20: Chapter 20 The First Step to Implementation (Part 1)

Chen Youhua sat at his temporary workstation, the project plan approved by Professor Zhang and passed through the laboratory's internal discussion spread out before him.

The clear steps and expectations on the paper were about to face the test of reality.

The core of the project was to study the effects of specially designed microstructured substrates on the dynamic behavior of intracellular microtubules.

The first step was to obtain these microstructured substrates—the chips he had ordered.

A few days later, a small delivery box arrived. After Senior Sister Li signed for it, she handed it directly to Chen Youhua.

"Chen Youhua, the chips you ordered from Supplier B."

Senior Sister Li pointed at the box.

"New consumables, especially custom parts like these, require a preliminary quality inspection before use. Come with me."

Chen Youhua followed Senior Sister Li to an ordinary optical microscope.

Senior Sister Li unpacked the box and took out several transparent thin sheets that appeared smooth and flat.

She took one out, added some mounting medium, carefully covered it with a coverslip, and fixed it onto the stage.

"It looks fine to the naked eye, but the microstructure needs to be confirmed."

Senior Sister Li adjusted the focus and motioned for Chen Youhua to observe.

"Mainly check for obvious scratches, contamination, or bubbles on the surface, as well as the uniformity and integrity of the microstructure."

A densely packed array of tiny cylinders appeared on the screen.

Senior Sister Li moved the stage.

"These are the specifications you requested; the diameter and spacing are both labeled.

Focus on the edge areas and random points to see if there are any structural defects."

Chen Youhua inspected it carefully.

Most areas met the requirements, but in one edge area, he found several rows of cylinders significantly shorter than the surrounding ones, and the arrangement was distorted.

"There's a defect here," he pointed out.

"Record the position and the specific problem."

Senior Sister Li nodded.

"Supplier B's process stability is slightly worse than Supplier A's. Localized flaws like this aren't uncommon in samples within this budget.

This is also why the initial purchase is for process validation. We'll use Supplier A's high-precision chips for the critical experiments."

Chen Youhua wrote down the details in a dedicated logbook.

"Sample No. B-003, Area C4, uneven pillar height, array distortion."

With the chips in hand, even if some were flawed, the next step was to culture cells on these substrates and fluorescently label the intracellular microtubules for long-term live-cell imaging observation.

Chen Youhua didn't try it directly on the chips; he first conducted pre-experiments in ordinary glass-bottom culture dishes, aiming to stably obtain clear dynamic images of the microtubules.

He strictly followed the operating procedures.

Preparing the culture medium, preheating, digesting cells, accurate counting, resuspending, plating... His movements were still a bit unpracticed, but the steps were correct.

However, the results were unsatisfactory.

First long-term imaging attempt, target duration: 3 hours.

Phenomenon: About half an hour after imaging began, the cells gradually detached from the substrate, their morphology changed, and the fluorescence signal continued to weaken until it disappeared.

Analysis: The cells were not firmly attached, possibly related to plating density or serum concentration. Additionally, laser irradiation caused photobleaching of the fluorescent dye.

Second attempt.

Adjustments: Reduced laser power and optimized plating conditions by increasing cell density and adjusting the serum ratio.

Phenomenon: Cell attachment improved, but the fluorescence signal of the labeled microtubules within the cells was weak and the images were blurry, making them unusable for subsequent tracking analysis.

Analysis: Low fluorescence labeling efficiency; there might be issues with staining time, dye concentration, or cell viability.

Third attempt.

Adjustments: Optimized the fluorescence staining steps by extending the staining time and changing the dye batch.

Phenomenon: The fluorescence signal strengthened, but non-specific fluorescent particle interference appeared in the background. After about an hour of imaging, the cell state significantly deteriorated and morphology contracted due to minor fluctuations in the culture environment, such as temperature or CO2 concentration.

Analysis: Poor control of staining background; environmental stability control needs to be further strengthened.

Chen Youhua closed the imaging software and looked at the unusable image sequences on the screen.

In the waste liquid jar nearby, several more discarded culture dishes had been added.

The steps for cell passage, fluorescence labeling, and live-cell imaging in the project plan were, in practice, full of uncertainties and details that required repeated debugging.

Wang Zhe walked over with a cup of coffee, catching a glimpse of Chen Youhua's solemn expression and the failed images on the screen.

"Still not working?"

"Yeah," Chen Youhua responded briefly.

"That's normal,"

Wang Zhe said calmly.

"When I first started with this, I failed just as many times, if not more.

Live-cell imaging is very demanding regarding cell state, environmental control, and parameter settings.

If any part isn't done well, the results won't be ideal."

He put down his cup and looked at the screen.

"What was the specific situation this time? Detachment? Weak signal? Or poor cell state?"

"I encountered all of those," Chen Youhua replied.

"Encountering them all is right; experience is gained through trial and error," Wang Zhe said.

"The key is to record in detail the parameters of each operation, the cell source, the medium batch, and the operational time points.

Failure itself is information; analyzing the cause is more important than blind repetition."

He left half a pack of energy bars.

"Get some energy. Take it slow and figure out every variable."

While debugging the cell state, Chen Youhua continued to practice operating the Confocal microscope under Wang Zhe's guidance, especially focusing on stability control for long-term time-lapse scanning.

"Your hand movements need to be steady, but don't be overly tense."

Wang Zhe stood nearby, watching Chen Youhua intently adjust the precision focus knob, his fingers slightly stiff from the effort.

On the screen, the internal structure of a cell expressing Green Fluorescent Protein-labeled microtubules was clearly visible.

Wang Zhe required him to practice setting up and executing continuous scans lasting over two hours.

"The core of long-term imaging is stability.

The instrument itself must be stable, the operating environment must be stable, and the operator must also be steady.

Field of view drift is a common problem that needs timely manual fine-tuning; you can't rely entirely on the auto-correction function.

The cells themselves also move, which adds uncertainty."

Chen Youhua held his breath and focused, trying to control his subtle hand movements while staring at the screen.

The mental pressure from maintaining high focus and precise control far exceeded his expectations.

During one practice session, a bright spot in the field of view moved rapidly. Chen Youhua's hand jerked instinctively, and the image blurred instantly.

"Pay attention to sudden situations,"

Wang Zhe reminded him.

"Don't panic; steady your hand and slowly adjust it back.

If necessary, pause the sequence and re-center the focus. Losing some time is better than having the entire segment of data be invalid."

Chen Youhua paused as instructed, repositioned, and started again.

The entire process required immense patience and concentration.