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 pore complex¹⁰, and membrane proteins¹¹. Its high-speed, non-invasive, and live-cell compatible capabilities allow for the tracking of molecular rotation dynamics with high temporal resolution^11,12. When integrated with bioforce probes, FPM not only maps force magnitudes at subcellular resolution but also measures force directions, thus advancing our understanding of biomechanical processes by revealing the direction of forces¹³.

In the past decades, super-resolution fluorescence polarization microscopy has undergone rapid evolution. A notable advancement in this field is single-molecule orientation localization microscopy, also termed SMOLM, which can localize both the position and orientation of fluorophores, thus enabling multi-dimensional localization. Polarization measurement in SMOLM can be performed using polarization excitation modulation⁷, multi-channel polarized detection³, or engineered polarization-sensitive point spread function (PSF)¹⁴. Despite SMOLM achieving spatial resolution on the order of tens of nanometers and measuring the polarization of single molecules, it suffers from prolonged imaging time. This is due to the repetitive cycles of fluorophore blinking and localization, which pose a challenge for video-rate imaging and live-cell applications.

In contrast, polarized structured illumination microscopy (pSIM) offers a spatial resolution of approximately up to 100 nm, coupled with the acquisition of polarization information with the same SIM dataset. Notably, pSIM can achieve video-rate imaging speeds and is highly compatible with live-cell imaging, without stringent requirements on fluorescent molecules. Recently, pSIM has successfully revealed the actin ring structure in the membrane-associated periodic skeleton (MPS)⁵ and enabled super-resolution mapping of biological forces¹³.

However, pSIM requires post-acquisition image reconstruction, which prevents real-time visualization of polarization results. This delay hinders the immediate observation of biological phenomenon, preventing researchers from quickly capturing biological phenomena of interest and making real-time adjustments to samples and imaging conditions. To address this limitation, we have developed an open-source implementation that facilitates image acquisition and real-time reconstruction and displaying of polarization results, based on the ImageJ and Micro-Manager platform (https://github.com/KarlZhanghao/live-pol-imaging).

Furthermore, while pSIM has been limited to providing 2D in-plane polarization information, we have recently extended its capabilities to achieve 3D orientation mapping using almost the same equipment¹⁵, termed as 3D orientation mapping (3DOM). This open-source software also provides the control, reconstruction, and visualization of 3DOM. The reconstruction and visualization modules are also compatible with the single molecule orientation tracking application. All these functionalities enhance the utility of polarization imaging in complex biological studies.

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 of three stripe directions by loading different patterns on spatial light modulator (SLM). Place and rotate a polarizer right after the objective and use a power meter to measure the laser power. For pSIM, rotate the wave plate to maintain the s-polarization of three directions with extinction ratio > 10.
3. For 3DOM, rotate the HWP2 to maintain the p-polarization of six directions. Replace the SIM spatial mask with the 3DOM spatial mask to allow 1-order beams of six directions to pass through (see Figure 1B).
   NOTE: s-polarization is required in pSIM while p-polarization is required in 3DOM. Detailed setup is included elsewhere for pSIM⁵ and 3DOM¹⁵.

2. Micro-Manager setup

1. Download Micro-Manager 2.0 version from the official website, install the software by following the instructions.
2. Prepare the corresponding device drivers and software to successfully connect to the Micro-Manager system. In this microscope system, use Micro-Manager to control the camera, translational stage, laser, DAQ board and microscope.
   NOTE: The supported instruments are listed in https://micro-manager.org/Device_Support. See the Table of Materials for details on the instruments to be connected. Install drivers or official/support software and connect the instrument to the computer using a USB cable.
3. Add the instruments to Micro-Manager using the Hardware Configuration Wizard.
   1. Open the software and select None from the initial dropdown list.
   2. Select Devices | Hardware Configuration Wizard… | Create new configuration and click Next to enter the configuration page.
   3. Locate the Camera / Laser / Stage / DAQ/ microscope plug-in in the dropdown hardware list and click Add | OK | Next.
   4. Go to Select default devices and choose auto-shutter setting. Set the Default camera, Default shutter, and Default focus stage. Click Next until Save configuration and exit is reached, and save the configuration file name. Click Finish.
4. Common template settings
   1. Save a template for live mode and SIM snap mode.
   2. Set the exposure time and trigger mode as follows: choose Group ' +' in Configuration settings, name the Group name as 'Mode', select Exposure and Trigger Mode, click OK.
   3. Choose Preset '+' to add different presets for the 'Mode' group. Name one preset 'live' for live mode exposure settings, and another 'SIM' for SIM mode. For example, in 'live' mode, set the exposure time to 15 ms, while for SIM snap mode, set 10 ms for exposure time and the trigger mode is Standard (Overlap).
      NOTE: A configuration file (MMConfig_psim_demo.cfg) is included in our code repository, which can be loaded directly by Micro-Manager 2.0.

3. System calibration with fluorescent beads

1. Immerse the coverslips in 75% ethanol, rinse the coverslips 3x with deionized water (ddH₂O), and dry them.
2. Vortex the 100 nm fluorescent beads (diluted to 1:1,000 with phosphate-buffered saline [PBS]) and pipette the suspension directly onto the coverslips. Incubate for 5-10 min in a dark environment, then gently wash with PBS.
3. Add 20 µL of mounting medium onto the microscope slide, and position the coverslip over it, making sure to avoid any bubbles. Maintain the temperature at 4 °C and keep the slide in the dark, so the slide with 100 nm fluorescent beads is prepared for System calibration.
4. For pSIM, acquire three images of the three different phases of fluorescent beads. A custom MATLAB code (Bead_calib.m) takes the images as input and outputs two calibration images (calib1.tif, calib2.tif) for further analysis.
   NOTE: As demonstrated earlier⁵, the pSIM experiment can be performed on most commercial systems. On either home-built or commercial SIM microscopes, system calibration is required to eliminate the measurement error of fluorescent dipoles, caused by the non-uniform illumination during three pattern directions. The calibration experiment assumes the isotropic polarization of fluorescent beads and only requires the raw images during SIM acquisition. Detailed calibration procedure is included in our original work⁵. The output of two calibration images calib1.tif and calib2.tif is required for pSIM reconstruction, which indicates the pixelwise illumination energy of pattern direction 2; 3 refers to pattern direction 1.

4. Sample preparation: actin in fixed cells

1. Immerse the coverslips in 75% ethanol, rinse the coverslips 3x with ddH₂O. Place the coverslips in a six-well cell culture plate.
2. Culture U2OS cells (ATCC HTB-96 cell line) in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) at 37 °C and 5% CO₂ on coverslips until they reach approximately 75% confluency.
3. Gently wash the cells 3x with PBS.For cell fixation, apply 4% paraformaldehyde for 15 min at room temperature. After fixation, washthe cells 3x again with PBS.
4. To enhance membrane permeability, treat the cells with 0.1% Triton X-100 in PBS for 5 min, then wash the cells 3x with PBS.
5. Dissolve fluorescent phalloidin with 150 µL of DMSO for stock solution (66µM). Prepare the Phalloidin dye working solution by diluting 5 µL of the stock solution in 995 µL of PBS. Incubate the cells in the dark with Phalloidin dye working solution at room temperature for ~1 h. Wash the cells for 3 x 3 min with PBS.
6. Add 20 µL of mounting medium onto the microscope slide. Carefully place the coverslip onto the slide without any bubbles. Store the slide at 4 °C and keep it in a dark place.

5. Sample preparation: in-vitro λ-DNA

1. Dissolve 0.5 g of Poly Methyl Methacrylate in 10 mL of denture base materials. After thorough dissolution,evenly apply the solution onto the surface of the slide, and then wait for it to evaporate to form a film.
2. Dilute 0.3 µL of λ-DNA and 32 µL of 1,000x SYTOX Orange Nucleic Acid Stain in 968 µL of PBS. Add 1 µL of the prepared solution to the slide and allow it to dry.
3. Seal the coverslip onto the slide with the mountant to protect and preserve the sample. Store the slide at 4 °C in a dark environment.

6. Imaging

1. Place the coverslip onto the sample holder and adjust to the focal plane in the live mode. Select a 512 x 512 pixels ROI and adjust the exposure time and laser intensity.
2. Open the independent control software of the spatial light modulator, and choose the appropriate illumination pattern sequence path for pSIM / 3DOM or live mode.
   NOTE: For pSIM, the illumination pattern includes three different polarizing angles and three different phases. For 3DOM, the illumination pattern includes six different polarizing angles.
3. Select the SIM capture mode, select Multi-D Acq., choose Time Points and set the Count (9 or 6 images for pSIM or 3DOM) and Interval (0 ms) in the open window. Click Acquire!
4. Click TriggerSLM.bsh (a configuration file is included in our code repository),the MyDaq acquisition card activates the spatial light modulator to take the pictures.

7. Live-view plugin of pSIM and 3DOM

1. Configure the FakeCamera and image access path: load the scrip (MMConfig_FakeCam_demo.cfg) by clicking on Devices | Load Hardware Configuration…. Configure the local directory of images in Devices | Devices Property Browser… | Camera-Path mask (Figure 2A-C).
   NOTE: The acquisition part of the script depends on the hardware setup, so we have provided a version (MMConfig_FakeCam_demo.cfg) using the FakeCamera device to read local images on hard disk for demonstration. Users can make changes in our version. Remember to change the image reading path (Camera-Path mask).
2. Set the Quick Access Panel by clicking on Tools | Quick Access Pannel | Create new panel. Click the Settings icon in the bottom left corner, drug 'Run Script' to the top part, which can load the 'psim.bsh' and '3DOM.bsh' (Figure 2D) .
   NOTE: The Quick Access Panel system enables users to create customized windows and easily access the controls in Micro-Manager, which is needed most frequently. 'Live', 'Snap,' or other channels can also be added , as shown in Figure 2D.
3. Taking pSIM as an example, perform real-time polarization image reconstruction by clicking on 'psim.bsh'; observe the real-time-reconstructed images with polarization results (Figure 2E). Remember to change the calibration images path in 'psim.bsh' file (calibPath = " path to calibration images file").
   NOTE: The live-view plugin is a beanshell script (psim.bsh and 3DOM.bsh), which can be run in the Script Panel. The plugin acquires nine images for pSIM or six images for 3DOM and calculates the orientation mapping results based on the wide field images. The orientation mapping images are displayed to provide real-time polarization information, which facilitates immediate inspection, despite its low resolution.

8. Data analysis with super-resolution reconstruction

1. Install MATLAB R2019b from the website (see the Table of Materials).
2. Open the software; for pSIM reconstruction, obtain nine images, consisting of three different phases for each of the three orientation angles. Ensure the names of the nine images are from '1.tif' to '9.tif', put them in the file named 'Input', and run the program named 'PSIM.m'. Observe the reconstructed Wide-Field image, SIM image, and pSIM image with the color wheel.
   NOTE: The reconstruction for pSIM needs two steps: a SIM step and a PM step. The data are analyzed by a custom-written MATLAB program for easier debugging. Get the reconstruct code from the 'reconsr' folder in GitHub.
3. For 3DOM, capture six different polarization-directions figures. Mergethe six images together, name the resulting file 'Raw_data.tif', and run the program named 'Recon_3DOM.m'. Observe the reconstructed Wide-Field image and 3DOM image with azimuthal results and the polar angle results.
   NOTE: During the reconstruction process, an error may occur'--'function or variable 'bfopen' is not recognized'. 'bfopen' is a function in the bio-formats library and is commonly used for opening and reading image files. Enter addpath ('path_to_bioformats_toolbox') in the MATLAB command window. Replace 'path_to_bioformats_toolbox' with the actual library folder path. We also added the Bio-Formats dependency to the GitHub repository.

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 h...

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

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