Domain: Natural Science — Cell & Developmental Biology
Target concept: Early apoptotic cells exhibit green fluorescence (Annexin V positive)

Video generation prompt: Early apoptotic cells stained with Annexin V-FITC/PI were imaged under a fluorescence microscope.

Evaluation guide:
(i) Target concept: Fluorescence-based discrimination of apoptotic stages via differential membrane phospholipid exposure and permeability.
Minimum mechanism: Annexin V-FITC must bind externalized phosphatidylserine on intact membranes; PI must be excluded from viable/early apoptotic cells but enter late-stage cells with ruptured membranes; green and red signals must originate from distinct biochemical states, not arbitrary labeling.
(ii)Storyline: 1. Static fluorescence microscope view of cell monolayer pre-staining (no signal). 2. Annexin V-FITC applied (visible droplet contact) → green signal emerges on morphologically intact cells. 3. PI applied (second droplet contact or sequential mixing) → red signal appears only on morphologically compromised cells. 4. Dual-channel overlay shows spatial segregation: green-only = early apoptotic, red-only = late/necrotic, green+red = transitional. 5. All events occur in fixed viewpoint, same focal plane, with no camera movement or perspective shift; observer sees only what a researcher would see through oculars or captured output.

Manual:
Experimental Procedure
1. Place adherent cell monolayer on microscope slide.
2. Add Annexin V-FITC reagent, incubate briefly.
3. Add PI reagent, mix gently.
4. Image immediately under fluorescence microscope using FITC & TRITC filters.
Observation of Experimental Phenomena
- Early apoptotic cells show bright green fluorescence (Annexin V bound) with intact morphology and no red signal.
- Late apoptotic/necrotic cells show red fluorescence (PI bound) with fragmented morphology.
- Viable cells show no fluorescence.
- Dual-positive cells (green rim + red core) indicate transitional stage.
Summary of Experimental Principles
Annexin V binds phosphatidylserine exposed on outer leaflet of plasma membrane in early apoptosis; PI enters only cells with loss of membrane integrity — thus distinguishing viable, early apoptotic, late apoptotic, and necrotic populations.

Prompt Grounding rubric:
level_1: No fluorescence microscope view is shown; no cell sample is visible; neither Annexin V-FITC nor PI reagent appears; the scene has nothing to do with an apoptosis fluorescence assay.
level_2: A microscope or lab bench is shown, but no cell sample sits on the stage, or only one reagent (Annexin V-FITC or PI) is shown in isolation (e.g., a labeled tube) without ever being brought into contact with a sample; no fluorescence signal of any color appears.
level_3: A fluorescence-microscope field of view containing a cell sample is shown, together with both the Annexin V-FITC and PI reagents somewhere in the scene (e.g., labeled tubes, pipette tips, or droplets near the sample); at least one frame shows a green signal on the sample, but a red signal is either absent, or the reagent-application step for PI is never shown.
level_4: A single, uninterrupted fluorescence-microscope field of view shows the cell sample throughout; the video depicts the Annexin V-FITC droplet (or pipette tip) making contact with the sample before a green signal appears, and separately depicts the PI droplet (or pipette tip) making contact before a red signal appears; both green and red signal are visible somewhere in that same field at some point, with no scene cut or reagent substitution (no unrelated stain shown).
level_5: All conditions of score 4 hold, and the procedural order and framing are unambiguous: Annexin V-FITC is applied and produces green signal before PI is applied and produces red signal, the field of view, focal plane, and illumination setting stay the same from first reagent contact through to the final dual-signal frame, and no unrelated object (hand, extra tube, on-screen label naming an outcome) intrudes on the shot.

Scientific and Causal Correctness rubric:
level_1: No green fluorescence and no red fluorescence appear anywhere in the field, cells look completely unstained, or the colors are swapped (green appears where red should be or vice versa), so early-apoptotic, late-apoptotic, and viable states are not visually distinguished at all.
level_2: Only one fluorophore color is shown (all green or all red) and it covers every cell regardless of shape; or green appears on cells that look shrunken/fragmented, or red appears on cells that still look round and intact, violating the rule that Annexin V marks externalized phosphatidylserine on an otherwise-intact membrane while PI only enters cells whose membrane has already broken down.
level_3: Both green and red signal appear on different cells in the field, but the assignment is not biologically consistent (for example, green appears on visibly fragmented/shriveled cells, or red appears on cells that still look round and smooth), so the fluorescence pattern does not track cell morphology in a way a viewer can recognize.
level_4: Green fluorescence appears only on cells that still look round, smooth-edged, and structurally intact (no green on any fragmented or ruptured-looking cell), and red fluorescence appears only on cells that look shrunken, blebbed, or fragmented (no red on any smooth intact-looking cell); any dual green+red cell shows an intermediate morphology (mild shrinkage) rather than either extreme, and no color appears outside a cell boundary.
level_5: All conditions of score 4 hold, and the signal pattern matches real Annexin V/PI staining in detail: green is concentrated at or near the outer edge of intact cells (consistent with surface phosphatidylserine binding) rather than filling the whole cell interior; red fills the cytoplasm and nucleus of membrane-compromised cells rather than sitting only at the rim; dual-positive cells show a green rim around a red-filled body; no green or red signal appears in the fluid/background outside any cell.

Spatiotemporal Consistency rubric:
level_1: Severe discontinuity: the video cuts between unrelated shots (e.g., microscope field to an unrelated close-up), the pipette tip appears already inside the sample with no approach shown, illumination flickers erratically between frames, or cells vanish and reappear without any visible motion causing it.
level_2: The focal plane changes abruptly mid-sequence (sharp to blurry to sharp with no smooth refocus shown), a reagent (Annexin V-FITC or PI) appears in the field with no visible droplet or pipette contact, green or red signal switches on/off between adjacent frames with no fade-in, or cell positions shift with no plausible motion path between them.
level_3: One small flaw remains: a brief dip in brightness or saturation occurs right as the channel switches from FITC to PI viewing, the field shows slight jitter for a moment, or the pipette-tip motion has one noticeable jump-cut, but no cell teleports and no structural element (cell boundary, field edge) breaks.
level_4: The pipette tip approaches and contacts the sample with continuous motion, green and red fluorescence each fade in gradually after their reagent's contact rather than switching on instantly, the focal plane stays sharp and stable throughout, brightness and hue shift consistently when the filter changes from FITC to PI, and no cell edge stretches, warps, or gets clipped by the frame.
level_5: All conditions of score 4 hold, and the sequence is fully coherent end to end: the field of view shows no drift or shake, the pipette motion has realistic deceleration as it reaches the sample, green signal appears and persists before red signal emerges later in the same field (matching the sequential Annexin V then PI application), no green bleeds into the red channel or vice versa when the filter switches, and every cell boundary stays sharp and stable with no frame-duplication stutter.