Invited Review Article: Pump-probe microscopy

Abstract

Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.

Raman spectroscopy: techniques and applications in the life sciences

“…While still requiring a more complex optical setup compared with spontaneous Raman imaging, much of the instrumentation from other nonlinear optical microscopy techniques can be utilized to construct an instrument [264]. Furthermore, progress has been made towards the goal of simpler, compact bench-top SRS microscopes or endoscopes involving fiber probes and lock-in free detection.…”

Rapid, 3D Chemical Profiling of Individual Atmospheric Aerosols with Stimulated Raman Scattering Microscopy

“…SRS is a third‐order nonlinear optical process with signal intensity proportional to the product of the pump and Stokes laser power intensities. Such a nonlinearity results in the intrinsic optical sectioning capability, similar to two‐photon fluorescence microscope, where the signal predominantly generates from the tight focus . By stepping through the z ‐axis of the samples with a step size of 0.4 µm, we could reconstruct the 3D chemical distributions of individual aerosol particles from multiple 2D images, as shown in Figure .…”

Emerging concepts in functional and molecular photoacoustic imaging

“…Pump-probe imaging of transient optical absorption is an emerging field in nonlinear microscopy [24,25]. Recently, this concept has been adopted by photoacoustic imaging for enriching the molecular contrast and refining the spatial resolution.…”