Advances of super-resolution fluorescence polarization microscopy and its applications in life sciences

Abstract

Fluorescence polarization microscopy (FPM) analyzes both intensity and orientation of fluorescence dipole, and reflects the structural specificity of target molecules. It has become an important tool for studying protein organization, orientational order, and structural changes in cells. However, suffering from optical diffraction limit, conventional FPM has low orientation resolution and observation accuracy, as the polarization information is averaged by multiple fluorescent molecules within a diffraction-limited volume. Recently, novel super-resolution FPMs have been developed to break the diffraction barrier. In this review, we will introduce the recent progress to achieve sub-diffraction determination of dipole orientation. Biological applications, based on polarization analysis of fluorescence dipole, are also summarized, with focus on chromophore-target molecule interaction and molecular organization.

Polarisation optics for biomedical and clinical applications: a review

“…Those parameters have been adopted in different polarimetric applications 16 , 20 , 22 , 23 , 32 , 80 , 81 . The polarisation angle (PA) and intensity of the linear SOP also can be defined, with respect to dipole orientation applications 153 – 155 (see Fig. 5a (iv)).…”

“… a Stokes vector based approaches: (i) DOP, DOLP and DOCP 2 – 5 ; (ii) Stokes vector projection approach 158 ; (iii) Stokes vector location approach 159 ; (iv) Dipole orientation differentiation approach. Polarisation angle (PA) and intensity are used as main parameters 154 , 155 . b MM based approaches: (i) MMPD method 52 : diattenuation (D), retardance (R) and depolarisation (Δ) (all of them maintain linear/circular components); and (ii) MMT method 57 : depolarisation (1-b; associated with small molecule scattering), level of linear anisotropy (t1), azimuth orientation of the anisotropy (α1), and more 179 ; (iii) PFP method 95 ; (iv) Property of symmetry and asymmetry 168 , 169 .…”

“…Much existing biomedical polarimetry research concerns sensing of bio-information in a label-free way without extraneous dyes 16 , 22 , 24 . In other areas, polarimetry can be used to characterise the vectorial information of fluorescence dyes, as the dipole orientation of the fluorophore is encoded in the polarisation state of the emitted light 154 , 155 . The SOP of such emission is always in a linear state; hence the polarisation angle (PA) and intensity of the linear SOP are quantities that can be harnessed, such as in biomedical applications in super-resolution microscopy 153 , 181 , 182 .…”

“…Here we briefly summarise common phantoms used for biomedical polarimetric techniques. These techniques include polarised wide-field microscopy 16 , 24 , 183 , polarised light spatial frequency imaging 184 , polarimetric endoscopy 185 – 190 , spectral light scattering polarimetry 18 , 82 , 191 – 193 , polarised fluorescence spectroscopy 194 – 196 , polarised confocal microscopy 197 , polarised Raman-spectroscopy 198 , 199 , polarised super-resolution microscopy 154 , 155 , polarisation sensitive optical coherence tomography 200 – 218 , non-diffraction beam polarimetry (such as Bessel beam based) 219 , polarisation-resolved nonlinear microscopy (including second/third harmonic generation) 220 – 226 , and polarised speckle imaging 213 , 227 (several techniques will be mentioned again in the Discussion). The relationship between incoherence and depolarisation of the light should be kept in mind when considering coherence based polarimetric techniques: they are different but related optical concepts.…”

Global fitting for high-accuracy multi-channel single-molecule localization

“…Compared to the single-channel SMLM, data analysis for all these methods is complicated by the fact that measures from two or more channels have to be combined to result in the additional information (color, z-position, polarization state, interference phase, etc.). Typically, this is achieved by first fitting the fluorophores individually in each channel to extract corresponding parameters, and then combining the returned parameters from different channels to obtain the extra information [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] . Separate fitting of an individual fluorophore present in two channels is not optimal, as we neglect the information that the fitting parameters (e.g., 3D positions and photons) are highly correlated.…”