Photons in - numbers out: perspectives in quantitative fluorescence microscopy for in situ protein counting

Abstract

The full understanding of cellular functions requires information about protein numbers for various biomolecular assemblies and their dynamics, which can be partly accessed by super-resolution fluorescence microscopy. Yet, many protein assemblies and cellular structures remain below the accessible resolution on the order of tens of nanometers thereby evading direct observation of processes, like self-association or oligomerization, that are crucial for many cellular functions. Over the recent years, several approaches have been developed addressing concentrations and copy numbers of biomolecules in cellular samples for specific applications. This has been achieved by new labeling strategies and improved sample preparation as well as advancements in superresolution and single-molecule fluorescence microscopy. So far, none of the methods has reached a level of general and versatile usability due to individual advantages and limitations. In this article, important requirements of an ideal quantitative microscopy approach of general usability are outlined and discussed in the context of existing methods including sample preparation and labeling quality which are essential for the robustness and reliability of the methods and future applications in cell biology.

Photobleaching step analysis for robust determination of protein complex stoichiometries

“…For higher occupancy, only the locations from the last iteration are considered, meaning we consider triple steps only where double steps yielded an improved posterior. All occupancies are reset to one in every iteration so that, for instance, [2,2,2] can be replaced by [1,3,2]. The result of this process is accepted and considered the new optimum, if a lower value of -log(P) is found at any point.…”

“…In the last two decades, different fluorescence based molecular counting methods have been developed. 3 Among them are methods relying on brightness calibration, 4 counting of photobleaching steps, 5,6 localization microscopy, [7][8][9][10] or photon antibunching. 11,12 To date, photobleaching step analysis (PBSA) and brightness estimation are most widely used in biological applications of molecular counting, due to their simplicity in data acquisition and the relatively straight-forward interpretation.…”

Embracing the uncertainty: the evolution of SOFI into a diverse family of fluctuation-based super-resolution microscopy methods

“…Apart from increasing the image resolution without the disadvantage of non-linear dynamic range expansion, bSOFI [136] allows to extract quantitative maps of parameters such as molecular state lifetimes, concentration and brightness distributions within the samples. As such, it can be used for quantitative fluorescence microscopy, for instance in situ protein counting, offering reasonably good temporal resolution of the order of 10 s and lower phototoxicity than some other methods used in this field [180]. bSOFI was used to measure the density of the protein paxilin in focal adhesions of mouse embryonic cells [181,182] and was used to study the nanoscale distribution and clustering of another protein, CD4 mutants, in the plasma membrane of T cells [181].…”

Photobleaching step analysis for robust determination of protein complex stoichiometries

“…It is highly specific, live-cell compatible, single-molecule sensitive, and therefore capable to resolve heterogeneities within ensembles in situ. In the last two decades, different fluorescence based molecular counting methods have been developed (Gruβmayer et al, 2019). Among them are methods relying on brightness calibration (Wu and Pollard, 2005), counting of photobleaching steps (Ulbrich and Isacoff, 2007), single-molecule localization microscopy (Jungmann et al, 2016;Lee et al, 2012;Puchner et al, 2013;Rollins et al, 2015), or photon antibunching (Grußmayer and Herten, 2017;Ta et al, 2010).…”