Computer Science Question

Description

Instructions

For this assignment, you will examine the data collection instruments used in two peer-reviewed articles on a technology topic and present the findings. Address all components for each article before moving on to the next article.

Your paper should include the following:

A discussion of the purpose of the instrument.
An overview indicating how the instrument was assessed for reliability (e.g., internal reliability analysis) and validity (e.g., confirmatory factor analysis).
An assessment of the strengths of the instrument.
An assessment of the limitations of the instrument.
Recommendations for improvements to the instrument.

Length: 5-7 pages, not including title and reference pages

References: Include a minimum of 5 scholarly resources

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sensors
Review
Trends in Single-Molecule Total Internal Reflection
Fluorescence Imaging and Their Biological Applications with
Lab-on-a-Chip Technology
Louis Colson 1 , Youngeun Kwon 2 , Soobin Nam 2 , Avinashi Bhandari 1 , Nolberto Martinez Maya 1 ,
Ying Lu 1 and Yongmin Cho 2, *
1
2
*
Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA;
[email protected] (L.C.); [email protected] (A.B.);
[email protected] (N.M.M.); [email protected] (Y.L.)
Department of Chemical Engineering, Myongji University, Yongin 17058, Republic of Korea;
[email protected] (Y.K.); [email protected] (S.N.)
Correspondence: [email protected]; Tel.: +82-31-330-6385
Abstract: Single-molecule imaging technologies, especially those based on fluorescence, have been developed to probe both the equilibrium and dynamic properties of biomolecules at the single-molecular
and quantitative levels. In this review, we provide an overview of the state-of-the-art advancements
in single-molecule fluorescence imaging techniques. We systematically explore the advanced implementations of in vitro single-molecule imaging techniques using total internal reflection fluorescence
(TIRF) microscopy, which is widely accessible. This includes discussions on sample preparation,
passivation techniques, data collection and analysis, and biological applications. Furthermore, we
delve into the compatibility of microfluidic technology for single-molecule fluorescence imaging,
highlighting its potential benefits and challenges. Finally, we summarize the current challenges and
prospects of fluorescence-based single-molecule imaging techniques, paving the way for further
advancements in this rapidly evolving field.
Citation: Colson, L.; Kwon, Y.; Nam,
Keywords: single-molecule imaging; TIRF; fluorescence; data analysis; microfluidics
S.; Bhandari, A.; Maya, N.M.; Lu, Y.;
Cho, Y. Trends in Single-Molecule
Total Internal Reflection Fluorescence
Imaging and Their Biological
Applications with Lab-on-a-Chip
Technology. Sensors 2023, 23, 7691.
https://doi.org/10.3390/
s23187691
Academic Editor: Sellamuthu Anbu
Received: 21 July 2023
Revised: 1 September 2023
Accepted: 3 September 2023
Published: 6 September 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Imaging techniques provide powerful tools to visualize and quantify molecular interactions, cellular dynamics, and tissue architecture and are therefore instrumental in advancing
our understanding of biological systems [1–12]. Certain imaging techniques can directly
observe individual biomolecules such as oligonucleotides, proteins, and protein complexes.
These single-molecule imaging techniques can provide information on the heterogeneity of the
system which can often be difficult to determine using other methods. In recent years, singlemolecule imaging with total internal reflection fluorescence (TIRF) has gained significant
popularity due to its accessibility and high sensitivity in probing the properties of biomolecules.
By enabling the visualization and tracking of individual molecules in exceptional spatial and
temporal resolutions, TIRF-based single-molecule imaging has opened up new avenues for
studying complex biological processes, including protein folding, protein–protein interactions,
DNA replication, and cellular signaling [13–22].
In this review, we specifically explore in vitro single-molecule imaging with
TIRF [11,23–25]. In addition to discussing the technical aspects of single-molecule imaging,
this review surveys and highlights several exemplary applications of TIRF-based singlemolecule imaging, especially microfluidic-based approaches. By showcasing the diversity of
biological questions addressed using this technique, we aim to demonstrate its broad impact
across various fields, including molecular biology, biophysics, and nanotechnology. Finally, we
address the potential prospects and challenges of fluorescence-based single-molecule imaging
Sensors 2023, 23, 7691. https://doi.org/10.3390/s23187691
https://www.mdpi.com/journal/sensors
Sensors 2023, 23, x FOR PEER REVIEW
Sensors 2023, 23, 7691
2 of 19
impact across various fields, including molecular biology, biophysics, and nanotechnology. Finally, we address the potential prospects and challenges of fluorescence-based sin2 of 20
gle-molecule imaging techniques. We also discuss the limitations and potential sources of
artifacts in single-molecule imaging experiments, as well as strategies to mitigate these
issues.
techniques. We also discuss the limitations and potential sources of artifacts in single-molecule
as well as strategies
to mitigate these
issues.
2.imaging
Optical experiments,
Systems for Single-Molecule
Fluorescence
Imaging
Fluorescence
single-molecule
imagingFluorescence
techniques rely
on the utilization of optical ra2. Optical
Systems
for Single-Molecule
Imaging
diationFluorescence
to probe individual
molecules
within
a
liquid
or
solid
sample.
To achieve
single-molecule imaging techniques rely on the
utilization
of opticalsuccessradiation
ful
single-molecule
imaging,
two
key
requirements
must
be
met:
(1)
ensuring
that resoto probe individual molecules within a liquid or solid sample. To achieve successful
singlenant
molecules
are
spatially
resolved
by
the
detector,
and
(2)
providing
a
sufficient
signalmolecule imaging, two key requirements must be met: (1) ensuring that resonant molecules are
to-noise
ratio
(SNR)by
forthe
the
single-molecule
signal within
a reasonable
averaging
time
[22].for
spatially
resolved
detector,
and (2) providing
a sufficient
signal-to-noise
ratio
(SNR)
Consequently,
a
fundamental
prerequisite
for
conducting
single-molecule
observations
is
the single-molecule signal within a reasonable averaging time [22]. Consequently, a fundamental
toprerequisite
dilute the concentration
of
the
target
molecule
of
interest
to
exceedingly
low
levels
(typfor conducting single-molecule observations is to dilute the concentration of the
ically
100 nM).ofThe
detection
of singlelow
molecules
via fluorescence-based
methods
detarget< molecule interest to exceedingly levels (typically < 100 nM). The detection of single mands careful optimization of the signal-to-noise signal requires molecules via fluorescence-based methods demands ratio. carefulMaximizing optimizationthe of the signal-to-noise the selection of an impurity the highest fluorescence quantum efratio. Maximizing the signalmolecule requires with the selection of anpossible impurity molecule with the highest ficiency. possible fluorescence quantum efficiency. This Thisapproach approachharnesses harnessesrecent recentadvancements advancementsininfluorescence fluorescenceimaging imagingtechniques, techniques, including TIRF microscopy [23–25], super-resolution microscopy, and single-molecule lo-loincluding TIRF microscopy [23–25], super-resolution microscopy, and single-molecule calization microscopy, calizationmicroscopy microscopy[26–32]. [26–32].TIRF TIRF microscopy,one oneofofthe themost mostcommonly commonlyemployed employed tools principle of of total totalinterintoolsininsingle-molecule single-moleculefluorescence fluorescencemicroscopy, microscopy, capitalizes capitalizes on the principle ternal reflection.This Thisphenomenon phenomenon occurs when aa laser nal reflection. laserbeam beamstrikes strikesthe theinterface interfacebetween between a amedium with a higher refractive index (typically glass) andand a medium withwith a lower remedium with a higher refractive index (typically glass) a medium a lower fractive index (such as a sample solution) at an angle greater than the critical angle (Figure refractive index (such as a sample solution) at an angle greater than the critical angle 1A). As a 1A). result, evanescent wave is generated, which excites fluorophores in the im-in (Figure Asan a result, an evanescent wave is generated, which excites fluorophores the immediate vicinity of the interface, facilitating the visualization of single molecules mediate vicinity of the interface, facilitating the visualization of single molecules near the near the sampleTIRF surface. TIRF microscopy is practically implemented using either a sample surface. microscopy is practically implemented by using by either a quartz quartz or the microscope to the generate the evanescent field and illuminate prism or prism the microscope objective objective to generate evanescent field and illuminate surfacesurface-immobilized (Figure 1B). TIRF microscopy offers exceptional optical immobilized moleculesmolecules (Figure 1B). TIRF microscopy offers exceptional optical sectioning sectioning and background suppression, leading to a high signal-to-noise ratio. and background suppression, leading to a high signal-to-noise ratio. Figure 1. 1. TIRF microscopy for for single-molecule fluorescence imaging. (A) Principle of TIRF Figure TIRF microscopy single-molecule fluorescence imaging. (A) Principle of microsTIRF micopy. (B) Types of TIRF microscopy: prism-type (P-TIRF) or objective-type (O-TIRF). croscopy. (B) Types of TIRF microscopy: prism-type (P-TIRF) or objective-type (O-TIRF). Theevanescent evanescent field intensity,I(z), I(z),atata aperpendicular perpendiculardistance distancez zfrom fromthe theinterface interfaceisis The field intensity, describedbybyEquation Equation(1). (1). described z I (z ) == I ( 00 (1) ) exp exp − d), , (1) λ0 2 2 −1/2 d == n 21 sin (2)(2) θ − n 2 ) / ,, 4π 4 where I (0) represents the evanescent field intensity at the interface (Figure 1A). The characwhere 0 represents the evanescent field intensity at the interface (Figure 1A). The charteristic penetration depth (Equation (2)), d, is determined by the wavelength of incident acteristic penetration depth (Equation (2)), d, is determined by the wavelength of incident light ( λ0 ), refractive index of the medium through which the light initially passes (n1 ) and in contact with the sample (n2 ), and incident angle (θ ). Typically ranging between 30 and 200 nm, the penetration depth defines the region within which fluorophores are effectively excited by the evanescent wave. Sensors 2023, 23, 7691 light ( , refractive index of the medium through which the light initially passes ( ) and in contact with the sample ( ), and incident angle . Typically ranging between 30 and 200 nm, the penetration depth defines the region within which fluorophores are effectively 3 of 20 excited by the evanescent wave. In addition, the detection of individual fluorophores is a critical aspect of single-molecule fluorescence imaging. Here, the numerical aperture (NA) is one of the key parameIn addition, the detection of individual is acollection critical aspect of singleters. High NA objectives are commonly used to fluorophores maximize light and detection moleculeThe fluorescence Here,on thethe numerical aperture is oneresolution of the key efficiency. specific NAimaging. value depends imaging setup and (NA) the desired parameters. NA objectives are commonlyfluorescence used to maximize light collection and and sensitivity.High For conventional single-molecule imaging, objectives with detection efficiency. The specific NA value depends on the imaging setup and the desired NA values ranging from 1.2 to 1.49 are frequently employed. These objectives offer a balresolution sensitivity. For conventional single-molecule fluorescence imaging, ance betweenand high light collection efficiency and reasonable working distances. They objecare tives with NA values ranging from 1.2 to 1.49 are frequently employed. These objectives suitable for imaging samples in various configurations, including liquid solutions, solid offer a balance between high light collection efficiency and reasonable working distances. surfaces, and biological specimens. They are suitable for imaging samples in various configurations, including liquid solutions, The choice of camera is another important factor in the detection of individual fluorsolid surfaces, and biological specimens. ophores. Ultimately, digital cameras capture the photons from individual fluorescent molThe convert choice ofthe camera is another important in the detection of individual fluecules and light into electrical signals. factor The cameras used for single-molecule orophores. Ultimately, digital cameras capture the photons from individual fluorescent TIRF imaging tend to have quantum efficiencies above 80%, spectral range between 300 molecules and convert the light into electrical signals. The cameras used for single-molecule and 1100 nm, low readout noise, and millisecond readout speeds [33]. Electron multiplyTIRF imaging tend to have quantum efficiencies above 80%, spectral range between ing charge coupled devices (EMCCDs) and the scientific complementary metal–oxide– 300 and 1100 nm, low readout noise, and millisecond readout speeds [33]. Electron multisemiconductor (sCMOS) devices are the most common types of cameras used in singleplying charge coupled devices (EMCCDs) and the scientific complementary metal–oxide– molecule imaging. Note that most of the biological applications discussed in Section 5 use semiconductor (sCMOS) devices are the most common types of cameras used in singlean EMCCD camera. Some recent laboratory advances in imaging technology may further molecule imaging. Note that most of the biological applications discussed in Section 5 use improve the performance of scientific cameras across the broadband spectrum [34–36]. an EMCCD camera. Some recent laboratory advances in imaging technology may further improve the performance of scientific cameras across the broadband spectrum [34–36]. 3. Sample Preparation Sample Preparation preparation is a critical step in single-molecule imaging of biological mole3. Sample cules for repeatable and reliable results. section, we define “sample preparation” Sample preparation is a critical stepIn inthis single-molecule imaging of biological molecules asfor therepeatable preparation of the imaging device (Figure 2) and any labeling of the biological and reliable results. In this section, we define “sample preparation”molas the ecules. Generally, biomolecules non-specifically adhere to the surfaces of the imaging depreparation of the imaging device (Figure 2) and any labeling of the biological molecules. vice. Thus, the preparation of the imaging device includes a passivation step to reduce Generally, biomolecules non-specifically adhere to the surfaces of the imaging device. Thus, non-specific binding andimaging false-positive Fluorophores provide readoutnon-specific for interthe preparation of the devicesignals. includes a passivation step toa reduce actions between molecules or the functions of theprovide reaction system.for Preparing biological binding and false-positive signals. Fluorophores a readout interactions between molecules for the experiments includes a labeling step to conjugate fluorophores to molecules or the functions of the reaction system. Preparing biological moleculesmolefor the cules of interestincludes and a strategy to constrain the location of the molecules of interest in the experiments a labeling step to conjugate fluorophores to molecules of interest and imaging region [37,38]. the There are methods that detect freely diffusing singleregion molecules a strategy to constrain location of the molecules of interest in the imaging [37,38]. [39,40], this review, we limit ourdiffusing scope to single single-molecule with immobiThere but are in methods that detect freely moleculesstrategies [39,40], but in this review, lized molecules. we limit our scope to single-molecule strategies with immobilized molecules. Figure Schematic representation imaging device. The construction imaging device Figure 2. 2. Schematic representation of of anan imaging device. The construction of of anan imaging device entails integration a microscope a coverslip, employing double-sided tape entails thethe integration of aofmicroscope slideslide and aand coverslip, employing double-sided tape for pre-for cision juxtaposition, followed by hermetic sealingsealing with epoxy resin. The holes the slide areslide usedare precision juxtaposition, followed by hermetic with epoxy resin. Theon holes on the as used the inlet and outlet solution exchange. as the inlet andfor outlet for solution exchange. 3.1. Surface Passivation 3.1. Surface Passivation Established passivation techniques prepare imaging device rely coating Established passivation techniques to to prepare thethe imaging device rely ononcoating chemically treated glass surfaces with biocompatible reagents such as polyethylene glycol chemically treated glass surfaces with biocompatible reagents such as polyethylene glycol (PEG), phospholipids, or Tween-20. In this section, we briefly describe the PEG, lipids, and Tween-20 passivation methods. These methods produce similar results and are described in depth elsewhere [41–43]. The PEG passivation method relies on the amino-silanization of the glass surface. Usually, researchers use KOH to form alcohol groups on the glass surface. Amino silane can react with these alcohol groups on the surface. This reaction (shown in Figure 3A) takes the following form: ROH + (OCH3 )3 SiOCH3 → CH3 OH. To complete Sensors 2023, 23, 7691 (PEG), phospholipids, or Tween-20. In this section, we briefly describe the PEG, lipids, and Tween-20 passivation methods. These methods produce similar results and are described in depth elsewhere [41–43]. The PEG passivation method relies on the amino-si4 ofon 20 lanization of the glass surface. Usually, researchers use KOH to form alcohol groups the glass surface. Amino silane can react with these alcohol groups on the surface. This reaction (shown in Figure 3A) takes the following form: + → the commercially availablecommercially PEG ester molecules silane groups passivation, . To complete the passivation, availablereact PEGwith esterthe molecules react (Figure to PEGylate the surface ofPEGylate the coverslip [41]. This passivation method with the3B) silane groups (Figure 3B) to the surface of covalent the coverslip [41]. This covacan protein denaturing conditions as 8 M conditions guanidinium chloride lent withstand passivationharsh method can withstand harsh proteinsuch denaturing such as 8 M (GdmCl) [44] and 4M urea [45]. For dichlorodimethylsilane guanidinium chloride (GdmCl) [44] the andTween-20 4M urea passivation, [45]. For theaTween-20 passivation, a (DDS)-treated glass surface forms a hydrophobic coating can be passivated the dichlorodimethylsilane (DDS)-treated glass surface forms that a hydrophobic coatingwith that can addition of the biocompatible surfactant Tween-20 [42]. For the passivation with phosphobe passivated with the addition of the biocompatible surfactant Tween-20 [42]. For the lipids, glasswith devices can be incubated with liposomes to form awith fluid lipid bilayer. Thea passivation phospholipids, glass devices can be incubated liposomes to form liposomes for passivation in single-molecule TIRF experiments have been made from fluid lipid used bilayer. The liposomes used for passivation in single-molecule TIRF experilipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and egg phosphatidylments such haveasbeen made from lipids such as 1,2-dioleoyl-sn-glycero-3-phosphocholine choline [46,47]. (DOPC) and egg phosphatidylcholine [46,47]. Figure 3. Chemical diagrams of surface preparation steps. (A) Diagram of amino silanization of Figure 3. Chemical diagrams of surface preparation steps. (A) Diagram of amino silanization of glass surface. (B) Diagram of PEG-ylation step with heterobifunctional biotin–PEG–succinimidyl glass surface. (B) Diagram of PEG-ylation step with heterobifunctional biotin–PEG–succinimidyl carbonate (SC) ester. (C) Diagram of the interactions between avidin and biotin. carbonate (SC) ester. (C) Diagram of the interactions between avidin and biotin. 3.2. Surface 3.2. Surface Functionalization Functionalization Typically, surface-immobilized avidin, streptavidin, neutravidin (from here on avTypically, surface-immobilized avidin, streptavidin, or or neutravidin (from here on avidin idin will be used interchangeably with any of these forms of avidin) tether biotinylated will be used interchangeably with any of these forms of avidin) tether biotinylated biomolecules biomolecules the imaging region. To immobilize avidinbiotins on the are surface, biotinsonto are the into the imagingtoregion. To immobilize avidin on the surface, introduced troduced ontoor the surface or during step. Formethod, the PEGapassivation surface before during thebefore passivation step. the Forpassivation the PEG passivation percentage method, a molecules percentagethat of passivate the PEG molecules passivate surface contain a biotin of the PEG the surfacethat contain a biotinthe moiety on the opposite end moiety on the opposite end from the ester group [43]. The ester group in the biotinylated from the ester group [43]. The ester group in the biotinylated PEG molecule reacts with the PEG molecule with the on the shown inpassivation Figure 3B.method, For the amine group onreacts the surface as amine showngroup in Figure 3B.surface For theas Tween-20 Tween-20 passivation method, researchers introduce to the imaging researchers introduce biotinylated BSA to the imagingbiotinylated device priorBSA to passivation. Thedebivice prior BSA to passivation. Thehydrophobic biotinylatedsurface BSA adheres to the hydrophobic surface before otinylated adheres to the before Tween-20 passivates the surface [42]. Tween-20 passivates the surface Forathe lipid of passivation either a fraction of For the lipid passivation method,[42]. either fraction the lipidsmethod, will be biotinylated [46] or avidin willwill be directly applied to theorimaging surface before passivation Avidin binds to the lipids be biotinylated [46] avidin will be directly applied to [47]. the imaging surface −15 M) [48]. This affinity comes from a number biotin with high affinity (KD~10 of beforemolecules passivation [47]. Avidin binds to biotin molecules with high affinity (KD~10−15 M) hydrogen formed aminoof acids of avidin’s biotin-binding site andthe theamino biotin [48]. This bonds affinity comesbetween from a the number hydrogen bonds formed between molecule as diagramed in Figure 3C. avidin forms a tetramer, avidin molecules bound acids of avidin’s biotin-binding site Since and the biotin molecule as diagramed in Figure 3C. to the avidin biotins forms on the aimaging surface still contain available bindingonsites immobilize Since tetramer, avidin molecules bound tobiotin the biotins theto imaging surbiotinylated molecules. face still contain available biotin binding sites to immobilize biotinylated molecules. 3.3. Protein Biotinylation 3.3. Protein Biotinylation One strategy One strategy to to directly directly biotinylate biotinylate proteins proteins of of interest interest involves involves introducing introducing an an AviTag [49–51]. The BirA ligase recognizes the 15 amino acid AviTag and conjugates AviTag [49–51]. The BirA ligase recognizes the 15 amino acid AviTag and conjugates biotin biotin the tag’s lysine residue [52,53]. BirA biotinylationoccurs occursthrough throughaa two-step two-step to the totag’s only only lysine residue [52,53]. BirA biotinylation reaction where biotin first reacts with ATP before the amine group of AviTag’s lysine residue attacks the ester in biotin (Figure 4C). Overexpressing BirA ligase can biotinylate the protein in vivo [53] or purified BirA can biotinylate the protein in an in vitro reaction system [53,54]. Other direct coupling methods include using biotinylated peptides [55] or ligating a biotinylated peptide to the C-terminus of a protein of interest [14]. Biotinylated peptides or molecules with a high affinity to fusion tags can also be used to introduce Sensors 2023, 23, 7691 reaction where biotin first reacts with ATP before the amine group of AviTag’s lysine residue attacks the ester in biotin (Figure 4C). Overexpressing BirA ligase can biotinylate the protein in vivo [53] or purified BirA can biotinylate the protein in an in vitro reaction sys5 of 20 tem [53,54]. Other direct coupling methods include using biotinylated peptides [55] or ligating a biotinylated peptide to the C-terminus of a protein of interest [14]. Biotinylated peptides or molecules with a high affinity to fusion tags can also be used to introduce biotin proteins of of interest interest[56]. [56].Biotinylated Biotinylatedantibodies antibodies [57,58] biotinylated nubiotin to to the the proteins [57,58] oror biotinylated nuclecleotide oligos [59–61] can indirectly couple proteins to the surface. Tethering the sample otide oligos [59–61] can indirectly couple proteins to the surface. Tethering the sample to to imaging surface does require high efficiency. majority of unbiotinythethe imaging surface does notnot require high efficiency. TheThe vastvast majority of unbiotinylated lated molecules will not stick to the passivated surface and at saturating conditions, most molecules will not stick to the passivated surface and at saturating conditions, most biotinbiotin-binding sites will bind to biotinylated biomolecules. binding sites will bind to biotinylated biomolecules. Figure4.4.Chemical Chemicaldiagrams diagramsof ofcommon commonprotein proteinlabeling labelingstrategies. strategies.(A) (A)Thiol–maleimide Thiol–maleimidereaction reaction Figure represents a common way to label cysteine residues in proteins. (B) NHS ester reaction with lysine represents a common way to label cysteine residues in proteins. (B) NHS ester reaction with lysine provides a way to label proteins. (C) The process of AviTag biotinylation as a way to tether proteins provides a way to label proteins. (C) The process of AviTag biotinylation as a way to tether proteins to immobilized avidin. to immobilized avidin. 3.4. Protein ProteinFluorescence FluorescenceLabeling Labeling 3.4. The requirements requirements for for fluorescent fluorescent labeling labeling are are more more stringent. stringent. One One of of the the most most comcomThe mon strategies conjugates fluorescent molecules with maleimide groups onto the thiol mon strategies conjugates fluorescent molecules with maleimide groups onto the thiol groupson onthe thecysteines cysteinesof ofproteins proteins(Figure (Figure4A). 4A).This Thisstrategy strategyworks worksbest bestfor forsmall small proteins proteins groups withoutessential essentialcysteine cysteine residues. residues. Similarly, Similarly, lysine lysine can can react reactwith withNHS NHSester estergroups groupson on without fluorophores(Figure (Figure4B). 4B).Recent Recentstudies studiesalso alsouse useunnatural unnaturalamino amino acids acids to toperform perform click click fluorophores chemistrywith withfluorophores fluorophores[54,62]. [54,62].This Thismethod methodworks workswell wellfor for large large proteins proteinsor orprotein protein chemistry complexesand andfor forproteins proteinsthat thatharbor harbor essential cysteine residues. Additionally, decomplexes essential cysteine residues. Additionally, thethe development of high-affinity protein fusion tags allows N- orNC-terminal fusionfusion tags totags provide a velopment of high-affinity protein fusion tags allows or C-terminal to prospecific and simple way toway bindtoa bind fluorophore to the target of interest [63]. Fluorescently vide a specific and simple a fluorophore to the target of interest [63]. Fluoreslabeled antibodies offer a potential alternative to these aforementioned methods. For all of cently labeled antibodies offer a potential alternative to these aforementioned methods. these, of fluorophores and the labeling strategy will depend on depend the biological For allthe of choice these, the choice of fluorophores and the labeling strategy will on the application. Roy et al. [33] provide a practical overview of the TIRF-based single-molecule experiments with Förster Resonance Energy Transfer (FRET). Sensors 2023, 23, x FOR PEER REVIEW Sensors 2023, 23, 7691 6 of 19 6 of 20 biological application. Roy et al. [33] provide a practical overview of the TIRF-based single-molecule experiments with Förster Resonance Energy Transfer (FRET). 4. Analysis Methods 4. Single-molecule data often exhibit exhibit inherent inherent noise noise stemming stemming from from both both the the system system Single-molecule under study study and and the the measurement measurement instrument. instrument. This noise can manifest in various forms, under including sample sample stage stage drift drift [64,65], [64,65], Gaussian Gaussian fluctuations fluctuations [66,67], non-Gaussian non-Gaussian variaincluding tions[68–70], [68–70],diffusive diffusivebehavior behavior [71,72], and even undefined sources [73,74]. Complications [71,72], and even undefined sources [73,74]. Complications tionsparticularly arise particularly when the nature of the underlying fluctuation is unknown, it arise when the nature of the underlying fluctuation is unknown, as it canas potentially follow follow either aeither Gaussian or non-Gaussian distribution. Consequently, extracting can potentially a Gaussian or non-Gaussian distribution. Consequently, exmeaningful information from single-molecule data poses significant challenges. In this tracting meaningful information from single-molecule data poses significant challenges. section, we willwe introduce key steps insteps the analysis with key examples (Figure (Figure 5). In this section, will introduce key in the analysis with key examples 5). Figure 5. Workflow of single-molecule imaging data analysis. Figure 5. Workflow of single-molecule imaging data analysis. 4.1. Point Spread Function Fitting 4.1. Point Spread high Function Fitting To achieve spatial resolution, it is essential to precisely localize the position Todetected achieve high spatial resolution, it is essential to precisely localize thea position of of each fluorophore. This localization is typically performed using technique called point spread function This (PSF)localization fitting, where the observed intensity distribution of a each detected fluorophore. is typically performed using a technique fluorophore fit to afunction mathematical the PSF. accurately determining the center called pointisspread (PSF) model fitting,ofwhere theBy observed intensity distribution of a of the PSF, the position of the fluorophore can be determined with sub-pixel precision, fluorophore is fit to a mathematical model of the PSF. By accurately determining the center enabling precise localization of single molecules. et al. [29] comprehensively of the PSF, the position of the fluorophore canSage be determined with sub-pixel evaluated precision, software for single-molecule localizationSage microscopy Many modules enabling packages precise localization of single molecules. et al. [29](SMLM). comprehensively evalufrom these software packages would be usable for TIRF-based single-molecule ated software packages for single-molecule localization microscopy (SMLM).fluorescence Many modimaging ules fromdatasets. these software packages would be usable for TIRF-based single-molecule fluorescence imaging datasets. 4.2. Extracting Information from Signal Among the most prevalent types of single-molecule imaging data are time series 4.2. Extracting Information from Signal signals characterized values ranging zero to an upper limit.data To extract desired Among the mostby prevalent types offrom single-molecule imaging are timethe series siginformation, several techniques have been developed to fit the noisy time series data to nals characterized by values ranging from zero to an upper limit. To extract the desired an idealized model involving discrete steps and dwell times [73]. One widely employed information, several techniques have been developed to fit the noisy time series data to an method is hidden Markov modeling (HMM) [75–79]. HMM enables the identification of idealized model involving discrete steps and dwell times [73]. One widely employed hidden (unobservable) states within a Markovian process, where the present and future method is hidden Markov modeling (HMM) [75–79]. HMM enables the identification of states depend solely on the current state, independent of the system’s prior states. The hidden (unobservable) states within a Markovian process, where the present and future idealized model