  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.
