Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Nikki M. Curthoys; Michael J. Mlodzianoski; Dahan Kim; Samuel T. Hess

doi:10.3791/50680

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Locali...

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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

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12:51 min

December 9, 2013

DOI: 10.3791/50680-v

Nikki M. Curthoys*¹, Michael J. Mlodzianoski*¹, Dahan Kim¹, Samuel T. Hess¹

¹Department of Physics and Astronomy,University of Maine

Overview

This study demonstrates the use of fluorescence photo activation localization microscopy (FPALM) to image multiple protein species within cells with nanometer precision. The technique allows for the localization of thousands of fluorescently labeled proteins in both fixed and living cells.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Imaging Techniques

Background

Fluorescence microscopy is a powerful tool for visualizing cellular components.
FPALM enhances the resolution beyond the diffraction limit of light microscopy.
This method allows for the study of protein interactions and distributions at a molecular level.
It can be applied to both fixed and live cell imaging.

Purpose of Study

To achieve simultaneous imaging of multiple protein species.
To provide high spatial resolution in cellular imaging.
To facilitate the study of protein localization dynamics in living cells.

Methods Used

Adjusting camera and optics for focused imaging.
Aligning laser beams onto the sample on the microscope stage.
Illuminating the cell sample expressing the desired proteins.
Acquiring datasets through FPALM by directing fluorescence to the camera chip.

Main Results

Successful localization of multiple protein species at the nanometer scale.
Demonstrated capability in both fixed and living cells.
Utilized wide field and total internal reflection fluorescence techniques.
Yielded high precision in imaging thousands of individual proteins.

Conclusions

FPALM is an effective method for studying protein localization.
The technique can be adapted for various cellular contexts.
It opens new avenues for research in cellular dynamics and interactions.

Frequently Asked Questions

What is FPALM?

Fluorescence photo activation localization microscopy (FPALM) is a technique used to achieve high-resolution imaging of fluorescently labeled proteins in cells.

Can FPALM be used on living cells?

Yes, FPALM can be applied to both fixed and living cells, allowing for dynamic studies of protein localization.

What is the resolution achieved with FPALM?

FPALM provides localization precision in the tens of nanometers range.

How does FPALM differ from traditional fluorescence microscopy?

FPALM surpasses the diffraction limit of light, enabling visualization of individual molecules with much higher resolution.

What types of samples can be imaged using FPALM?

FPALM can be used to image a variety of samples, including fixed tissues and live cells expressing fluorescent proteins.

What are the applications of FPALM?

FPALM is used in research to study protein interactions, localization, and dynamics within cellular environments.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers within single cells.

The overall goal of this procedure is to image multiple protein species simultaneously with nanometer precision in fixed or living cells. This is accomplished by first adjusting the position of a camera and optics until a focused image from the microscope is projected onto the camera chip. The second step is to arrange laser beams so they're directly aligned onto a sample on the microscope stage.

Next, the prepared cell sample is illuminated and a cell expressing the desired proteins is chosen. The final step is to image the cell with fluorescence photo activation localization microscopy by illuminating the sample with lasers, and then directing the cell fluorescence to the camera chip to acquire a dataset set. Ultimately, fluorescence photo activation localization microscopy is used to show localization of multiple protein species at the nanometer spatial scale in fixed or living cells and in either wide field or when using total internal reflection fluorescence to isolate a thin region of the sample.

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