Domain: Healthcare — Pathophysiology
Target concept: Mitochondrial Morphological Changes in Hypoxia

Video generation prompt: Primary cultured hepatocytes were incubated in an hypoxia chamber (oxygen concentration 5%) for 1 hour, and mitochondrial morphology and fluorescence intensity were observed by fluorescent staining.

Evaluation guide:
(i) Target concept: Hypoxia-induced mitochondrial fragmentation and depolarization in primary hepatocytes.
Minimum mechanism: Functional mitochondrial membrane potential (ΔΨm) is required for accumulation of potential-sensitive cationic dyes (e.g., TMRM, MitoTracker Red CMXRos); loss of ΔΨm under hypoxia reduces dye retention, and activation of fission machinery (DRP1, FIS1) causes structural fragmentation; both necessary for observed phenomena.
(ii)Storyline: [Frame 0–2] Hepatocytes in culture dish placed inside sealed hypoxia chamber (visible gas port/O₂ display); [Frame 3–5] Chamber remains closed, no external intervention; [Frame 6–7] Dish removed, dye applied (liquid contact visible), brief incubation; [Frame 8–10] Fluorescence imaging reveals intracellular punctate structures with lower intensity than expected for healthy mitochondria; all observed from fixed upright microscope perspective, no zoom/pan, no occlusion of cell layer, mitochondria never outside cytoplasm.

Manual:
Experimental Procedure
1. Place primary cultured hepatocytes in a transparent culture dish.
2. Transfer dish into hypoxia chamber set to 5% O₂ for 1 hour.
3. Remove dish, apply a membrane-potential-sensitive mitochondrial fluorescent dye (e.g., TMRM or MitoTracker Red CMXRos), incubate briefly.
4. Image mitochondria using fluorescence microscopy.
Observation of Experimental Phenomena
Hypoxia-exposed hepatocytes show fragmented (punctate) mitochondrial morphology and reduced fluorescence intensity compared to normoxic controls.
Summary of Experimental Principles
Hypoxia triggers mitochondrial fission via DRP1 activation and reduces membrane potential, decreasing uptake and retention of potentiometric fluorescent dyes — resulting in morphological fragmentation and diminished signal.

Prompt Grounding rubric:
level_1: No hypoxia chamber appears anywhere in the clip; no hepatocyte culture dish is shown; no fluorescent dye or stained mitochondria appear; the video contains none of the core elements described in the prompt.
level_2: A chamber or a dish appears, but not both, and the missing piece is essential: for example, cells sit in an open dish with no hypoxia chamber ever shown, or a sealed chamber appears with no visible cells inside it; no fluorescent staining or mitochondrial structures are ever visible.
level_3: A sealed hypoxia chamber and a culture dish of hepatocytes are both shown, and fluorescent dye is applied to the cells (a visible color such as orange-red or green appears), but no mitochondrial shapes (tubular networks or small puncta) can actually be made out in the fluorescence image.
level_4: The hypoxia chamber (with a visible O₂ readout or gas line indicating a low-oxygen setting), the hepatocyte culture dish, and dye-stained mitochondria inside the cells are all shown; however the order is slightly off: for instance the dye appears to be added before the chamber exposure, or the mitochondria are shown partly outside the cell boundary at some point.
level_5: The hypoxia chamber visibly holds a reduced-oxygen environment (gas line and/or numeric O₂ display reading roughly 5%), the same hepatocyte culture dish is shown before being placed in the chamber and again after removal, the fluorescent dye is applied only after the hypoxia exposure (never before), and every mitochondrion is rendered inside a hepatocyte, never floating in the surrounding dish or chamber; the same camera framing and object scale are used throughout.

Scientific and Causal Correctness rubric:
level_1: No mitochondria are visible, or the puncta shown are mislabeled as another organelle (e.g., called nuclei or lysosomes), or mitochondria are shown floating free in the culture medium outside any cell, or they glow without any dye having been applied.
level_2: The mitochondria shown after hypoxia exposure still look like an intact, elongated tubular network rather than fragmented puncta, and/or the fluorescence looks bright and undiminished: nothing about the image suggests reduced membrane potential or fission; or the emission color contradicts the dye actually used (e.g., a dye described as emitting orange-red is rendered glowing green or blue for no stated reason).
level_3: Mitochondria inside the hepatocytes show some visible change from an intact network toward shorter or fewer tubules, and the fluorescence looks somewhat dimmer than a healthy, fully polarized mitochondrial network would appear, but the fragmentation is incomplete (long tubules still dominate) or the dimming is only partial or inconsistent across cells.
level_4: Mitochondria inside the hepatocytes appear clearly fragmented into short rods or round puncta rather than a connected network, and the fluorescence signal is visibly reduced compared to what a healthy, energized mitochondrial network would show; these changes are confined to the hepatocytes shown undergoing the 5% O₂ exposure, without stray fragmentation appearing in any unexposed comparison if one is shown.
level_5: Mitochondria are exclusively intracellular, and after the hypoxia exposure they present as clearly separated puncta or short fragments (no continuous tubular network remains) while the fluorescence intensity across essentially all visible mitochondria is dim and uniform (no bright outliers suggesting an unaffected subpopulation); nothing in the image contradicts the expected hypoxia-driven fragmentation and dye-retention loss for this dye and cell type.

Spatiotemporal Consistency rubric:
level_1: The culture dish or hypoxia chamber vanishes and reappears between shots, or suddenly appears in a different spot; mitochondrial puncta blink on and off with no visible movement in between; the lighting jumps from bright to dark in a single frame with no fade or transition.
level_2: The chamber's lid appears to vanish or teleport open/closed instead of visibly opening; the hepatocyte layer's density or shape changes abruptly without a shown cause; the fluorescent signal switches on instantly with no dye-application step shown beforehand; mitochondria change from tubular to punctate shape within a single frame with no intermediate stage.
level_3: There is a brief flicker in the fluorescence channel, a slight shift in the dish's position, or a moment where something opaque briefly covers part of the mitochondria, but the hepatocyte layer stays intact, the chamber stays sealed, and mitochondrial shape changes gradually rather than jumping straight from tubular to punctate.
level_4: The chamber, dish, and hepatocyte layer all stay in the same place and keep their shape and density throughout; any mitochondrial movement is a slow drift within the cell, not a jump; there is no visible distortion, resizing, or lighting change over time, and the dish rim and cell membranes stay sharp and continuous rather than breaking apart.
level_5: The chamber seal, the dish, and the cell layer look completely stable and continuously present across the whole clip; mitochondrial puncta emerge gradually through visibly shortening and beading tubules rather than appearing suddenly; the fluorescence dims smoothly over time rather than dropping in a single jump; there is no visible misalignment, warping, or ghosting between consecutive frames anywhere in the video.