Confocal Microscopy and Neuronal Differentiation Lab Notes

Microscopic Techniques and Confocal Imaging Procedures

• Confocal Microscopy Utilization: The students and the instructor, Matt, are utilizing a confocal microscope to inspect cell cultures. Matt emphasizes that the microscope is "fire" (excellent) but notes the complexity of its operation.
• Optical Orientation and Inversion: When looking through the microscope, the image is inverted. Matt explains the orientation as looking at mirrors, where looking from "left first and then right" is the standard approach to identify the position of the cells.
• Targeting and Focusing Strategy: To save time and ensure accuracy, the instructor uses a visual approach before focusing through the lens:
  • He looks visually at the slide to identify "cloudy bits."
  • These cloudy bits represent the location of the cells.
  • Once a "nice spot" is identified visually, he proceeds to focus the lens on that specific area.
• Magnification Levels and Troubleshooting: Observations are made at $10\times$ and $40\times$ magnification.
  • A specific issue was noted where cells were visible at $10\times$ but disappeared or became difficult to see at $40\times$.
  • Potential cause: Two coverslips might have stuck together from the beginning of the process. If coverslips are not separated, they can stick together, creating an optical thickness that prevents proper focusing at higher magnifications like $40\times$.
• Oil Immersion and Lens Care: When moving the slide, one must be careful not to move too far, as the immersion oil might not maintain contact across the entire area.
• Digital Imaging and Software Operations:
  • The system uses a laser, specifically referencing $488\text{ nm}$, and a DAPI system.
  • Imaging involves "normal line sequential" sequences.
  • Settings include adjusting "analog" versus "counting" modes.
  • Live imaging ("fast live") is used to find cells before capturing the final image.
  • Parameters such as "intensity," "gain," and "z-position" (focal plane) are manually adjusted to optimize image quality.
  • The software allows for viewing scales in nanometers ($\text{nm}$).
  • Files are saved in specific directories: Users > Lily Chen Lab Training or the Compass 2026 folder.

Cell Staining, Markers, and Visualization

• DAPI (4',6-diamidino-2-phenylindole):
  • DAPI is used for nuclear staining to visualize DNA.
  • The DAPI is contained within the mounting medium (the "mounting lid" or "fluoro").
  • It allows researchers to see exactly where the DNA is located within the cell and identify nuclei.
• Map 2 (Microtubule-Associated Protein 2):
  • Map 2 is a specific dendritic cell marker used for neurons.
  • It is used to identify differentiated cells.
  • In the experiment, Map 2 was labeled with a green fluorescent dye.
  • Dilution Factor: A specific dilution factor was used for Map 2, which resulted in a dimmer signal compared to other markers like Oct 4.
• Oct 4 (Octamer-binding transcription factor 4): Mentioned as a marker used for undifferentiated cells, typically appearing much brighter than Map 2 in those specific states.
• Experimental Differentiation Results:
  • Undifferentiated Cells: These show very little to no expression of Map 2. When switching from the DAPI channel (which shows many cells are present) to the Map 2 channel, the background appears mostly empty.
  • Differentiated Cells: These show bright Map 2 expression, highlighting the formation of dendrites.
  • Dendrites: The imaging shows cells forming dendrites to communicate with each other, often attempting to "touch" neighboring cells. These might appear on different $z$-levels.

Experimental Contamination and Incubator Maintenance

• Bacterial Contamination (E. coli):
  • A student named Charlie had a "worst-case scenario" where his wells were contaminated with rod-shaped bacteria, likely $E.\,coli$.
  • $E.\,coli$ divides rapidly, approximately every $30\text{ minutes}$. Over a single weekend, this leads to a "ton" of growth, completely filling the wells.
  • Contaminated wells show a mix of dead cells and "little rods."
• Environmental Contaminants:
  • Yeast and Fungi: These are common contaminants.
  • Spores: Yeast spores travel easily through the air.
  • Incubator Filters: If the filters in the incubator are failing, it increases the likelihood of finding yeast or black mold, which can spread very quickly.
• Mitigation and Prevention: Contamination often occurs when materials are taken out of the laminar flow hood and not properly sprayed with disinfectant before being returned, or if the hood's sterile environment is breached.

Laboratory Procedures and Troubleshooting

• Slide Preparation: Mounting involves placing the coverslip onto the slide using a mounting medium containing DAPI. Participants discussed the possibility of accidentally using "inspection coverslips" versus regular ones.
• Drying Cells: A student (Amira) mentioned potentially damaging her cells by pressing a Kimwipe down directly on the whole thing while drying, which could have "seared" or flattened the cells.
• Passaging Cells: The instructor notes that cells should not be allowed to become too "crowded," as this can kill them. Passaging involves transferring a portion of the cells to new media to maintain growth.
• Organelle Visualization: While DNA and general cell outlines (cytoplasm) are visible, specific organelles like vacuoles are harder to distinguish, appearing only as faint outlines or marked circles within the cell. Some cells may be captured in the process of mitosis.

Academic and Professional Backgrounds

• Graduation Timelines:
  • Ruff is entering his second year and expects to graduate in $2\text{ years}$.
  • Jake is graduating in the spring (of the next year).
• Institutional Comparisons:
  • The group compares San Jose State University (SJSU) to UC Berkeley.
  • Matt suggests that SJSU is more "fun" and offers better opportunities for undergraduates, whereas graduate studies at both institutions are fairly similar.
  • The group notes that "dumb people" or "out of touch" students (e.g., those who do not know what the BART is at age $20$) exist at all levels of university, including prestigious ones like Berkeley or UPenn.
• Military Service:
  • Matt served in the Marine Corps for a $4\text{ year}$ contract.
  • He describes talking to old veterans as a "hella fun" experience.
  • He discusses the Veterans Affairs (VA) hospitals, comparing the Sacramento facility (which he describes as "amazing" but often chaotic with emergency room incidents) to the San Francisco facility (which he describes as "pretty light" and "really nice").

Personal Anecdotes and Socio-Cultural Discussions

• Pop Culture Breakup: The group discusses the breakup of Ariana Grande and Ethan Slater (referred to as "the ginger guy" who played Spongebob in the musical).
• Regional Stereotypes: There is a discussion about low education metrics in states like Mississippi, Louisiana, Arkansas, and Oklahoma. Matt mentions meeting people from "bum pucks" (rural areas) in West Virginia during his military service who seemed to lack "depth" due to limited exposure to the world.
• Experience of Poverty vs. Wealth: A dialogue occurs regarding being "money tone-deaf." One student describes a friend who thinks traveling is "cheap" because her parents pay for everything, while another student contrasts her life in an apartment with a friend who claims to be poor while living in a house.
• Voting: The group mentions an upcoming vote in November involving a "top two" system where parties are not the primary filter; rather, the top two candidates move forward regardless of affiliation.

Questions & Discussion

• On DAPI use during inspection:
  • Question: "Did you use DAPI for the inspection too?"
  • Response: Yes, it was in the mounting lid/fluoro applied to the outside.
• On the Spongebob Musical:
  • Question: "There's a Spongebob musical?"
  • Response: Yes, it featured Ariana Grande's boyfriend (Ethan Slater).
• On Military Service Value:
  • Question: "Was it worth it?" (Regarding the Marines).
  • Response: "Yeah. It's a good time. It's good."
• On Personal Safety and Graduation:
  • Context: A joke was made about shooting Jake if he is seen outside of campus after graduation.
• On VA Hospital Experiences:
  • Dialogue: Matt explains that the Sacramento VA is next to the emergency room, leading to situations where patients "rage out" or believe people are trying to kill them, requiring police intervention.
• On Cell Differentiation Markers:
  • Question: "Why do you think [the Map 2 stains] are dimmer?"
  • Response: Because Map 2 is dendritic-specific and the dilution factor used was different than for other markers. Also, undifferentiated cells will not express it highly.
• On Racism in Education:
  • Dialogue: A student shares an experience of overt racism in high school where a peer straight-up said, "I don't like your kind," and that individual now attends UPenn while his friends are at Berkeley.