The emergence of fluorescence microscopes has made the observation of organelles possible. To observe finer targets, single-molecule fluorescence imaging is required. Today, we share a single-molecule fluorescence imaging study conducted this year using a TIRF (Total Internal Reflection Fluorescence) microscope: exosome secretion kinetics are controlled by temperature.
01 Why Use a TIRF Total Internal Reflection Fluorescence Microscope?
TIRF Total Internal Reflection Fluorescence Microscope MF53-TIRF
The TIRF total internal reflection fluorescence microscope utilizes the evanescent wave generated by total internal reflection of light to observe fluorescence in an extremely thin region. Compared with conventional fluorescence microscopes (wide-field fluorescence), this microscope has significantly lower background fluorescence, enabling higher signal-to-noise ratio and more detailed fluorescence imaging. It is particularly suitable for dynamic observation of membrane substances.
Evanescent Wave ①
The evanescent wave is an optical phenomenon. When the excitation light is incident at a specific angle, total internal reflection occurs, and all excitation light is reflected. An excitation light with a depth of only a few hundred nanometers and an exponentially decaying intensity is formed near the sample surface of the reflection plane, known as the evanescent wave.
Comparison Between Ordinary Fluorescence Imaging and TIRF Imaging ①
Using the evanescent wave, the TIRF total internal reflection fluorescence microscope can confine the excitation range to an extremely thin region of the sample surface, avoiding the blurred halo formed by fluorescence excitation outside the focal plane of traditional fluorescence microscopes and greatly improving the signal-to-noise ratio and resolution. Due to the exponential decay of evanescent wave intensity, it is most suitable for membrane-related research.
02 Exosome Secretion Kinetics Are Controlled by Temperature
Let’s look at a research paper case to understand the application advantages of TIRF total internal reflection fluorescence microscopy: super-high resolution and dynamic observation.
Visualizing pH-Sensitive Proteins Using CD63-pHluorin
CD63-pHluorin is used to visualize the fusion process of exosomes with the plasma membrane. The TIRF total internal reflection fluorescence microscope can achieve single-molecule dynamic tracking observation, requiring a high-frame-rate and high-sensitivity microscope camera, such as the back-illuminated sCMOS scientific camera MSH12.
Distinguishing Different Exosome Activities by Imaging Analysis ②
Single-molecule fluorescence imaging studies usually involve data statistics and analysis, often requiring algorithm design for automated analysis and quantitative processing. For example, this paper uses MATLAB scripts, which can be downloaded from GitHub.
Reliability Verification of Imaging Analysis to Exclude Lysosome or Vesicle Transport ②
Imaging analysis of CD63-pHluorin is used to visualize exosome fusion with the plasma membrane, excluding lysosome or vesicle transport.
Exosomes Exhibit Multiple Kinetic Modes During Fusion with the Plasma Membrane ②
Algorithm analysis shows that exosomes have multiple kinetic modes during fusion with the plasma membrane.
Exosome-Plasma Membrane Fusion Events Are Controlled by Temperature ②
Analysis of different kinetic modes shows that exosome-plasma membrane fusion events are controlled by temperature.
Model Validation ②
A model is used to validate and explain the kinetics observed in the experiment.
Further Kinetic Analysis ②
xosomes dock before fusing with the plasma membrane.
In summary, the TIRF total internal reflection fluorescence microscope MF53-TIRF is an ideal instrument for dynamic observation of cell surface substances, such as molecules fixed on the coverslip or cell membrane. Based on TIRF, MINGMEI also offers dSTORM super-resolution imaging solutions. Interested teachers are welcome to contact us.
If you are interested in this paper or wish to obtain the MATLAB automatic analysis and processing scripts used in the paper, please refer to the information in the application sources ②.
Citation Sources:
① Fish KN. Total Internal Reflection Fluorescence (TIRF) Microscopy. Curr Protoc. 2022 Aug;2(8):e517. doi: 10.1002/cpz1.517. PMID: 35972209; PMCID: PMC9522316.
② Mahmood A, et al. Exosome secretion kinetics are controlled by temperature. Biophys J. 2023 Apr 4;122(7):1301-1314. doi: 10.1016/j.bpj.2023.02.025. Epub 2023 Feb 22. PMID: 36814381; PMCID: PMC10111348.