescence lifetime data

Fluorescence spectroscopy at the single-molecule scale has been indispensable for studying conformational dynamics and rare states of biological macromolecules. Single-molecule two-dimensional (2D) fluorescence lifetime correlation spectroscopy is an emerging technique that holds promise for the study of protein and nucleic acid dynamics, as the technique is 1) capable of resolving conformational dynamics using a single chromophore, 2) resolves forward and reverse transitions independently, and 3) has a dynamic window ranging from microseconds to seconds. However, the calculation of a 2D fluorescence relaxation spectrum requires an inverse Laplace transform (ILT), which is an ill-conditioned inversion that must be estimated numerically through a regularized minimization. Current methods for performing ILTs of fluorescence relaxation can be computationally inefficient, sensitive to noise corruption, and difficult to implement. Here, we adopt an approach developed for NMR spectroscopy (T1-T2 relaxometry) to perform one-dimensional (1D) and 2D-ILTs on single-molecule fluorescence spectroscopy data using singular-valued decomposition and Tikhonov regularization. This approach provides fast, robust, and easy to implement Laplace inversions of single-molecule fluorescence data. We compare this approach to the widely used maximal entropy method.