Domain: Healthcare — Basic Immunology
Target concept: T lymphocyte

Video generation prompt: Isolate human peripheral blood mononuclear cells (PBMCs), which are predominantly lymphocytes including T lymphocytes, by density gradient centrifugation. Adjust the cell concentration to 2×10⁶ cells/mL and prepare a thin smear on a glass slide; air-dry the smear. Apply Wright's staining solution directly to the dried smear, then add buffer solution to the slide for counterstaining. Observe the morphology of the lymphocytes under an optical microscope.

Evaluation guide:
(i) Target concept: Visual confirmation of lymphocyte (predominantly T-cell) morphology after standardized PBMC isolation and slide-based Wright-Giemsa staining.
Minimum mechanism: Density gradient separates the PBMC layer from granulocytes/erythrocytes by buoyancy; Wright's stain, applied to an air-dried smear, binds DNA/RNA (purple-blue) and basic proteins (blue); buffer controls pH to prevent eosin over-staining; optical microscopy resolves sub-10µm nuclear details.
(ii)Storyline: [0–2s] Static overhead shot: centrifuge tube shows a layered gradient with a sharp interphase band containing the PBMC layer. [2–4s] Close-up: a thin smear of the interphase cells is spread onto a glass slide and left to air-dry, with no cracks or clumping. [4–6s] Purple-blue Wright's staining solution is applied directly onto the dried smear on the slide. [6–8s] Clear buffer solution is dispensed onto the same stained slide, causing immediate uniform color stabilization. [8–10s] Seamless transition to microscope eyepiece view: in-focus field showing predominantly small round cells with purple-blue nuclei and pale blue cytoplasm, few if any granulocytes; all shots share fixed viewpoint, consistent lighting, and continuous motion.

Manual:
Experimental Procedure
1. Perform density gradient centrifugation on peripheral blood to isolate the PBMC interphase band (predominantly lymphocytes, including T lymphocytes).
2. Adjust cell concentration to 2×10⁶ cells/mL and prepare a thin smear of the interphase cells on a glass slide; air-dry the smear.
3. Apply Wright's staining solution (purple-blue) directly onto the dried smear.
4. Add buffer solution (colorless) onto the slide for counterstaining.
5. Observe under optical microscope: small round cells with dark purple-blue nuclei and pale blue cytoplasm.
Observation of Experimental Phenomena
- Centrifuge tube shows sharp interphase band containing small mononuclear cells.
- Wright's stain imparts purple-blue color to nuclei and faint blue to cytoplasm.
- Buffer addition stabilizes stain without precipitation or fading.
- Slide displays even monolayer of uniform small round cells.
- Microscope view reveals high nuclear-to-cytoplasmic ratio, smooth nuclear membrane, no granules.
Summary of Experimental Principles
PBMCs — predominantly small, non-granular mononuclear lymphocytes including T lymphocytes — are isolated from peripheral blood via density gradient centrifugation. Once smeared on a slide and air-dried, Wright's stain differentially colors nuclear chromatin (purple-blue) and cytoplasm (pale blue), enabling morphological identification under light microscopy.

Prompt Grounding rubric:
level_1: No centrifuge tube with a layered PBMC gradient is shown; no glass slide with a cell smear appears; no Wright's staining solution or buffer solution is shown applied to a slide; and no microscope-view shot appears.
level_2: At least one required object appears (centrifuge tube, glass slide, staining bottle, or microscope) but the action order is scrambled relative to the prompt, for example, Wright's stain or buffer is shown being added while cells are still in the tube rather than on the slide, or the slide is stained before it is smeared and air-dried.
level_3: All required objects appear (a centrifuge tube with a visible density-layered interface, a glass slide, purple-blue Wright's staining solution, a clear buffer solution, and a microscope-view shot), and the five actions occur in the correct relative order: (1) centrifuge to form the interphase band, (2) smear interphase cells onto the slide and air-dry, (3) apply Wright's stain to the dried smear, (4) add buffer to the stained slide, (5) observe under the microscope; however, at least one object is visually mislabeled, e.g., the 'buffer' bottle is shown as an obviously colored liquid instead of clear.
level_4: Everything at level 3 is shown correctly, and in addition every object is correctly colored (interphase band visible in the tube, Wright's stain purple-blue, buffer colorless), the stain and buffer are each applied directly onto the slide rather than into a separate tube, and the five actions run in strict sequential order without the camera cutting away mid-action.
level_5: Everything at level 4 is shown correctly, and in addition the interphase band in the centrifuge tube sits at the expected mid-height location, the slide smear looks even and covers a defined area of the glass, the buffer is shown mixing into the stain on the slide before the microscope shot begins, and the whole five-step sequence runs as one continuous, uncut take; the only framing change allowed is a smooth push-in/focus pull from the bench-level view into the microscope-eyepiece view, with no hard cuts or jump cuts anywhere in the sequence.

Scientific and Causal Correctness rubric:
level_1: The video shows outcomes that contradict basic Wright-Giemsa staining or hematology: e.g., large granular cells resembling neutrophils dominate the microscope field, the stained smear is uniformly pink/red with no purple-blue nuclei visible, or the centrifuge tube shows no distinguishable density layer at all.
level_2: At least one scientifically invalid causal link appears: e.g., Wright's stain is applied to unspun whole blood rather than to the isolated PBMC smear, buffer is added to the slide before the stain, the microscope field is dominated by spindle-shaped cells or intact red blood cells rather than lymphocytes, or the centrifuge tube shows a uniformly red liquid with no interphase band.
level_3: The centrifuge tube shows a distinct interphase band; the stained smear shows purple-blue nuclei and pale blue cytoplasm; the buffer step leaves the color stable with no fading or visible precipitate; the microscope field is dominated by small, round, single-nucleus cells rather than granulocytes, but at least one detail is scientifically off, e.g., many nuclei look pink instead of purple-blue, or more than a third of visible cells have lobed nuclei suggesting neutrophils.
level_4: The interphase band contains visibly small, round, non-granular mononuclear cells; the stained smear shows purple-blue nuclei with a thin pale blue cytoplasmic rim and no red/orange cytoplasmic granules; the buffer step leaves no purple precipitate or background haze; more than 80% of the cells in the microscope field are small round lymphocyte-like cells with a high nuclear-to-cytoplasm ratio; only minor deviations remain, such as one or two larger monocyte-like cells or slight background staining at the smear's edge.
level_5: The interphase band is sharply defined and contains only small, uniformly sized round cells; the stain produces clean purple-blue nuclei with no reddish cytoplasmic tinge anywhere in the field; the buffer step causes even, immediate color stabilization with no streaking; the finished smear shows an even monolayer with no clumping, rupture, or drying cracks; the microscope field shows essentially all visible cells as small round lymphocytes with condensed chromatin, a smooth round or oval nucleus, and a thin pale blue cytoplasmic rim, with no vacuoles or granules in any cell.

Spatiotemporal Consistency rubric:
level_1: The centrifuge tube, slide, or microscope view changes abruptly between unrelated-looking shots (e.g., the tube's contents reset to unmixed liquid, or the microscope view jumps to a completely different specimen); the camera viewpoint cuts or tilts suddenly mid-action; or the lighting on the bench changes drastically between consecutive frames with no visible cause.
level_2: The viewpoint stays fixed, but at least one clear continuity break remains: e.g., the slide appears fully stained before the smearing/air-drying step is shown, the buffer bottle pours while the stain bottle is still mid-pour, or the microscope eyepiece view appears before the slide is ever placed on the stage.
level_3: The centrifuge tube, slide, staining bottle, buffer bottle, and microscope stay in consistent positions on the same bench surface for the whole clip; each action (centrifuging, smearing, staining, buffering, viewing) follows the previous one without the camera cutting away; but one small continuity slip occurs, such as the tube briefly leaving frame during a hand movement for slightly more than an instant.
level_4: Everything at level 3 holds, and in addition no object changes size or shape unexpectedly between frames, the hand/pipette motions delivering stain and buffer enter and exit the frame smoothly rather than cutting in mid-air, and the transition into the microscope-eyepiece shot is a smooth focus change rather than an instant cut.
level_5: Everything at level 4 holds, and in addition every object (tube, slide, bottles, microscope) keeps the same size, angle, and position relative to the bench throughout, the hand motions moving liquid from bottle to slide look like one continuous physical reach, and the final microscope view arrives via a gradual focus pull rather than a jump cut, with no visible parallax mismatch between the bench-level and close-up shots.