Challenges in long-term imaging and quantification of single-cell dynamics

Abstract

Continuous analysis of single cells, over several cell divisions and for up to weeks at a time, is crucial to deciphering rare, dynamic and heterogeneous cell responses, which would otherwise be missed by population or single-cell snapshot analysis. Although the field of long-term single-cell imaging, tracking and analysis is constantly advancing, several technical challenges continue to hinder wider implementation of this important approach. This is a particular problem for mammalian cells, where in vitro observation usually remains the only possible option for uninterrupted long-term, single-cell observation. Efforts must focus not only on identifying and maintaining culture conditions that support normal cellular behavior while allowing high-resolution imaging over time, but also on developing computational methods that enable semiautomatic analysis of the data. Solutions in microscopy hard- and software, computer vision and specialized theoretical methods for analysis of dynamic single-cell data will enable important discoveries in biology and beyond.

Shaping development by stochasticity and dynamics in gene regulation

“…One common strategy to achieve this goal is via long-term imaging of cultured live cells with integrated fluorescence biosensors [106]. Recent technological advances from several aspects have greatly expanded the application of this approach, in particular for mammalian cells.…”

Challenges and emerging directions in single-cell analysis

“…During this process, cells are isolated from their local environment and destroyed prior to profiling. These “snapshots” lose important contextual information regarding both a cell’s spatial environment and position within a trajectory of dynamic behavior [25]. Both sources of information are crucial to interpret the precise state of a cell at the time point of its isolation (and usually destruction).…”

“…These dynamic behaviors greatly complicate the interpretation of snapshot single-cell analyses because individual cells will differ in their molecular state not only from other cells, but even from themselves if analyzed at a different time point [25]. Importantly, these dynamics may not represent noise, but rather a basis for important regulatory mechanisms controlling cell identity, so it is important to quantify dynamic changes and to understand their relevance [53].…”

“…These differ from tools available to analyze snapshot data and often require self-made custom developments. This holds true for the required theoretical algorithms and for user-friendly implementation [25]. …”

Nanoscale imaging and force probing of biomolecular systems using atomic force microscopy: from single molecules to living cells

“…6 Investigating biological activities at the single-cell level can reveal novel regulatory mechanisms for diverse cellular processes, which has significant implication in the field of biomedicine. 3 In fact, in recent years researchers have performed single-cell studies to investigate various biological issues, including the epigenetic regulation of single cells, 7 single-cell extraction for molecular analyses, 8 single-cell whole-genome analyses, 9 single-cell dynamics, 10 and stem cell behaviors, 11 providing novel ways to address biological issues in the life sciences. Down to the single-cell level, it is known that the fulfillment of cell functions is based on a complex set of molecular interactions which control signaling pathways to influence ultimate cell fates, including proliferation, differentiation, and apoptosis.…”