Abstract
A recent challenge in the field of bioimaging is to image vital, thick, and complex tissues in real time and in non-invasive mode. Among the different tools available for diagnostics, nonlinear optical (NLO) multi-photon microscopy allows label-free non-destructive investigation of physio-pathological processes in live samples at sub-cellular spatial resolution, enabling to study the mechanisms underlying several cellular functions. In this review, we discuss the fundamentals of NLO microscopy and the techniques suitable for biological applications, such as two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG-THG), and coherent Raman scattering (CRS). In addition, we present a few of the most recent examples of NLO imaging employed as a label-free diagnostic instrument to functionally monitor in vitro and in vivo vital biological specimens in their unperturbed state, highlighting the technological advantages of multi-modal, multi-photon NLO microscopy and the outstanding challenges in biomedical engineering applications.
Highlights
One of the most fascinating advancements in bioengineering is the possibility to observe and control the microscopic universe of cells in order to understand the biological mechanisms involved in physiological and pathological phenomena of life (Vo-Dinh, 2003)
In order to underline the importance of live imaging in the investigation of fundamental biological processes, we provide a background on the physical principles of the different nonlinear optical (NLO) microscopies and we discuss the state of the art and new advanced applications of label-free multi-modal microscopy in living organisms
Membrane inserts of transwell culture plates made by PTFE, PET and PC were tested: PTFE resulted more durable during Coherent anti-Stokes Raman scattering (CARS)
Summary
One of the most fascinating advancements in bioengineering is the possibility to observe and control the microscopic universe of cells in order to understand the biological mechanisms involved in physiological and pathological phenomena of life (Vo-Dinh, 2003). You et al (2018) presented an innovative NLO multi-modal imaging system based on four photodetectors to speed up the acquisition time by sequentially collecting autofluorescence signals of FAD with TPEF, NAD(P)H with three-photon fluorescence, extracellular matrix (ECM) with SHG, and interfaces through THG microscopy on intravital mouse model of mammary gland tumor (Figure 5).
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