Intravital imaging by simultaneous label-free autofluorescence-multiharmonic microscopy

Area of Science:

• Biomedical Imaging
• Microscopy
• Biophysics

Background:

• Intravital microscopy (IVM) is crucial for studying biological processes in vivo.
• Label-free multiphoton IVM offers non-perturbative imaging but faces limitations in contrast and modality integration.
• Existing label-free techniques struggle with efficiency and simultaneous acquisition of diverse signals.

Purpose of the Study:

• To introduce a novel, high-speed, label-free imaging platform for intravital microscopy.
• To overcome the contrast and integration challenges of current label-free IVM methods.
• To enable simultaneous acquisition of multiple nonlinear optical signals from living tissues.

Main Methods:

• Developed Simultaneous Label-free Autofluorescence-Multiharmonic (SLAM) microscopy.
• Utilized a single excitation source at 1110 nm with shaped ultrafast pulses at 10 MHz.
• Enabled simultaneous acquisition of autofluorescence (FAD, NADH) and second/third harmonic generation (SHG/THG).

Main Results:

• Achieved a 2-orders-of-magnitude improvement in imaging speed.
• Enabled fast, simultaneous, and efficient acquisition of multiple label-free signals.
• Successfully imaged diverse cellular and extracellular components (tumor cells, immune cells, vesicles, vessels) in vivo with low excitation power (14 mW).

Conclusions:

• SLAM microscopy is a versatile and efficient advance in label-free intravital microscopy.
• This platform significantly enhances the capability for tracking cellular events in vivo.
• SLAM microscopy overcomes previous limitations, enabling extended time-lapse investigations of living tissues without perturbation.