Circular Intensity Differential Scattering for Label-Free Chromatin Characterization: A Review for Optical Microscopy

Abstract

Circular Intensity Differential Scattering (CIDS) provides a differential measurement of the circular right and left polarized light and has been proven to be a gold standard label-free technique to study the molecular conformation of complex biopolymers, such as chromatin. In early works, it has been shown that the scattering component of the CIDS signal gives information from the long-range chiral organization on a scale down to 1/10th-1/20th of the excitation wavelength, leading to information related to the structure and orientation of biopolymers in situ at the nanoscale. In this paper, we review the typical methods and technologies employed for measuring this signal coming from complex macro-molecules ordering. Additionally, we include a general description of the experimental architectures employed for spectroscopic CIDS measurements, angular or spectral, and of the most recent advances in the field of optical imaging microscopy, allowing a visualization of the chromatin organization in situ.

Keywords: DNA organization; biopolymers; microscopy; polarization.

Figure 1

Block diagram of a general MM polarimeter. PSG: Polarization States Generator. PSA: Polarization States Analyzer. PSD: Polarization States Detector. ${\overrightarrow{S}}_{in}$ and ${\overrightarrow{S}}_{out}$ are the input and output polarization states described by the Stokes vectors.

Figure 2

Block diagram of general CIDS setups using one PEM in the PSG (a) with a single detector and (b) with a two channel detection based on the DoA method. LP: Linear Polarizer. PEM: Photoelastic Modulator. PSA: Polarization States Analyzer. LA: Lock-in Amplifier. BS: Beamsplitter. D: Detector.

Figure 3

Block diagram of a general complete MM polarimeter using (a) two PEMs and (b) four PEMs with a single detection coupled with an electronic synchronization. The output intensity temporal spectrum exhibits multiple frequencies $k.{\omega }_1$ and $k^{\prime }.{\omega }_2$, expressed as a mathematical product between Fourier amplitudes $A({\omega }_1,{\omega }_2)$ and the Bessel functions $J_1$ and $J_2$, leading to the combination of the MM elements ($m_{ij}$). The resulting set of equations allow recovering the full MM. LP: Linear Polarizer. PEM: Photoelastic Modulator. LA: Lock-in Amplifier. D: Detector. DAQ: Data Acquisition board. FPGA: field-programmable gate array.

Figure 4

Block diagram of general CIDS experimental setups for (a) spectral and (b) angular measurements. PSG: Polarization States Generator. PSA: Polarization States Analyzer. $\theta$: Scattering angle. LA: Lock-in Amplifier. D: Detector.

Figure 5

Block diagram of different optical microscope architectures allowing the imaging of the pixel-by-pixel CIDS signal. (a) Optical microscope in wide-field configuration using two Pockels cells, inspired by [124,125]. (b) Optical microscope using one photoelastic modulator synchronized with a lock-in amplifier and an XY translating sample holder, inspired by [100,126,127]. (c) Optical scanning microscope using a photoelastic modulator synchronized with a lock-in amplifier, inspired by [128,129,130]. PC: Pockels Cell. PEM: Photoelastic Modulator. F: monochromatic filter. LP: Linear Polarizer. LA: Lock-in Amplifier. PMT: Photo-multiplier tube. GS: galvanometric scanner.

Figure 6

(a) Normalized CIDS image of an isolated HEK nucleus after extraction. (b) Fluorescence image of the same isolated HEK nucleus labeled with Hoechst. (c) Merge of images (a,b). (d) Intensity plot from the orange arrow in (c). The blue plot is the intensity profile from the CIDS image (a), and the green plot is the Hoechst profile from Image 4. (b). The dashed lines indicate the estimated area of the nucleus. Reproduced with permission from [130].

Figure 7

Schematic principle of the scattering process into the PSF volume (a) without digestion and (b) after n hours of digestion. (c) Principle of the SNR decreasing and imaging contrast quality improvement after the expansion process. Reproduced with permission from [140].

Figure 8

Images of HEK cells labeled with Hoechst in CIDS (a,e,i) and fluorescence (b,f,j) modalities. A merged image of both modalities is presented in (c,g,k), and a line profile relative to the yellow line in those pictures is shown in (d,h,l). Here, CIDS is represented in blue, while the fluorescence signal is represented in green. Reproduced with permission from [140].