# Calibration nanotemplates as universal standards for determining protein copy number in super-resolution microscopy

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2021 · $365,625

## Abstract

Project Summary
In this proposal, we will develop new methods to overcome one of the main challenges in super-resolution
microscopy and enable quantification of protein copy number at the nanoscale level. Super-resolution
microscopy is an enabling tool that reveals the subcellular organization of molecular complexes with
unprecedented spatial resolution. The nanoscopic organization of these complexes into functional units is
highly important for regulating their subcellular activity in a spatially and temporally controlled manner.
However, it has been very challenging to quantify the protein copy number composition of multi-protein
complexes. Protein copy number is highly important for regulating or mis-regulating protein function. Proteins
may be functional below a certain oligomeric assembly and gain toxic function when their oligomeric
composition crosses a critical threshold leading to diseases. Therefore, the ability to properly quantify the sub-
cellular copy number distribution of proteins within molecular complexes is important for gaining mechanistic
insight into healthy and diseased function of these proteins. The main challenge, in doing so, is to overcome
the artefacts arising from the unknown labeling stoichiometry and complex fluorophore photophysics.
We have made important leaps towards overcoming this challenge by developing calibration nanotemplates for
super-resolution microscopy. However, the lack of standard, easy-to-use methods and reagents that account
for variability in experimental conditions has made it difficult for non-experts to adapt these developments.
Therefore, there is an immediate need for highly standardized methodologies that allow protein copy number
quantification independent of experimental conditions. The goal of this proposal is to address this big challenge
and establish a versatile, easy-to-use and universal calibration method that can be adapted by the scientific
community to quantify the copy number distribution of any protein of interest. Our aims are: (i) to develop
calibration nanotemplates for both small and large protein complexes using DNA origami as well as novel
nanotemplates that use designer protein nanocages, (ii) to acquire calibration data for diverse, super-resolution
compatible labeling strategies and identify calibration functions, (iii) to develop the innovative concept of
standardization based on novel use of benchmarking standards and methods that can transform the calibration
functions among different experimental conditions and (iv) to develop an all-integrated, user-friendly, open-
source, modular software that incorporates all the steps from single molecule localization to protein copy
number determination.
This proposal has the potential to advance super-resolution microscopy from a mainly descriptive tool into the
era of quantitative and mechanistic cell biology. As one specific example, the methods developed here will
make it possible to reveal the sub-cellular distribution an...

## Key facts

- **NIH application ID:** 10225515
- **Project number:** 5R01GM133842-03
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Melike Lakadamyali
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $365,625
- **Award type:** 5
- **Project period:** 2019-09-20 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10225515

## Citation

> US National Institutes of Health, RePORTER application 10225515, Calibration nanotemplates as universal standards for determining protein copy number in super-resolution microscopy (5R01GM133842-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10225515. Licensed CC0.

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