Measuring CAR T potency with label-free imaging

Efficient CAR T cell development demands functional assays that provide rapid, reproducible insights into T cell potency, specificity, and mechanism of action. By eliminating the need for labels or endpoint staining, and leveraging AI-based automation, the LIVE T Cell Assay empowers scientists to assess the efficacy and specificity of CAR T clones, supporting data-driven decision-making. In this edition, we share case study data investigating four aspects of CAR T cell function; cytotoxicity, activation, interactions, and serial killing. Finally, we highlight March 2026’s key publications featuring label-free live cell imaging and AI analysis in other fields including mitochondrial transplantation and analysis of cell morphology during differentiation.

Label-free CAR T assessment 

Nanolive’s unique high content data combined with its automated digital assay distinguishes between CAR-T and tumor cell responses in co-culture with automated, AI-powered analysis of quantitative label-free images:

• Quantify T cell activation, early AICD (activation-induced cell death) and target cell behavior, all with a single click

• Perform rapid, reproducible assessment of CAR-T clone specificity and potency

• Prioritize the most effective candidates and de-risk early development with functional evidence

For a printable poster overview of label-free analysis of T cell activity and target cell killing, click here

CAR T cytotoxicity assay 

This AI-powered CAR T cytotoxicity assay detects and quantifies target cell death, as shown in the figure below. This experiment measured the CAR-T cells’ ability to specifically kill tumor cells expressing a target epitope. Results confirmed that the tested CAR-T clone mediated highly specific and efficient killing only of cells expressing the target epitope. Moreover, these results highlight the applicability of the LIVE T Cell Assay for screening CAR-T cells for on-target effects, a critical step in early-stage CAR-T candidate selection.

Assessing CAR-T on-target effects. Target cells shown digitally colored in green (living) and orange (dead), and a plot of the target cell death over time for negative control cells (green) and for cells expressing the CAR-T target epitope (orange).

CAR T activation assay 

CAR T cell activation state can also be predicted from their morphology using label-free imaging and ML-powered analysis. For the same experiment, the LIVE T Cell Assay’s activation analysis revealed that CAR-T cells exhibited clear functional activation only in the presence of their specific target epitope. Activated cells were automatically detected based on their elongated shape, while inactive and dead cells remained rounded or disintegrated. The percentage of activated CAR-T cells increased rapidly in the target epitope condition, suggesting exposure to the target epitope triggered T cell activation. In contrast, cells exposed to the off-target epitope showed minimal activation throughout the acquisition, demonstrating the high specificity of the tested CAR construct. Interestingly, activation levels began to decline after approximately one hour in the target epitope condition, accompanied by a rise in T cell death, suggesting activation-induced cell death (AICD) was detected, a phenomenon with direct implications for persistence and in vivo efficacy.

Label-free time-lapse images processed with the LIVE T Cell Assay show AI-driven segmentation and classification of individual CAR-T cells. CAR-T cell activation and viability digital overlays (a-b) and plots of the %Active T cells (c) and %Dead T cells (d) over time in the control (green) and CAR-T target epitope (orange) conditions.

For more detail on this experiment, download the Application Note.

Measuring CAR T interactions with tumor cells 

Nanolive’s label-free assay segments label-free quantitative holotomographic images and detects areas of interaction between CAR T cells and tumor cells. This provides additional insight into the ability of CAR T cells to recognise and bind their targets. 

Digital segmentation of tumor cells (blue), CAR T areas interacting with tumor cells (yellow), non-interacting areas of CAR T cells (red). Areas of higher refractive index appear brighter for example, the bright dots appearing particularly in the bottom right cells (lipid droplets).

CAR T serial killing 

Label-free microscopy of CAR T cells allows us to assess their activity without bias or toxicity and, in comparison to endpoint assays, we can explore investigate cell behavior at an even closer level. This acquisition captured the extraordinary serial killing of seven tumor cells in seven hours by a single CAR T cell. The high temporal resolution also reveals that one cell was particularly resistant to the CAR T cell’s attack, highlighting the heterogeneity of tumors that complicates the development of immunotherapies.

To hear leading CAR T scientist Dr Roddy O’Connor discuss his research using Nanolive’s imaging and LIVE T Cell Assay, you can watch his webinar on demand here.

Explore the Nanolive workflow 

In case you missed it, the online tour of Nanolive’s software is now available on demand! Join our Field Application Specialist Matthias as he demonstrates how easy it is to transform label-free images directly into valuable insights.

Discover how a single high content experiment provides: 

• More insights into drug mechanism of action and side effects

• More biologically relevant conclusions

• Dynamic, real-time data, to move beyond the limits of endpoint assays

• Noninvasive monitoring of sensitive cells to preserve their natural behavior

Reduce data silos and complex analysis with EVE Explorer, find out how here.

Latest publication highlights with Nanolive imaging:  

• Mitochondrial morphology analysis: Schnurr, T. M. et al., (2026) ‘Colocalization and functional analyses identify GBE1 as a gene linking muscle strength and cardiometabolic fitness’, Endocrinology and metabolism, https://doi.org/10.1152/ajpendo.00470.2025  

• Mitochondria in mitosis: Yuhasz, D. et al., (2026) ‘LabelFree Quantification of Mitochondrial Dynamics Through Mitosis Using Holotomographic Microscopy’, The Journal of Precision Medicine: Health and Disease, https://doi.org/10.1016/j.premed.2026.100029  

• Cell morphology analysis: Li, L. et al., (2026) ‘Morphodynamic cellular changes prior to and during cell fusion related to osteoclast formation’, SSRN, http://dx.doi.org/10.2139/ssrn.6337624  

• Mitochondrial transfer: Song, R. et al., (2026) ‘Cytoskeletal remodeling promotes tunneling nanotube formation and drives cardiac resident cell mitochondrial transfer in sepsis’, Science Advances, https://doi.org/10.1126/sciadv.adz3266  

• Mitochondrial transplantation: Bian, N. et al., (2026) ‘Astrocytic Mitochondria Transplantation Rescues Neuron Loss and Dendritic Injuries in Acute Cerebral Ischemic Stroke Mouse Model by Flexibly Regulating Mitochondria Dynamics’, Annals of Neurology, https://doi.org/10.1002/ana.78197  

Find over 400 publications featuring Nanolive imaging here.

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