Background-free deep imaging by spatial overlap modulation nonlinear optical microscopy

Abstract

We demonstrate how the resolution and imaging depth limitations of nonlinear optical microscopy can be overcome by modulating the spatial overlap between two-color pulses. We suppress out-of-focus signals, which limit the imaging depth, by a factor of 100, and enhance the lateral and axial resolution by factors of 1.6 and 1.4–1.8 respectively. Using spatial overlap modulation, we demonstrate background-free three-dimensional imaging of fixed mouse brain tissue at depths for which the signals of the conventional technique are swamped by background noise from out-of-focus regions.

Advances and challenges in label-free nonlinear optical imaging using two-photon excitation fluorescence and second harmonic generation for cancer research

“…This could be due to accompanying motion artefacts and increased tissue scattering from thicker biological sample present during in vivo imaging. These limitations may however be addressed by future technological advancements like (a) spatial overlap modulation NLOI [84] to reduce background noise, (b) use of advanced motion compensations and adaptive optics during optical imaging [85] to reduce motion artefacts from the specimen or the imaging setup itself, (c) use of lasers with longer wavelengths >1000 nm [86] to enable deeper imaging and (d) incorporation of photon counting detection to improve signal-to-noise ratio [87]. These advancements may enable label-free NLOI to be a real time non invasive diagnostic tool in the clinic.…”

Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters

“…30,31 A similar technique was also reported for multiphoton excitation. 32 In this study, we report the depth imaging properties of saturated excitation (SAX) microscopy, in which SAX effectively suppresses the background fluorescence signal primarily from the out-of-focus planes, and demonstrate the fluorescence imaging of 3-D-cultured cells with improved spatial resolution. Recently, studies using 3-D cell cultures have emerged, since specimens in 3-D culture can more closely mimic important natural cellular functions and structures.…”

Biological imaging with coherent Raman scattering microscopy: a tutorial

“…Many tissue abnormalities manifest themselves at tissue layers deeper than 0.5 mm, emphasizing the need to push the technical capabilities beyond the current numbers. Research in the area of adaptive optics 85 and the development of modulation techniques 86 for improving the SNR deeper in the tissue are examples of efforts in this direction. An alternative strategy is the use of excitation frequencies in a spectral window where tissue scattering is inherently small.…”