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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session D

Poster F4 - Nonlinear Structured Illumination Microscopy with rsEGFP2 and Liquid Crystal Variable Retarder

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Ashley Saju

Undergraduate Student Researcher
University of Illinois at Urbana-Champaign
Mount Prospect, Illinois, United States

Co-Author(s)

Shaoheng Li, MS

Graduate Student
University of Georgia, United States

Primary Investigator(s)

PK

Peter Kner

Associate Professor
University of Georgia, United States

Introduction: Super-resolution microscopy enables scientists to study subcellular structures and dynamics with nanoscale detail. Among the various super-resolution techniques, structured illumination microscopy (SIM) stands out for its compatibility with live-cell imaging. SIM doubles the diffraction-limited resolution to approximately 120 nm but is restricted to a resolution improvement factor of two. Nonlinear SIM (NSIM) utilizes reversible fluorescent proteins to generate a nonlinear response, allowing for the collection of higher spatial frequency information and theoretically extending the resolution infinitely. To maximize the nonlinear response from the emission light, the polarization of the illumination beams must be optimized to enhance the modulation depth of the illumination pattern. However, the dichroic mirror introduces a phase shift in the illumination light that affects the polarization of the beams. We propose correcting this phase shift by using a Liquid Crystal Variable Retarder (LCVR) in the illumination path, thereby improving the nonlinear response of the system.

Materials and

Methods: Image reconstruction: For SIM reconstruction, the fairSIM plugin in ImageJ was used, and for NSIM reconstruction, custom Python code from Kner’s lab was used.

Setups: LCVR testing setups (fig.1), Microscope setup (fig.2)

LCVR Testing Material: Thor Labs PM100D power meter, LCVR from Meadowlark Optics, half-waveplate (HWP), linear polarizer (LP).
Dichroic mirror: 1mm multi-bandpass, 3mm 488nm single-bandpass, 1mm 655nm single-bandpass, and 3mm 660nm single-bandpass
OBIS laser: 488nm, and 647nm
Lens: 50mm, 20mm, and 170mm.

LCVR Testing

Methods: The voltage of LCVR was set (0V-10V) to correct the phase shift in the system. The HWP and LP were used in all tests for determine the polarization of the system. A couple of preliminary tests were done to determine the setup layout:
- Laser beam size: By changing L2 (20mm lens vs. 170mm lens) while keeping L1 fixed (50mm). Bigger beam size (L2=170mm) has slightly better correction.
- LCVR placement: before vs. after dichroic mirror (fig.1C&1D)). LCVR placed before the dichroic mirror has slightly better correction.

With the layout fixed, we investigated the performance of different dichroic and the effects of LCVR’s orientation (fast vs. slow axis horizontal to table). The linearity of the polarization is obtained for all tests by rotating the LP to find the maximum and minimum laser intensities.
Control tests:
- The ideal case (fig.1A): Test with no dichroic mirror or LCVR. This represents the best polarization state of the system.
- The worst case (fig.1B): Test with dichroic mirror. This represents the polarization state after the dichroic mirror.

Results, Conclusions, and Discussions: LCVR Testing Results (fig.3): The LCVR was best able to correct the phase shift introduced by the 1 mm thick dichroic mirror although this dichroic had a greater base phase shift than the 3mm dichroic mirror (fig.3A). We tested at two wavelengths, and the 3 mm dichroic consistently performed worse than the 1 mm dichroic – Fig. 3A and 3B. Regarding the LCVR’s orientation (fig. 3B & 3C), the tests were inconclusive because each dichroic mirror had better correction with different orientations.

Widefield vs. SIM without LCVR vs. SIM with LCVR (fig.4): 100nm fluorescent beads were imaged with (fig.2B) and without the LCVR (fig.2A) using the 1 mm multi-bandpass dichroic mirror. Imaging with SIM, the structured illumination pattern without the LCVR has a modulation depth of 0.346 (fig.4B), and with the LCVR, has a modulation depth of 0.557 (fig.4C). There is a clear improvement in SIM resolution when the LCVR is used (fig.4D).

The LCVR seems to be an effective approach to correcting the phase shift that occurs from the dichroic mirror. With a 1mm dichroic mirror and large laser beam size, the LCVR placed before the dichroic mirror can clearly increase the modulation depth and therefore the resolution. The next step is to image U2OS live cells with actin labeled by rsEFGP2. Then reconstruct the image with NSIM with and without the LCVR to see if the results are consistent for NSIM.

Acknowledgements (Optional): This material is based upon work supported by a National Science Foundation Research Experiences for Undergraduates (REU) site program under Grant No. 1950581.