In fluorescence microscopy images what is meant by the term "puncta"?

Question

I am reading papers where confocal fluorescence microscopy images were analysed. In many of the papers I see the term "puncta" being used when researchers analyse the colocalisation between 2 proteins. For example, in the linked paper:

In addition, many C1q-positive puncta in the IPL were associated with synaptic puncta identified by double immunostaining with synaptic markers such as PSD-95

I am not sure what is meant by the term "puncta" in the context of immunocytochemistry. I have read that the general definition for puncta is a small, distinct point. However, I was wondering whether puncta is a synonym for clusters of a protein (e.g. in the example above, does synaptic puncta refer to clusters of the PSD-95 protein?)

Any insights are appreciated.

Answer 1

I have read that the general definition for puncta is a small, distinct point.

That's correct. From my understanding, puncta are dots.

However, I was wondering whether puncta is a synonym for clusters of a protein

In the context of fluorescence microscopy, a punctum (plural puncta) does not specifically refer to the aggregation of proteins. Any small, distinct points in the field of view may be called puncta. I like to think of puncta in contrast with foci, which I generally understand to be regions of interest, though, in practice, the two terms are often used interchangeably. Here are some examples from the literature:

The phosphorylation of H2AX at Ser¹³⁹, resulting in the formation of γH2AX puncta in the nuclei, is an early event in the cellular response to DNA damage.

^{Chiu YH, Hsu SH, Hsu HW, Huang KC, Liu W, Wu CY, Huang WP, Chen JY, Chen BH, Chiu CC. Human non‑small cell lung cancer cells can be sensitized to camptothecin by modulating autophagy. Int J Oncol. 2018 Nov;53(5):1967-1979.}

It is well known that histone H2AX becomes phosphorylated at Serine139 to γH2AX immediately after irradiation, and involves a large chromatin region of up to ~ 2 Mbp, thus forming distinct μm-sized foci at the sites of DSBs.

^{Memmel S, Sisario D, Zimmermann H, Sauer M, Sukhorukov VL, Djuzenova CS, Flentje M. FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy. BMC Bioinformatics. 2020 Jan 28;21(1):27.}