Fibre-coupled multiphoton microscope with adaptive motion compensation

Abstract

Abstract:To address the challenge of sample motion during in vivo imaging, we present a fibre-coupled multiphoton microscope with active axial motion compensation. The position of the sample surface is measured using optical coherence tomography and fed back to a piezo actuator that adjusts the axial location of the objective to compensate for sample motion. We characterise the system's performance and demonstrate that it can compensate for axial sample velocities up to 700 µm/s. Finally we illustrate the impact of motion compensation when imaging multiphoton excited autofluorescence in ex vivo mouse skin.

In vivo multiphoton microscopy using a handheld scanner with lateral and axial motion compensation

“…Our axial motion compensation approach has been shown previously to compensate for axial sample velocities up to 700 μm s −1 . In this paper, we demonstrated in Figures and that our method for measuring and then correcting for lateral sample motion worked for a sample velocity of up to approximately 65 μm s −1 and that this led to a reduction in the normalised cross‐correlation coefficient between temporally adjacent speckle frames to approximately 0.8.…”

“…A variety of methods have been applied to address motion of the sample, including physical immobilisation, acquiring a 3‐dimensional image stack and using the image data to monitor and correct for sample motion , gated imaging approaches and the use of optical sensors to monitor the axial sample position. The latter have included off‐axis illumination and spectral domain optical coherence tomography (OCT) . Generally, previous work addressing lateral motion compensation has employed image‐based correction methods, for example using pairwise rigid transformations or using more advanced approaches that model the intra‐frame motion using methods such as Hidden‐Markov‐Models , the Lucas‐Kanade framework , or algorithms based on Lie groups .…”

“…We have previously demonstrated a handheld multiphoton scanner including axial motion compensation utilising OCT, which was demonstrated using ex vivo samples . We have also previously demonstrated the use of a negative curvature fibre (NCF) for delivery of tunable ultrafast infrared light without the need for pulse pre‐chirp .…”

“…have included off-axis illumination [13] and spectral domain optical coherence tomography (OCT) [14][15][16]. Generally, previous work addressing lateral motion compensation has employed image-based correction methods, for example using pairwise rigid transformations [17] or using more advanced approaches that model the intra-frame motion using methods such as Hidden-Markov-Models [18], the Lucas-Kanade framework [19], or algorithms based on Lie groups [20].…”

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

“…Axial sample motion in and out of the focal plane cannot be corrected post-imaging. In this case, it may be possible to use real-time correction approaches, where an optical coherence tomography (OCT)-based tracking system was used to adjust the position of the objective in real-time (Sherlock et al, 2015(Sherlock et al, , 2017.…”