A team at Nagoya University’s Institute of Transformative Bio-Molecules has developed a marker molecule that bypasses the problem of photobleaching in STED microscopy. It’s allowing an unprecedented view of live mitochondria, which could help researchers better understand, diagnose, and potentially cure human mitochondrial disease. The new marker molecule — MitoPB Yellow — is absorbed by the inner membrane of mitochondria, which includes the cristae (fold-like structures), and has demonstrated a long lifetime under a STED beam. According to the researchers, existing marker molecules succumb to photobleaching when they degrade under intense STED beams and stop fluorescing, making it nearly impossible to obtain a time-lapse sequence of images “over any decent length of time.” Additionally, damaged marker molecules can become toxic to cells. In the study, mitochondria was placed in conditions known to cause structural changes. Until now, the researchers said, these have only been observed using transmission electron microscopy, which cannot be used on live cells. The mitochondria were treated with a reagent that suppresses DNA replication, inducing dysfunction, to observe their survival and dying processes. Inner membranes of live mitochondria under a STED microscope imaged using the MitoPB Yellow fluorescent marker molecule created by researchers at the Institute of Transformative Bio-Molecules (ITbM) at Nagoya University. The outer membranes of the mitochondria are invisible. The marker molecule can withstand the STED beam for a relatively long time, which allows time-lapse imaging of the live subject. The mitochondria have been treated with a reagent that suppresses DNA replication, inducing dysfunction, in order to see their survival (left) and dying (right) processes. Being able to see the dysfunction processes occurring inside mitochondria could lead to a better way of diagnosing human mitochondrial disease and perhaps even a cure. Courtesy of ITbM, Nagoya University. Next they used time-gated STED microscopy to create still images at 60-nm resolution, along with time-lapse image sequences that showed the mitochondria responding to a deprivation of nutrients by changing form to survive. According to the researchers, the long image sequences — containing up to 600 images over about 7 minutes — are “the first ever made of mitochondria at the relatively high spatial resolution of 90 nm.” The team found that the inner mitochondrial structure changed dramatically. The inner membranes of some neighboring mitochondria fused together into one, and two cristae fused together within a single mitochondrion. There was also an increase in the number of cristae, which were elongated. According to the researchers, the increase in and elongation of cristae could boost “the efficiency of energy production (ATP synthesis) while protecting the mitochondrium from ‘autophagosomal degradation’ — a programmed death whose purpose is to remove unnecessary or dysfunctional components from the cell and allow the orderly degradation and recycling of cellular components.” In response to being deprived of nutrients, mitochondria fuse together and increase the number of cristae. (a) Frames from a time-lapse sequence showing two separate mitochondria fusing together to form a single mitochondrion. The outer membranes of the mitochondria are invisible: We are seeing the inner membranes fusing together. (b) Frames from a time-lapse sequence showing two cristae inside a single mitochondrion fusing together. The scale bars represent 2 mm. Courtesy of ITbM, Nagoya University. Upon further study, and with the same fluorescence intensity, the researchers saw the inner membranes of some mitochondria split into globules that swelled and lost cristae, while some globules ruptured altogether. Others formed concentric spheres. The researchers noted that the cristae and membranes remained sharply imaged, “which indicates that the cause of the mitochondrion’s death is not toxicity due to degradation of the marker molecule under the beam. The extremely strong STED laser might have damaged the mitochondria.” The team plans to continue the study, noting that multicolor STED imaging with a single STED laser is also possible. The researchers agreed that fluorescent markers similar to MitoPB Yellow could find a wide range of applications in other superresolution techniques as well. The research was published in the journal PNAS (https://doi.org/10.1073/pnas.1905924116). Live mitochondria imaged in unprecedented detail — for an unprecedented length of time — using the MitoPB Yellow fluorescent marker. The marker molecule is designed to be absorbed by only certain membranes within each mitochondrion and retains its fluoresescence under the STED microscope for a very long time. This video was shot at 1.5 fps and a resolution of 90 nm. Still images were captured at 60 nm resolution. In response to being deprived of nutrients, mitochondria fuse together and increase the number of cristae. This time-lapse sequence shows events such as two separate mitochondria fusing together to form a single mitochondrion; and a single mitochondrium fusing together. Note that the outer membranes of the mitochondria are invisible: we are seeing the inner membranes fusing together. Courtesy of ITbM, Nagoya University.