High‐speed microscopy for in vivo monitoring of lymph dynamics

Abstract

The lymphatic system contributes to body homeostasis by clearing fluid, lipids, plasma proteins and immune cells from the interstitial space. Many studies have been performed to understand lymphatic function under normal conditions and during disease. Nevertheless, a further improvement in quantification of lymphatic behavior is needed. Here, we present advanced bright-field microscopy for in vivo imaging of lymph vessels (LVs) and automated quantification of lymphatic function at a temporal resolution of 2 milliseconds. Full frame videos were compressed and recorded continuously at up to 540 frames per second. A new edge detection algorithm was used to monitor vessel diameter changes across multiple cross sections, while individual cells in the LVs were tracked to estimate flow velocity. The system performance initially was verified in vitro using 6- and 10-μm microspheres as cell phantoms on slides and in 90-μm diameter tubes at flow velocities up to 4 cm/second. Using an in vivo rat model, we explored the mechanisms of lymphedema after surgical lymphadenectomy of the mesentery. The system revealed reductions of mesenteric LV contraction and flow rate. Thus, the described imaging system may be applicable to the study of lymphatic behavior during therapeutic and surgical interventions, and potentially during lymphatic system diseases.

Emerging Role of Lymphatics in the Regulation of Intestinal Lipid Mobilization

“…has been documented (Breslin et al, 2007;Choe et al, 2015). New and improved imaging techniques are being developed to specifically assess mesenteric lymphatic functions in relation to lipid drainage (Kassis et al, 2012;Sarimollaoglu et al, 2018). However, challenges remain in this aspect due to technical difficulties for such measurements (Bouta et al, 2018).…”

Doxorubicin Activates Ryanodine Receptors in Rat Lymphatic Muscle Cells to Attenuate Rhythmic Contractions and Lymph Flow

“…The lymph vessel outer diameter was recorded as described earlier (Souza-Smith et al, 2011). Lymph flow was measured continuously using high speed in vivo flow cytometry and an inverted microscope (Olympus, Tokyo, Japan) equipped with a monochrome high-speed CMOS camera (model MV-D1024-160-CL8; Photonfocus AG, Lachen SZ, Switzerland) to acquire high-speed images of moving cells with minimum distortion at relatively high speeds (#7 mm/s) (Galanzha et al, 2007a;Sarimollaoglu et al, 2018). Videos of lymph flow were then analyzed using custom software developed by the UAMS Nanomedicine Center.…”

“…Videos of lymph flow were then analyzed using custom software developed by the UAMS Nanomedicine Center. Details of the instrumentation and software used to calculate lymph flow were published recently by us (Sarimollaoglu et al, 2018). Briefly, lymph flow velocity initially was analyzed as bidirectional flow, whereby cell movement from distal to proximal (positive flow) is characterized as a positive deflection, and cell movement from proximal to distal (negative flow) is characterized as a negative deflection.…”

“…Cell velocity and diameter measurements were used to calculate positive volumetric lymph flow. This innovative method for detection of moving cells in lymph vessels in vivo does not require fluorescent or infrared dyes and allows for continuous data acquisition rather than discrete measurements (Sarimollaoglu et al, 2018).…”

Recent advances in fluorescence-based in vivo flow cytometry

“…To further capture the morphological features of circulating cells, Lee et al developed an image°ow cytometer that visualizes and counts°owing cells rapidly in circulatory system. [13][14][15][16][17] Wei et al developed an image°ow cytometer in combination with arti¯cial intelligence technology for the counting and imaging of speci¯c cell populations in the blood circulation, such as CTCs and immune cells. Compared with conventional IVFC, it can provide the morphology and structure information of cells, blood vessels and surrounding tissues, which is helpful for studying the hydrodynamic and behavioral characteristics of cells in blood.…”