This is based on opinion. Please don't list it on a work's trope example list.
Tear Jerker / Ace Lightning
Sparx's death in "Unidentified Flying Superhero", blasted by her own sword and returns to the game. She get better.
Lady Illusion's Heroic Sacrifice in the finale to save Ace from Lord Fear. The final exchange between the lovers is a tearjerker.
Lady Illusion: Oh, Ace. Those human emotions, they made you weak. Before I go, I have the power to take them away. My last gift to you.
Ace Lightning: No. Emotions are what I'm feeling for you right now. That's the real gift. (He then sheds a tear).
Lady Illusion dies in Ace's arms, and Sparx and Random Virus come to his side to comfort him.
Random Virus as a character. He is a Lightning Knight and Ace's friend. However, an unidentified accident left him severely injured and mentally damaged. He became a cyborg suffering from multiple personality disorder, being either a self-loathing hero or a violent destructive psychopath who has a twisted sense of justice.
The pilot has a musical montage showing Mark struggling to adapt to high school, and looks longingly to the UK on a map in geography class. We then cut to Ace touching a computer screen and bowing his head in sadness, implying he misses his home too.
Mark and Samantha closing the book to their relationship so Mark can pursue Kat. Both give each other a thankful kiss, but when Mark leaves, Samantha looks heartbroken.
Chuck's near death experience after a crazed Ace zaps him with a lightning bolt. This plays back to a previous episode when Chuck was hit by a deflected lightning bolt and temporarily gained superpower.
Ace Lightning: Good thing it was a deflected shot, or he would have sizzled.
The episode "Nobody's Hero" where Mark's normal life is collapsing around him, he is stalked by Lord Fear, and he is growing tired of lying to his loved ones. So when badass Sparx arrives on the scene and belittles Mark, he just snaps and quits the hero business. He comes back the next episode, but Ace is deeply saddened by his departure, not really appreciating the effort and stress his sidekick went through.
There is some pity for Duff Kent and Mr. Cheseborough, whose lives are invaded by the video game characters. Duff's livelihood is effected by Lord Fear, and Mr. Cheseborough goes from a somewhat Sadist Teacher to a paranoid wreck believing he was abducted by aliens.
Lord Fear's defeat in the first season's finale has him going down on his knees begging for Lady Illusion to save him. She refuses, revealing her loyalties to Ace. The likely realisation the love of his life had betrayed him for his arch nemesis hit Lord Fear hard judging by his hatred towards her in the second season.