For decades, the spatial resolution of conventional far‐field optical imaging has been constrained due to the diffraction limit. The emergence of optical super‐resolution imaging has facilitated biological research in the nanoscale regime. However, the existing super‐resolution modalities are not feasible in many biological applications due to weaknesses, like complex implementation and high cost. Recently, various newly proposed techniques are advantageous in the enhancement of the system resolution, background suppression, and improvement of the hardware complexity so that the above‐mentioned issues could be addressed. Most of these techniques entail the modification of factors, like hardware, light path, fluorescent probe, and algorithm, based on conventional imaging systems. Particularly, subtraction technique is an easily implemented, cost‐effective, and flexible imaging tool which has been applied in widespread utilizations. In this review, the principles, characteristics, advances, and biological applications of these techniques are highlighted in optical super‐resolution modalities.

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