how-to-quantify-fraction-photoactivated-fluorescent-proteins-bulk

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells ...

assessing-protein-interactions-live-cells-with-fret-sensitized

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

Stanford University School of Medicine

Photoactivatable fluorescent proteins (PA-FPs) have been widely used to assess the dynamics of cell biological processes. In addition, PA-FPs enabled single-molecule based super-resolution imaging (photoactivated localization microscopy) and thereby provided unprecedented structural insight. For the lack of tools, however, the fraction of PA-FPs that is, actually being switched on to fluoresce, that is, the photoactivation efficiency, has been difficult to assess. Uncertainty about photoactivation efficiency has hampered an understanding of the absolute amount of PA-FPs, that is, being examined. Here, we present internal rulers to assess photoactivation efficiencies of photoactivatable proteins. These internal rulers comprise a PA-FP that is genetically directly coupled to a spectrally distinct always-on fluorescent protein. Thus, these fluorescent proteins will be expressed in the bacterial and mammalian cell in a one-to-one ratio. With these tools, we describe photoactivation efficiencies of PA-GFP and PA-Cherry in intensity-based ratiometric ensemble studies and on the single-molecule level. In ratiometric ensemble studies, we show that photoactivation efficiency depends on how the PA-FPs are exposed to 405 nm light. Using a laser-scanning microscope, hundreds of iterative low-level exposures are up to four times more efficient than a short high-power exposure. Using wide-field illumination, photoactivation was similarly efficient and instantaneous. These findings suggest that the repetitive or stochastic exposure to photons of 405 nm light results in more efficient photoactivation than a continuous flow of photons. Because of the differential photoactivation efficiency, it is crucial to assess photoactivation efficiency for any given experimental set-up. The tools we provide can be applied to any genetically encoded photoactivatable protein. Determination of photoactivation efficiency is essential for an understanding of absolute molecule numbers in ensemble studies and, most importantly, quantitative superresolution imaging. © 2017 International Society for Advancement of Cytometry.

Internal rulers to assess fluorescent protein photoactivation efficiency.

To evaluate practice patterns among gynecologic oncologists with regard to sentinel lymph node injection and biopsy in endometrial cancer.

Sentinel Lymph Node Biopsies in Endometrial Cancer: Practice Patterns among Gynecologic Oncologists in the United States.

Although photoactivated localization microscopy offers the potential to interrogate protein interactions in the physiological environment of a cell, uncertainties in the detection efficiency of photoactivatable proteins lead to complications with data interpretation. Here, we present a numerical model that provides probabilities to detect neighboring molecules dependent on their oligomerization status, density, detection efficiency, and radius, and can be used to assess oligomeric states or detection efficiencies of two molecular species. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Dissecting Oligomeric States with Photoactivated Localization Microscopy: A Numerical Model.

Cancer cell metastasis determines disease prognosis. During cancer cell metastasis, the cancer cell and the cancer cell nucleus have to undergo extreme shape changes. To monitor shape changes of cancer cells and cancer cell nuclei and the positioning of the cancer cell nucleus during cancer cell invasion, a customized invasion assay with 8-μm pores and reconstituted basal membrane was imaged using fluorescence live-cell microscopy.

Malte Renz

Gynecologic Oncology Division

Stanford University School of Medicine

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Malte Renz

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Gynecologic Oncology Division

Stanford University School of Medicine

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Gynecologic Oncology Division