Castelian (NES)/any%(ntscj)

The route shown here is for the Japanese version, played on Hero difficulty . This is the fastest combination for beating the game.

Tower 1

Taking the elevator past the first molecule on Hero difficulty only works on the JP-version and is frame perfect (= 3 actual frames since the game runs at 20 fps).
With a well times jump (around 0:28-0:29 in the video), it's possible to time the spawn of the vertical moving enemy at the second destroyable ball so it bounces back off the ball, eliminating any waiting time. It saves a little under a second. However, this depends on other enemy positions that are difficult to gauge in real-time and therefore adds quite a bit more risk.
It's important to walk, and not jump, onto the elevator leading to the aforementioned vertical moving enemy and the second bouncing ball (at around 0:30). This ensures that the bouncing ball can be destroyed before other enemy blocks it and will also set up a better timing of the last molecule appearance.

Note the jump at 0:16. It would have been possible to jump one step further down to scale the stairs faster. However, jumping on this step will spawn the enemy above as early as possible. This will allow you to push on a little earlier when it's blocking the way a little later on.
Walking (and not jumping) up the stairs after the jump mentioned above and allowing the character to turn in the wrong direction at 0:24 (by not holding right) work as a buffer strat for jumping past the enemy at 0:26 pretty much as early as possible. Arriving here earlier will otherwise only lead to waiting for the enemy to move out of the way and then judging when it's safe to jump.

By shooting just out of range of the bouncing ball at the beginning, it's often possible to manipulate the ball to delay its start of movement. This allows you to enter the opening, which otherwise is not possible without shooting the enemy. By avoiding to destroy it, 6 frames of countdown are saved at the end of the level.

The timing and time window for passing beneath the first molecule is tight. A well-timed jump (at 0:11) is needed to make the molecule spawn when you're at the top of the jump. The molecule always appears at the same time after the level begins, but since it can be difficult to have consistent movements, it's not always easy to gauge when to jump with the required precision. This trick is however important as failing it will cost several seconds both here and also set up the molecule in an unfavorable way later on.
If the blinking pellet to the right is destroyed at 0:39, you can continue to the left at 0:45. However, that route is slightly slower.
There is a risk that the bouncing ball at 1:19 will not start moving if you push on too fast. This can be mitigated by adding an intentional pause before shooting (see enemy movements for more details). Postponing shooting until after the jump is both risky (if the ball starts moving towards you) and slower. Once the ball is destroyed, the molecule timer will start ticking down. The more it ticks down, the more time saved at the very end when waiting for getting knocked down.
It's slightly faster to jump (and not walk) onto the last lift that leads to the top of the tower.

Try to jump off the blinking pellet at 0:08. There is not much room to do so, but it will trigger the enemy above earlier if successful. The time save is free (at worst you'll just walk up to the next step), but also very minor though.
Turning around at 0:17 (instead of immediately activating the elevator) gives the best pattern for the circling enemy above.
The jump at 0:43 was to trigger the spawning of the enemy that was passed beneath at 0:57.
The horizontal moving enemy that spawned around 1:02 will occasionally collide with one of the vertical moving enemies and then create a different pattern. Basically all the enemy movements are linked from this point until the end of the level. So when this happens, it's difficult to predict the outcome. However, most of the time the horizontal moving enemy will not collide.

The final section is a bit more complex and will be described based on the following illustration:

While standing at position 'A' (1:30), the molecule timer won't run. It's important to manipulate the molecule to appear at the right time. The time when to move two steps to the right to position 'B' (1:33) is therefore critical. Watch the enemy positions for the queue when to start moving.
'2' will always bounce off '1', like in the video. This is assuming that the route until this point has been followed and that you didn't get bad luck with a collision between the enemies around 1:05.
The last stretch with the two bouncing balls ('4' and '5') and the two vertical moving enemies ('6' and '7') is difficult to fully control in real-time.
'7' will appear when '4' is destroyed. If you destroy '4' too early, it can be tight to squeeze past '7'.
'6' will appear when '3' (the molecule) disappears. Ideally, '6' should bounce off '5' before it's shot. This requires:

- not shooting too early

- that '5' starts moving in a timely fashion (see enemy movements for more details)

- that '3' was manipulated to appear (and therefore disappear) at the right time for '6' to spawn.

It should be mentioned that this level has the most theoretical potential for improvement. In TAS-conditions, the enemy manipulations can be pushed a few steps further to save several seconds.
It's not possible to jump down the pit end the bonus level early. You have to walk into it.

It's worth noting the jump at 0:20 to despawn the enemy below for a small time save.
At 0:48, it's not necessary to take the elevator up to jump onto the stack of destroyable floor blocks. Jumping directly from the elevator at its lowest position is pixel perfect and saves a little less than a second. Initially it saves a bit more time, but the enemy positions are worse near the end and eat up some of that time.
It's not possible to jump down the pit end the bonus level early. You have to walk into it.

Shooting the bouncing ball on the top of the tower is optional, but is overall a few frames faster by reducing lag.
It's not possible to jump down the pit end the bonus level early. You have to walk into it.

The pause at 0:33 was only to better adjust for the next jump (pixel perfect) and was not related to any enemy manipulation.
The enemy that was jumped over at 0:44 can spawn at other times than what's shown in this video. This is because it will take over a memory allocation of one of the 4 enemies below. Since they're moving in a tight space, one of the might or might not be despawned during one of your jumps. This will have an impact on how to pass the enemy (jump over, under or have to wait for it), but can also impact the timing of the rest of the level. This branches out to several possitibilites that have not yet been explored for this guide.
It's sometimes possible to have enough time at 1:44 to turn around and shoot the blinking pellet without getting knocked down by the molecule. However, this is the exception when following the route in the video in a real-time attempt.