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Quant. Biol.    2016, Vol. 4 Issue (3) : 177-191    https://doi.org/10.1007/s40484-016-0083-0
REVIEW
Single molecule fluorescence spectroscopy for quantitative biological applications
Ruchuan Liu1,Yuliang Li2,Liyu Liu1,2()
1. College of Physics, Chongqing University, Chongqing 401331, China
2. Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China
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Abstract

Single molecule techniques emerge as powerful and quantitative approaches for scientific investigations in last decades. Among them, single molecule fluorescence spectroscopy (SMFS) is able to non-invasively characterize and track samples at the molecular level. Here, applications of SMFS to fundamental biological questions have been briefly summarized in catalogues of single-molecule counting, distance measurements, force sensors, molecular tracking, and ultrafast dynamics. In these SMFS applications, statistics and physical laws are utilized to quantitatively analyze the behaviors of biomolecules in cellular signaling pathways and the mechanisms of biological functions. This not only deepens our understanding of bio-systems, but also provides a fresh angle to those fundamental questions, leading to a more quantitative thinking in life science.
Author Summary   

It is essential and fundamental to understand life science at the molecular level. Single molecule techniques are able to uncover the mystical insights of the biological systems and quantitatively measure the important parameters, and thus become extensively applied in biology. The unique advantage of fluorescence detection makes single molecule fluorescence spectroscopy (SMFS) especially suitable in this field, and plenty of applications of SMFS to locate and track biomolecules have supplied us tremendous new quantitative knowledge. Here, we summarize the applications in five catalogues to show that SMFS brings the new angle of view and more quantitative thinking to life science.
Keywords single-molecule fluorescence spectroscopy      biomolecule detection      molecular tracking      molecular dynamics      molecular mechanism     
PACS:     
Fund: 
Corresponding Author(s): Yuliang Li,Liyu Liu   
Just Accepted Date: 13 July 2016   Online First Date: 29 August 2016    Issue Date: 07 September 2016
 Cite this article:   
Ruchuan Liu,Yuliang Li,Liyu Liu. Single molecule fluorescence spectroscopy for quantitative biological applications[J]. Quant. Biol., 2016, 4(3): 177-191.
 URL:  
https://academic.hep.com.cn/qb/EN/10.1007/s40484-016-0083-0
https://academic.hep.com.cn/qb/EN/Y2016/V4/I3/177
Fig.1  Scheme of Single-molecule fluorescence.

(A) An energy level diagram of a fluorescent molecule and (B) a trajectory of a single fluorescent molecule showing the intensity decrement in one step.

Fig.2  Magnetic tweezers used to measure the binding events.

The Ni-NTA (labeled N) grafted slides containing the talin rod fixed through its 6×His N terminus (labeled H) to the glass and with the avidinated magnetic bead bound to its biotinylated C terminus (labeled B) was placed over the objective. Alexa 488–vinculin head was added to the slides for the period of the incubation. The talin rod and vinculin head structures are represented in green and yellow, respectively. The arrow shows the direction of the movement of the beads when they are pulled using the magnetic tweezers.

Fig.3  Diagram of photobleaching events of Alexa 488-vinculin head bound to (A) talin rod, (B) dimeric tandem talin rod, and (C) α-actinin.

Histograms show the number of beads per photobleaching event. In all cases, blue, gray, and green colors correspond with no force, 2-pN force, and 12-pN force applied, respectively. The talin rod, talin dimeric tandem (positive control), and α-actinin (negative control) showed maximally 1 and 3, 2 and 6, and 1 and 1 photobleaching events (black arrows) when no force and 12 pN force, respectively, was applied.

Fig.4  EGFP-Talin1-mCherry as a tool to measure talin 1 length.

(A) Schematic of stretching of labeled talin molecules: unfolding from a 51 nm length in the relaxed state. This can be measured in focal adhesions under stretch where the separation of the termini is apparent in two channel TIRF microscopy. Using the same edge detector criterion for both the GFP and mCherry signal reveals the dislocation of the ensemble. (B) Verification that EGFP-Talin1-mCherry distributes as expected for talin. Distribution of EGFP, mCherry, and antibody (alexa 647) staining shows colocalization of the modified and the endogenous talin (green for EGFP, red for mCherry, and blue for alexa 647 in the superposition panel) (this is under conditions of high expression but the same colocalization is observed at low expression). The image was adapted from [42] with permission.

Fig.5  Displacements after suppression.

(A) Time evolution of the stretching after adding Blebbastatin or Y-27632, respectively. The total displacement and the amplitude of the stretching are decreased very noticeably. Even though for the Y-27632, it is still clearly larger than the error of observation. (B) Vinculin head added: The vinculin head construct stretched out the talin molecule and left it stretched. There are still observable oscillations in stretching, but the amplitude of change is dramatically suppressed. The image was adapted from [42] with permission.

Fig.6  A scheme of two-state interaction model of binding of calmodulin and C28W and single-molecule FRET results.

(A) calmodulin bind with C28W in two states: the C-terminal of calmodulin binds to the N-terminal domain of C28W; calmodulin tightly binds to C28W through both domains. (B) distributions of single-molecule FRET efficiency; and (C) calculated relative donor-acceptor distance (R/R0). The blue and red curves are the Gaussian components used to fit the distributions, and the black curve is fitted results. Reprinted with permission from Ref. [44]. Copyright 2006 American Chemical Society.

Fig.7  The cartoon of molecular tension sensor and the force calibration.

The sensor consists of mTFP1, elastic linker (GPGGA)8 and venus (A) without force and (B) once a force is applied the extension of the linker can lower the FRET efficiency. (C) The force is calibrated as a function of FRET efficiency. The image was adapted from [62] with permission

Fig.8  Time schedule of various molecular process (Reproduced from Ref. [111] with permission from The Royal Society of Chemistry).
Fig.9  Two color fs phase control of individual LH2 complexes at room temperature. with permission (The image was adapted from [113])
SMFS,single-molecule fluorescence spectroscopy
APD,avalanche photodiode
PMT,photomultiplier tube
EMCCD,electron-multiplying charge-coupled device
GFP,green fluorescent protein
EGFP,enhanced green fluorescent protein
NTA,Nitrilotriacetic acid
PALM,photo-activated localization microscopy
PSF,point spread function
E. coli,Escherichia coli
FRET,Förster Resonance Energy Transfer
smFRET,single-molecule FRET
TSMod,tension sensor module
mTFP,Clavularia teal fluorescent protein
SMT,single-molecule tracking
HoH,hand-over-hand
LH2,light harvesting 2
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