Total Internal Reflection-Fluorescence Correlation Spectroscopy

Nanoparticles With Aggregation-Induced Emission for Monitoring Long Time Cell Membrane Interactions

“…In the last decade, along with the thriving of research in the single molecule and subwavelength level, the TIRFM system has been utilized and developed extensively. TIRFM has been used in technical innovations such as smFRET [17], TIR-FCS [10], SPT [9] for single molecule detection and SIM [18], STORM [19] for fluorescence super-resolution microscopy and these reflect its flexibility in the related research. The combination of both advantages of AIE nanoparticles and the TIRFM system provides a fresh optional cell tracing tool and allows for more detailed studies of biological process in this platform.…”

“…In microscopy systems, TIRFM is commonly realized in two types: the prism-based type and the objective-based type [7]. Though comparatively complicated, the objective-based type shows its advantages with sample-top accessibility, higher light collection efficiency and better image quality [10], which makes it more flexible and stable in biological application. In this geometry, an objective lens of high numerical aperture (> 1.4) [11] is utilized for both excitation and signal collection.…”

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

“…For example, TIRF can be combined with fluorescence correlation spectroscopy to measure binding at, and diffusion near surfaces, a technique known as TIR-FCS [35,36]. It can also be combined with single-molecule mechanical measurements [37] by optical tweezers [38] or a variant of AFM [39].…”

Quantitative fluorescence nanospectroscopy of nucleotide excision repair : from single molecules to cells

“…After some technical improvements (mainly a function of precisely autopositioning along z-axis, i.e., optical path), the quantitative on-line confocal FCS can be the most important method to construct the interfacial architecture of molecular interactions since it is, to the best of our knowledge, a unique technique that can measure molecular mobility both in solution and at interface. In contrast, the TIRF FCS technique [303][304][305][306][307][308][309] based on the total internal reflection fluorescence microscopy can only perform FCS measurements at surface (< 200 nm depth into solution).…”

“…Compared with laser scanning fluorescence confocal (LSFC) microscope, obvious advantages in TIRF microscopy for NSL are as follows: 1) high temporal resolution allows fast kinetic image acquisition and simultaneous observation for many molecular events in a large field of view of microscope; 2) localization accuracy can be theoretically predicted and is dependent on Poisson distributed photon noise and normally Gaussian distributed background noise from camera. In contrast, the position precision of piezo stage in LSFC microscope is dependent on scanning step size and can induce additional localization error for NSL; 3) Very thin excitation volume of the order of 100 nm depth (it is ~1.2 µm for LSFC microscope) is ideal for capturing high resolution images with very high signal to noise ratio, but it can also be a drawback for the study of molecular interaction especially in TIRF-FCS [303][304][305][306][307][308][309] since surface-molecule interaction or other interfacial effects (see Chapter 2) can play a significant role at glass-solution interface and thus has to be minimized or eliminated prior to the actual experiments for real molecular interaction systems. Current software platform of our TIRF microscope is commercial Andor Solis, which only has basic function for image acquisition from the two EMCCD cameras.…”