| 1. PicoSIM – The idea |
|
As a wide-field technique, SIM is potentially significantly faster than scanning techniques such as confocal microscopy. However, the requirement to acquire three images reduces its speed.
Polarised illumination coded Structured Illumination Microscopy (PicoSIM) encodes the three individual light patterns needed for conventional SIM in the polarisation of the illumination light.Benefit: Acquisition of complete SIM data in one single exposure.
This would allow ultrafast optical sectioning with high spatial resolution in all three dimensions. |
|
|
|
|
2. How it works
|
|
Illumination
| SIM |
PicoSIM |
|
|
| 3 illumination images with sinusoidal intensity pattern, arbitrary polarisation. |
1 illumination image with constant intensity and varying linear polarisation. |
|
|
|
Effective Illumination
| If the light emitted by the sample retains at least some of the illumination polarisation, the orientation of a polarising analyser in the emission pathway can retroactively alter the effective position of the illumination pattern. |
|
|
|
|
|
3. Performance
|
|
| Because of fluorescence anisotropy the full degree of input polarisation cannot be preserved. Hence, picoSIM images recorded after polarisation filtering correspond to conventional SIM images with reduced illumination pattern contrast. This leads to a reduced signal-to-noise ratio as compared to conventional SIM. |
|
|
4. Setup
|
|
|
|
|
Illumination
- Principle similar to conventional SIM
- Incoherence aperture guarantees that diffraction orders can be separated
- 0 order is blocked
- +1st and -1st orders are right- & left-circularly polarised respectively: Their interference in the sample yields the desired polarisation distribution
|
Detection
- Emitted light is split into 3 identical components
- 3 components are filtered using polarization analysers oriented at 0°, 60°, 120°
- 3 components are simultaneously detected on different areas of the same camera
|
|
|
|
5. Preliminary results
|
|
Data acquired using coherent laser illumination and an additional magnification on the illumination side of the system. Furthermore, just one analyser instead of three was used on the detection side, i.e. the emission light was not yet split into 3 components, but the analyser was successively rotated.
(Objective: 100x, 1.4 NA oil immersion) |
|
| Analyser 0˚ |
Analyser 60˚ |
Analyser 120˚ |
sample: Fluorescent plane |
|
|
| PicoSIM reconstruction |
Wide-field |
sample: Adult rat cardiomyocytes |
|
