Open-source Micro-Manager Plugin for Live-view Imaging of Fluorescent Dipoles

Summary

We developed an open-source Micro-Manager plugin, which enables live-view observation of fluorescent dipoles on a structured illumination microscope. The plugin supports observation of both 2D and 3D dipole orientation.

Abstract

Fluorescence polarization microscopy (FPM) can image the position and dipole orientation of fluorophores. Despite the achievements of super-resolution fluorescence polarization microscopy, their reliance on post-acquisition hinders real-time observation. Polarized structured illumination microscopy (pSIM) offers super-resolution imaging of fluorescent dipoles with fast imaging speed and is well-suited for live-cell applications. We developed an open-source implementation for real-time reconstruction of polarization images and display of the fluorescent dipoles. Additionally, we extended the method to achieve 3D orientation mapping (3DOM), broadening its utility for complex biological studies.Furthermore, we have presented a thorough introduction to extending an existing SIM microscope on polarization imaging and provided a detailed configuration guide of Micro-Manager 2.0 to control the microscope, enabling real-time preview of polarized imaging. Additionally, we have provided the MATLAB code for full reconstructionencompassing both pSIM and 3DOM. This comprehensive guide aims to assist beginners in quickly mastering and easily getting started with the operations.

Introduction

Fluorescence polarization microscopy (FPM) has emerged as a powerful technique for simultaneously imaging both the position and dipole orientation of fluorophores, offering profound insights into biological imaging^1,2. By facilitating direction observation of biomolecules' orientations, FPM unveils the intricate arrangement of macro-molecules such as actin^3,4,5, microtubule⁵, septin⁶, DNA filament^7-9, nuclear....

Protocol

1. Extending an existing structured illumination microscope for polarization imaging

1. Prepare a SIM microscope.
   NOTE: We assume that readers have a certain foundation in microscope setup and already possess a structured light microscope. If you lack relevant experience, refer to this paper, which provides a detailed description of how to build a SIM microscope¹⁶. The polarization control unit of "HWP+WQP+LCVR" can be replaced by a pizza vortex wave plate in our pSIM work⁵ or a pizza half wave plate¹⁷.
2. Measure polarization extinction ratios ....

Results

The pSIM method can be performed on SIM microscopes based on the interference using s-polarized laser beams. s-polarization interference is the mostly widely used type of SIM and generates high-contrast illumination stripes. The academic prototype of a microscope setup is included in the original work of pSIM⁵. Briefly introduced in Figure 1, a spatial light modulator (SLM) generates the ±1 order of diffractive beams and a pizza half w.......

Discussion

In our study, we developed a plugin that allows real-time preview of two polarization imaging techniques, pSIM and 3DOM. Both technologies can be performed in an existing SIM system with slight modification. We have provided the detailed steps to install the pSIM and 3DOM microscope and set up Micro-Manager to control the microscope and demonstrate how to obtain the live-view polarization results. The experimental results include the actin filament imaged by pSIM and λ-DNA imaged by 3DOM.The orientation of the phall.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 nm Fluorescent beads	Invitrogen	F8801
4% Formaldehyde solution	Invitrogen	R37814
Camera	Tucsen	Dhyana 400BSI V3	https://www.tucsen.com/download-software/
Denture base materials (Type I Thermally setting type, liquid)	New Century Dental	N/A
Dulbecco’s Modified Eagle’s Medium	Gibco	C11995500BT
Eclipse TE2000 Inverted Microscope	Nikon	TE2000 E
Fetal Bovine Serum	Gibco	10099141C
MATLAB R2019b	MathWorks	Version R2019b	https://ww2.mathworks.cn/downloads/
MetroCon V4.0	Kopin	Version 4.0	Software of Spatial light modulator
Micro-Manager 2.0	μΜanager	Version 2.0	Download Micro-Manager Latest Release
MS-2000 XYZ Automated Stage	Applied Scientific Instrumentation	MIM3	https://www.asiimaging.com/support/downloads/usb-support-on-ms-2000-wk-controllers/
myDAQ	National Instruments	781325-01	Software and Driver Downloads - NI
OBIS 561 nm LS 20 mW Laser	Coherent	1325777
Phalloidin-AF568	Invitrogen	A12380
Phosphate buffered saline	Corning	21-040-CV
Poly Methyl Methacrylate	Solarbio	M9810
ProLong Diamond	Invitrogen	P36980
Spatial light modulator	Kopin	SXGA-12
SYTOX orange nucleic acid stain	Invitrogen	S11368
Triton X-100	Invitrogen	HFH10
Trypsin	Gibco	25200056
λ-DNA	Invitrogen	S11368