Splinter's back-story. People familiar with previous versions would know the details, but to make it worse, Splinter also had a daughter.
It gets worse: the daughter didn't die. Shredder raised her as his own and raised her to hate Splinter.
When Donatello pleads his case, Splinter looks at a picture of him and his previous family and we hear in Splinter's mind a fire , wooding creaking and falling and finally Splinter screaming in pain. This scene reminding Splinter of the worst night of his life
Michelangelo sounded so hurt when he found out that he was being used by Bradford. He so badly wanted to prove he coul be friends with a human, and instead ends up captured by the Foot for it. "Rad Brad..."
Shredder looking at a picture of him and Splinter showing a time when they were the best friends and not the bitterest of enemies.
Splinter realizes that everything he had is gone, his family, his ninja clan, and even his humanity.
Not just that, but what sends Splinter over the edge—what makes him really lose it—is the thought of his children no longer needing him. He's watched his wife die, and lost his daughter, but the idea of losing his sons is what makes him give up hope. And the worst part is, the boys ARE going to grow up. Them not needing Splinter could very well become a reality one day.
You can hear the crack and despair in his voice as he says this Soliloquy
Rat king using splinter's memory of the night he lost everything
There's something sad about Leonardo feeling weighed down by the Chains Of Commanding, and the only people who give him any kind of real sympathy are Mikey and Karai. Even his father says that leadership is about responsibility and that he should just tough it out.
Pulverizer, wanting so bad to be a hero, intentionally exposes himself to the mutagen. Instead of making him into an humanoid animal, it painfully turns him into a blob monster, who mindlessly lashes out at his surroundings until the turtles can trap him. Yes, he was an idiot and it was his own fault, but geez man, he didn't deserve that! Things like that shouldn't happen to comic relief!
April finding out that the foot have brainwashed her father
Splinter's mission against the Shredder is chalk full of these. Perhaps the biggest one of all being that his own daughter considers him her worst enemy because she thinks he killed her mother. The look of pain on his face is easily palpable as he flees.
Also, his look of shock when the Shredder tells him about how he stole his daughter away.
The worst part for one moment Karai almost recognized her birth name when Splinter says Miwa but then tells Splinter her name is Karai.
And to add salt to the wound Shredder is laughing at pain he is causing, at what could be the second worst night of Hamato Yoshi's life.
Not to mention the pain and anger in Karai's voice when Splinter retreats and she yells "Coward! Why won't you fight me!?" Her Missing Mom situation has really affected her emotionally. One can only imagine her reaction if she ever finds out the truth, realizes that she's been lied to her entire life, and that she tried to kill her real father.
There's something inherently sad about seeing Splinter embrace his inner rat to defeat Shredder. Half a season ago, his turtle sons brought him back by reminding him of his humanity away from rats. And now, in a moment of rage, he embraced the rat just so he could survive.
Kirby O Neil getting transformed into a bat monster and April trying in vain to talk to him
Then April getting angry at the turtles (for being indirectly responsible for Kirby becoming a monster) and saying she never wants to see them again.
After He's been transformed and gone feral he attacks April who's forced to hit her own father in self defense and he stops the human side regaining control and you can see in his eyes that he can't believe he just attacked his own daughter.
Leo: Donnie, whatever, Pulverizer...Timothy, was, is gone now.
All segments with him in general this episode. After hearing Donnie lamenting how he misses April, he breaks free and tries to go find her. But the attacks from The Police, Casey, and The Turtles only made him go more crazy.
Donnie's final words to him in the episode.
Donnie: Don't worry my friend. One day, I'll find a cure.
Pulverizer/Mutagen Man's final words are heartbreaking. Donnie manages to finally take him down with a sort of petrifying goop that was disguised as mutagen, as as he freezes he seems to regain some of his sanity.
Pulverizer: Donnie, there's something wrong...!
April still being upset at the turtles and even refusing herself to help them fight Mutagen Man.
Every scene in which Donnie deals with April meeting Casey, especially the first time he sees them together and we see an Imagine Spot of his whole body shattering, and later during the fight with Mutagen Man when Leo casually refers to Casey as April's boyfriend, making Donnie wince. It's painful to watch, especially for anyone who was ever the geeky awkward friend that had to watch his unrequited crush meet someone else.
Slash falling off the building with Raph's anguished Say My Name. Later, it turns out he survived the fall and disappeared. Then we see Raph holding a leaf he fed Spike in sorrow. The look on his face shows how heartbroken he is that his pet turned into a savage beast and left him.
You want to know how this can be even sadder some owners see their pets as their kids. In a way Raph lost his child when he lost Spike.
The fact that he lost Spike by having him turn against him parallel to Splinter with Karai.
Splinter's advice to Raphael reveals that he still feels the pain of losing his family
Also a tearjerker if one looks at it from Slash's perspective. It's clear that his actions make him come off more as a Well-Intentioned Extremist. However after Raph calls him out on what he did to Donnie and Mikey, there is a slight pause before he lets out a monstrous roar signifying a prime of example of Then Let Me Be Evil as Slash only did what he Raph's sake and is hurt that the person that treated him as a friend for so many years essentially called him a monster.
Metalhead's Shoot the Shaggy Dog-ness in the episode overall. Metalhead has finally been upgraded with intelligence in order to work alongside the Turtles, only for them to not trust him due to how much of a 3rd wheel he winds up becoming. Realizing this, Metalhead leaves on his own to take care of the mission by himself to show the turtles he was on their side the whole time, not only rescuing ALL the mutants and the turtles, but also destroying himself in order to ensure the Kraang would not find another way to get back to Earth. The ending implies that Donatello will fix him again, but it really makes the viewers feel for Metalhead's loyalty to his team and creator.
Leo's constant raging on Metalhead sure didn't help, by the time Metalhead leaves the lair. It seemed like Leo had been waiting for a chance to force Donnie to dismantle him.