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Colson, L.; Kwon, Y.; Nam, S.; Bhandari, A.; Maya, N.M.; Lu, Y.; Cho, Y. Application of Lab-on-a-Chip Techniques for Single-Molecule Fluorescence Imaging. Encyclopedia. Available online: https://encyclopedia.pub/entry/49530 (accessed on 09 July 2024).
Colson L, Kwon Y, Nam S, Bhandari A, Maya NM, Lu Y, et al. Application of Lab-on-a-Chip Techniques for Single-Molecule Fluorescence Imaging. Encyclopedia. Available at: https://encyclopedia.pub/entry/49530. Accessed July 09, 2024.
Colson, Louis, Youngeun Kwon, Soobin Nam, Avinashi Bhandari, Nolberto Martinez Maya, Ying Lu, Yongmin Cho. "Application of Lab-on-a-Chip Techniques for Single-Molecule Fluorescence Imaging" Encyclopedia, https://encyclopedia.pub/entry/49530 (accessed July 09, 2024).
Colson, L., Kwon, Y., Nam, S., Bhandari, A., Maya, N.M., Lu, Y., & Cho, Y. (2023, September 22). Application of Lab-on-a-Chip Techniques for Single-Molecule Fluorescence Imaging. In Encyclopedia. https://encyclopedia.pub/entry/49530
Colson, Louis, et al. "Application of Lab-on-a-Chip Techniques for Single-Molecule Fluorescence Imaging." Encyclopedia. Web. 22 September, 2023.
Application of Lab-on-a-Chip Techniques for Single-Molecule Fluorescence Imaging
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This entry is adapted from the peer-reviewed paper 10.3390/s23187691

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. Lab-on-a-chip techniques have emerged as valuable tools in single-molecule fluorescence imaging, offering numerous advantages and enabling new possibilities for experimental design and analysis.

single-molecule fluorescence imaging lab-on-a-chip techniques

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][2][3][4][5][6][7][8][9][10][11][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, single-molecule 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][14][15][16][17][18][19][20][21][22].

2. Optical Systems for Single-Molecule Fluorescence Imaging

Fluorescence single-molecule imaging techniques rely on the utilization of optical radiation to probe individual molecules within a liquid or solid sample. To achieve successful single-molecule imaging, two key requirements must be met: (1) ensuring that resonant molecules are spatially resolved by the detector, and (2) providing a sufficient signal-to-noise ratio (SNR) for the single-molecule signal within a reasonable averaging time [22]. Consequently, a fundamental prerequisite for conducting single-molecule observations is to dilute the concentration of the target molecule of interest to exceedingly low levels (typically < 100 nM). The detection of single molecules via fluorescence-based methods demands careful optimization of the signal-to-noise ratio. Maximizing the signal requires the selection of an impurity molecule with the highest possible fluorescence quantum efficiency.
This approach harnesses recent advancements in fluorescence imaging techniques, including TIRF microscopy [23][24][25], super-resolution microscopy, and single-molecule localization microscopy [26][27][28][29][30][31][32]. TIRF microscopy, one of the most commonly employed tools in single-molecule fluorescence microscopy, capitalizes on the principle of total internal reflection. This phenomenon occurs when a laser beam strikes the interface between a medium with a higher refractive index (typically glass) and a medium with a lower refractive index (such as a sample solution) at an angle greater than the critical angle (Figure 1A). As a result, an evanescent wave is generated, which excites fluorophores in the immediate vicinity of the interface, facilitating the visualization of single molecules near the sample surface. TIRF microscopy is practically implemented by using either a quartz prism or the microscope objective to generate the evanescent field and illuminate surface-immobilized molecules (Figure 1B). TIRF microscopy offers exceptional optical sectioning and background suppression, leading to a high signal-to-noise ratio.
Figure 1. TIRF microscopy for single-molecule fluorescence imaging. (A) Principle of TIRF microscopy. (B) Types of TIRF microscopy: prism-type (P-TIRF) or objective-type (O-TIRF).
The evanescent field intensity, I(z), at a perpendicular distance z from the interface is described by Equation (1).
I z = I 0 exp ⁡ ( − z d ) ,
d = λ 0 4 π ( n 1   2 s i n 2 θ − n 2 2 ) − 1 / 2 ,
where 𝐼(0) represents the evanescent field intensity at the interface (Figure 1A). The characteristic 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.
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 parameters. High NA objectives are commonly used to maximize light collection and detection efficiency. The specific NA value depends on the imaging setup and the desired resolution and sensitivity. For conventional single-molecule fluorescence imaging, objectives with NA values ranging from 1.2 to 1.49 are frequently employed. These objectives offer a balance between high light collection efficiency and reasonable working distances. They are suitable for imaging samples in various configurations, including liquid solutions, solid surfaces, and biological specimens.
The choice of camera is another important factor in the detection of individual fluorophores. Ultimately, digital cameras capture the photons from individual fluorescent molecules and convert the light into electrical signals. The cameras used for single-molecule TIRF imaging tend to have quantum efficiencies above 80%, spectral range between 300 and 1100 nm, low readout noise, and millisecond readout speeds [33]. Electron multiplying charge coupled devices (EMCCDs) and the scientific complementary metal–oxide–semiconductor (sCMOS) devices are the most common types of cameras used in single-molecule imaging. 

3. Application of Lab-on-a-Chip Techniques for Single-Molecule Fluorescence Imaging

Lab-on-a-chip techniques have emerged as valuable tools in single-molecule fluorescence imaging, offering numerous advantages and enabling new possibilities for experimental design and analysis [34][35][36][37][38][39][40]. Lab-on-a-chip devices provide unparalleled control and manipulation capabilities, enabling precise management of fluid flow and sample handling in single-molecule imaging experiments through the utilization of a laminar flow regime. The controlled flow within microfluidic channels not only facilitates efficient sample processing but also aids in reducing background noise by removing unbound or non-specifically bound molecules, thereby enhancing the SNR. Moreover, lab-on-a-chip platforms leverage miniaturized channels and chambers to create controlled microenvironments for the delivery, mixing, and incubation of samples, as well as the manipulation of individual molecules.

3.1. Enhance Signal-to-Noise Ratio

One advantage of single-molecule imaging over ensemble studies is its superior time domain resolution for investigating molecular dynamics. However, this advantage can be compromised by the limited photostability of singlet exciton emission, which is prone to bleaching and blinking due to factors like O2 and intersystem crossing. The stochastic fluctuations resulting from blinking are unrelated to the underlying biological behavior. To overcome these challenges, microfluidics has been employed by incorporating oxygen scavengers and triplet quenchers into the imaging buffer [41][42]. By carefully designing the setup, this approach has recently facilitated the shortest observation durations [43]. It effectively addresses the limitation of time domain resolution posed by fluidic speed, particularly during fluidic mixing. A different method was shown [44], wherein the imaging channels were integrated with those consistently supplied with nitrogen ventilation. Furthermore, sophisticated microfluidic architectures can reduce flow velocities immediately after mixing, enabling longer optical interrogations [45].

3.2. Increase Sample Concentration

As mentioned, single-molecule fluorescence imaging techniques are limited to using pico- to nanomolar concentrations to ensure that only single molecules are resonant within the laser-probed volume and provide a sufficient SNR. However, many biologically relevant processes occur at micromolar level concentrations, necessitating a reduction in the conventional observation volume by three orders of magnitude. Here, arrays of zero-mode waveguides (ZMWs) consisting of subwavelength holes in a metal film provide a means to increase sample concentrations to the micromolar range while confining the observation volume to zeptoliter dimensions [46][47][48]. This breakthrough enables studies in the physiological concentration range and has been successfully applied in real-time, protein–protein interactions [48]. ZMWs have also been utilized to investigate ribosome-mediated translation processes, allowing the observation of tRNA transit in real-time at physiological concentrations [49]. Additionally, ZMWs have demonstrated versatility in studying biomolecular interactions, protein receptor diffusion, and oligomerization on living cell membranes [50].

3.3. On-Chip Single-Molecule Manipulation

On-chip devices have been developed with the capability to spatially modulate individual molecules with nanometer or even sub-nanometer sensitivities. A notable example is the microfluidic-based “DNA curtain”, which has recently emerged as an elegant on-chip tool for investigating DNA–protein interactions [16][17][18][51][52]. This technique involves driving DNA molecules that are tethered to a fluidic lipid bilayer on the surface. These molecules drift downstream under the influence of flow until they encounter a thin layer of metal, which serves as a diffusion barrier. Consequently, the DNA molecules align with each other, forming what is referred to as a DNA curtain [51]. This fluidic-chip setup has proven highly successful in unraveling the searching modes of a DNA repair complex at DNA damage and elucidating the disruption of a transcription complex by a DNA translocase at the single-molecule level [18]. In addition, Alwan et al. utilized a microfluidics-based single-molecule live cell fluorescence imaging to study the spatiotemporal dynamics of selectin ligands on the membrane tethers and slings during cell rolling [53].

3.4. Microenvironment Control

Lab-on-a-chip technology also affords precise control over the microenvironment surrounding single molecules. Variables such as temperature, pH, and chemical gradients can be precisely manipulated within microfluidic devices, providing valuable insights into the impact of different conditions on the behavior and functionality of biomolecules. This level of control allows for the investigation of dynamic processes under various physiological or pathological conditions, mimicking complex biological environments. For example, Zhang et al. studied the in situ conformational response of single biomolecules such as DNA to a change in environmental solution conditions [54]. This level of control allows researchers to probe biomolecular interactions, enzymatic activities, and other dynamic processes with exceptional temporal resolution.
Moreover, lab-on-a-chip devices possess the remarkable capability of automation when integrated with other techniques. This integration not only minimizes experimental bias but also facilitates high-throughput screening, data acquisition, and analysis, which are indispensable for conducting large-scale single-molecule studies. By automating microfluidic processes, researchers can streamline their experiments, achieve consistent and reliable results, and analyze vast amounts of data efficiently.

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Subjects: Engineering, Biomedical
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Louis Colson
,
Youngeun Kwon
,
Soobin Nam
,
Avinashi Bhandari
,
Nolberto Martinez Maya
,
Ying Lu
,
Yongmin Cho
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Update Date: 26 Sep 2023
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