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I have beaten Isaac the Cathedral level once, and when I returned to the title screen, there was a rectangle with what appeared to be a piece of a puzzle in it. I infer that I can complete the puzzle by beating Isaac with each character. Is this the way to complete the puzzle, and what happens when I complete it?

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up vote 2 down vote accepted

Your guesses are correct. The puzzle is completed by beating the Cathedral six times.

Here it is after my second victory against Isaac:

unlock 2

Completing the puzzle unlocks the Polaroid trinket, an item that shields Isaac when brought to 1/2 heart, but that also

unlocks the final level, the Chest, if you beat Isaac while equipping it. This is the only way to access it and get to the TRUE final boss, ???. Beating Isaac with it also unlocks Dad's Key.

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I am pretty sure that spoiler is totally incorrect. I have gotten that boss a few times without getting the Polaroid and now bringing the Polaroid to Isaac lets you access the chest. – murgatroid99 Jul 4 '12 at 3:40
@murgatroid99 The spoiler was correct when it was written due to a bug present in v1.1. It has been fixed since then. I've corrected my answer. – Kappei Jul 4 '12 at 4:57
You dont have to beat Isaac boss in the Cathedral with every character in order to unlock the Polaroid trinket, you just have to beat him six times, with whoever character you want to. – user62512 Nov 27 '13 at 12:15
@brianakias thanks for the correction, I've updated the answer. – Kappei Nov 28 '13 at 8:48

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