Real-time tissue offset correction system for intravital multiphoton microscopy

Abstract

The development of multi-photon intravital microscopy, in particular two-photon microscopy (2PM), has been a breakthrough technique for deep-tissue imaging of dynamic cell behavior inside live organisms and has substantially advanced the field of immunology. However, intravital time-lapse imaging over prolonged time periods is complicated by slow tissue drifts caused by vital activity, leading to shifting fields of views and making the acquired image sequence partially or completely unanalyzable. To solve this issue, we have established a system that performs continuous drift offset correction in real time using fine pattern matching during 2PM acquisition. We incorporated an extensive use of graphical processing unit (GPU) for high-speed computing required for real time correction during data acquisition. This allowed us to perform prolonged acquisitions and increase the proportion of analyzable datasets to nearly 100% in lymphoid and non-lymphoid tissues. Considering the straightforward implementation of our newly developed system, we anticipate that it will be applicable for other users interested in improving the quality of live imaging data acquisition.

Salivary gland macrophages assist tissue-resident CD8+T cell immune surveillance

“…2PM was performed with an Olympus BX50WI microscope equipped with a 20X Olympus (NA 0.95) or 25X Nikon (NA 1.0) objective and a TrimScope 2PM system controlled by ImSpector software (LaVisionBiotec). Some of the image series were acquired using an automated system for real-time correction of tissue drift ( 84 ). For 2-photon excitation, a Ti:sapphire laser (Mai Tai HP) was tuned to 780 or 840 nm.…”

Intravital microscopy: Imaging host–parasite interactions in lymphoid organs

“…This requires good surgical skills, and for some organs, including the LNs, dedicated microscope stages. To overcome potential problems regarding tissue motion, a recent technical development is real‐time drift offset correction, which adjusts the positioning of the stage based on pattern matching, allowing certain anatomical landmarks to remain in the same position despite tissue shifts during image acquisition (Vladymyrov et al ).…”

“…This requires good surgical skills, and for some organs, including the LNs, dedicated microscope stages. To overcome potential problems regarding tissue motion, a recent technical development is real-time drift offset correction, which adjusts the positioning of the stage based on pattern matching, allowing certain anatomical landmarks to remain in the same position despite tissue shifts during image acquisition(Vladymyrov et al 2016).The popliteal LNs are located within the popliteal fossa, or knee joint. Various protocols have been developed for surgical exposure and IVM of this LN(Liou et al 2012;Mempel et al 2004;Stein & Gonzalez, 2017).…”

VivoFollow 2: Distortion-Free Multiphoton Intravital Imaging

“…Previously, we have developed the VivoFollow feedback microscopy system that performs tracking of the sample drift in real time and adjusts the imaging position directly during the acquisition [20]. VivoFollow has thus solved the issue of severe tissue drift.…”