Our protocol describes the use of conventional BODIPY dyes for super-resolution microscopy using their sparse, red-shifted states. This enables us to study organelles and biomolecules in living cells with 30-nanometer resolution. The advantage of this method is its simplicity and the versatility of different available BODIPY conjugates that provide a long-lasting source of single molecule signals.
Our demonstrated application could become important to gain insights in diseases such as fatty liver disease or type 2 diabetes by resolving lipid droplets and fatty acid below the diffraction limit. We gain insight in fatty acid and lipid droplet biology in yeast and mammalian cells. However, this technique is applicable to all other transparent cell types with low background fluorescence.
When using the technic for the first time, make sure to optimize the di concentrations and laser powers, to observe bright single molecule signals. A visual demonstration of this technic is important to see how the di concentration and optimization of laser power results in bright single molecule signals. Maintain the mammalian U2OS cells in non-fluorescent DMBM with 10%fetal bovine serum four millimolar glutamine, one millimolar sodium pyruvate, and one percent penicillin streptomycin antibiotics in a T-25 flask.
Split the cells at 70 to 80%confluency to one to five, and pipette in a single well of an eight well plate. Alter the cells in the eight well plate for 12 to 24 hours. Ten minutes prior to imaging it, add a BODIPY-C12 Lysotracker green or and the other BODIPY conjugate, add a final concentration of 100 nanomolar.
Mount the appropriate filter sets in the emission path based on the emission color of BODIPY being used. Turn on the microscope. Microscope stage, 488 nanometer and 561-nanometer lasers, as well as the camera.
Add a drop of emerging oil on the microscope objective. Open the HAL4000 software that controls the LED light for bright field imaging, laser powers, laser shutters, and camera settings for imaging. Set the EMCCD gain to 30 and the camera temperature to minus 68 degrees Celsius.
Prepare the camera and corresponding software to record movies at 20 Hertz. Turn on the microscope stage heater and set it to temperature of 37 degrees celsius and to a carbon dioxide level of five percent. Adjust the objective correction color accordingly.
Mount the sample on the microscope stage and focus until the focusing system engages. Move the stage using the stage controller until healthy cells appear in the field of view. Load laser shutter sequences for the excitation of monumus as well as dimmers, to whom laser power of the 561-nanometer laser to between 821 kilowatts per square centimeters for a single molecule localization microscopy, such that, single molecule fluorescence pores are detected in the red-shifted emission channel.
Adjust the laser power between point 035 and point 07 watts per square centimeter for the 488-nanometer laser, so that conventional fluorescence appears in the green emission channel. Choose a destination folder for movies and record 5000 to 20000 acquisition frames to collect enough localizations for reconstructing super resolution images. Move to different fields of view and repeat to collect data for more cells.
Load the movie into a single molecule localization microscopy analysis software. Visually screen the movie and adjust contrast settings, such that single molecule fluorescent blinking is visible. To set single molecule identification parameters for fitting the 2D Gaussian, PSFs.
Visually screen through some example frames to check the identification parameters, and reliably detect the distinct single molecule fluorescent bursts. Press analysis to perform single molecule localization microscopy imagine analysis with the optimized identification parameters. And then, render each single molecule as a 2D Gaussian whose width is weighted by the inverse square root of the detected number of photons.
Assess the quality of the data. Use restricted frame ranges to observe single molecule distributions at more specific instances and time. This accounts for organelle movement during data acquisition.
In this study, we presented an optimized sample preparation, data acquisition and analysis procedure with single molecule localization microscopy using BODIPY conjugates. To demonstrate an example of the workflow for acquiring and analyzing single molecule localization microscopy data, BODIPY in yeast was employed to resolve lipid droplets below the optical diffraction limit. Examples of the different multicolor imaging modes of BODIPY in conjugation with other probes such as GFP and mEos2 are shown here.
BODIPY-C12 formed in mobile non-lipid droplet clusters in cell periphery upon fasting, in contrast to their incorporation into lipid droplets under fed conditions. To further extend the single molecule localization microscopy capability of BODIPY conjugates to mammalian cells, BODIPY-C12 and Lysotracker green in live U2OS cells were imaged. Optimizing BODIPY concentration as well as exciting the laser powers are critical steps in order to visualize bright single molecule signals, and to reconstruct super resolution images.
This method can be used with any other BODIPY conjugate to gain high resolution insight, in the special temporal distribution of specific bio-molecules inside living cells. Our technic paved the way, to further interrogate lipid droplet and fatty acid biology on the nanoscopic lens scale. However, this application goes far beyond the specific philopectetes due to the availability of various functional BODIPY conjugates.
Please be sure to follow the standard operating procedures for handling the biological samples and dyes, and also be our obliged hedgers and follow the laser safety procedures.
