As I am setting up a 2-photon microscope in my lab, I decided to leave my note here. The system should be significantly cheaper (say, 50%) and probably better than any commercial 2-photon systems, but obviously you need to commit more time and efforts to build.
Materials
- Microscope: I use olympus upright microscope, IX51, because their fluorescence microscope is featured with extremely low background light, so that one can even visualize single molecule without any special setting. I asked them to make a hole (~35 mm) in the side of a filter cube turret, and screw holes around the hole that fits to microconstruction system from Thorlabs. Also, I use a dichroic mirror holder that reflects light to the side (Olympus now provide this cube). This way, we can put photo-multiplier (PMT) to the side of microscope. It should be noted that we need to put PMT as close as possible to the objective to get maximum number of photons in light-scattering tissues. (~$40K)
- mirrors, lens, posts, holders: Thorlabs and Newport (~$1K)
- Optical table: I use big table (12 x 4 feet) that can hold two rigs. Because lasers are so expensive, it is cost effcient to split one laser light and feed into two rigs. It is a little bit tricky to do electrophysiology in two rigs in a table, but still doable. (~$8K)
- Scanner: Cambridge technologies provides fairly good and cheep scanner (6201H). Their lead time is ridiculously long (9 weeks). Their driver board get very hot during opperation. One should not put a lid on this. (~$2K)
- Scan lens: I purchased scan lens for a confocal microscope from Olympus. If one wants to reduce cost, it is good enough with a standard achromatic lens. Singlet lens could also work. (~$1K)
- Machining: One needs to machine an adaptor to connect scanner to the scan lens.
- Cage: One needs complete darkness for 2-photon unlike confocal system. I made a box (size = hight 36''x width 30''x depth 30'') made of angled and slotted bars (McMaster.com, 4664T51) and thin alminum plates (89015K71
). Inside of the cage is black-painted with a car painting spray. Make several holes for wiring and laser light. Metal cage also reduces electrical noise. (~$0.2K)
- Signal amplification and filtering: It is necessary to filter photon signal from PMT with ~300KHz to sample with AD converter. I use stanford amplifier SR-570.(~$2K)
- AD-DA converter: NI6110, National Instruments ($3K). This is both for scan control and data acquisition.
- Photomultiplier: Hamamtsu R3896 Select for QY>20%, Dark Current < 15nA. 4 PMT for 1 microscope. (Gree/red channels for epi/trans fluorescence)
