Single Molecule Imaging
Single Molecule Tracking (SMT) is the method of choice for quantification of the dynamics of molecules moving through the cytoplasm or nucleus, or associated with intracellular targets such as DNA, transcription factories, nuclear pores. Other microscopy methods, such as Fluorescence Recovery After Photobleaching (FRAP), and Fluorescence Correlation Spectroscopy (FCS), are based on population analysis and mathematical modeling, which adds additional difficulties in data analysis. The powerful technique of SMT derives data from direct observation of individual molecules. SMT requires bright photostable fluorophores suitable for in vivo labeling of the proteins and special types of illumination increasing the signal to noise ratio in the sample.
OMC adapts and develops SMT techniques for live cells. This includes the development of imaging and analysis protocols and Matlab-based routines for tracking and estimation of biophysical parameters, such as diffusion rate or the binding rate of molecules. Three types of imaging for SMT, complementing each other, are currently available at OMC. (1) Two custom-built microscopes are based on HILO illumination producing Highly Inclined Laminated Optical sheet. They are equipped with EMCCD cameras allowing acquisition of the movies of fluorescently labeled molecules with time resolution as low as 10 ms. (2) Lattice Light Sheet (LLS) microscope is available for single molecule tracking in 3D.This type of illumination reduces photobleaching and allows prolonged tracking of single molecules. This technique is currently under development. (3) MINFLUX nanoscope, based on a state of the art technique of precise localization by a minimum of fluorescence, allows acquisition of 2D and 3D molecular tracks with nano-precision and high temporal resolution as high as 0.1 ms. Imaging technique and analysis of MINFLUX data are under development.
New technology MINFLUX is based on excitation of the fluorescent molecules with doughnut-shaped beam, and photon-counting. Exact position of the molecule is detected by a minimum of fluorescence; thus, it requires low photon count and imaging is fast and non-photodamaging. Resolution is not dependent on wavelength and molecular tilt. This system may reach a level of resolution up to 3 nm, bridging the gap between the light and electron microscopy. MINFLUX may be used for molecular mapping of the intracellular structures such as organelles and high molecular complexes (centromeres, centrosomes, Cajal bodies, etc.).MINFLUX of OMC is oe of the first in USA, this is a brand-new technique. Currently, imaging is limited to two colors detected in a single infra-red channel and differentiated ratiometrically. OMC works on expanding the color palette for multicolor imaging, and adapting the techniques to a wide range of samples.