Non-invasive detection of regulatory T cells with Raman spectroscopy.

Regulatory T cells (Tregs) are a type of lymphocyte that is key to maintaining immunological self-tolerance, with great potential for therapeutic applications. A long-standing challenge in the study of Tregs is that the only way they can be unambiguously identified is by using invasive intracellular markers. Practically, the purification of live Tregs is often compromised by other cell types since only surrogate surface markers can be used. We present here a non-invasive method based on Raman spectroscopy that can detect live unaltered Tregs by coupling optical detection with machine learning implemented with regularized logistic regression. We demonstrate the validity of this approach first on murine cells expressing a surface Foxp3 reporter, and then on peripheral blood human T cells. By including methods to account for sample purity, we could generate reliable models that can identify Tregs with an accuracy higher than 80%, which is already comparable with typical sorting purities achievable with standard methods that use proxy surface markers. We could also demonstrate that it is possible to reliably detect Tregs in fully independent donors that are not part of the model training, a key milestone for practical applications.

Compressed sensing laser scanning microscopy.

We present a measurement and reconstruction method for laser-scanning microscopy based on compressed sensing, which enables significantly higher frame rates and reduced photobleaching. The image reconstruction accuracy is ensured by including a model of the physical imaging process into the compressed sensing reconstruction procedure. We demonstrate its applicability to unmodified commercial confocal fluorescence microscopy systems and for Raman imaging, showing a potential data reduction of 10-15 times, which directly leads to improvements in acquisition speed, or reduction of photobleaching, without significant loss of spatial resolution. Furthermore, the reconstruction model is also robust to noise, and effective for low-light applications. This method has promising applications for all imaging modalities based on laser-scanning acquisition, including fluorescence, Raman, and nonlinear microscopy.

Single-shot, simultaneous incoherent and holographic microscopy.

In this work, we demonstrate single-shot, simultaneous recording and subsequent retrieval of one incoherent and two holographic (intensity and phase) images from the same camera frame. Demultiplexing of incoherent and holographic signals in the spatial frequency domain is made possible by carrier frequency modulation and spatial oversampling intrinsic to the off-axis digital holographic configuration. In particular, we show applications to combined fluorescence and digital holographic microscopy, as well as combined bright field and holographic second harmonic generation microscopy.