"System Name" "FVMPE-RS"
"System Version" "2.3.1.163"

High speed, high sensitivity, deep imaging multiphoton microscope

Instrument startup

Before startup, the correct filter sets need to be installed. This involves knowing the colors being imaged and understanding which detectors will be used. Practically, the filters need to be inserted into the system and they will be programmed into the software after startup. We envision that a few users will be trained to do this for their projects (and they will be required to follow the steps for documenting all hardware changes made). Most users will need to schedule ahead with staff. At the beginning of new projects or when characterizing new samples, variations of hardware settings may need to be evaluated before settling on a standard protocol.

If looking at fluorescence by eye to screen sample, U-HLPS metal halide lamp on front first. Press power button and let go quickly (holding for three seconds will turn off). If you hold too long or turn off accidentally, there is a five minute wait time built in to the circuit.

If computer was shut down, power up at any time.

CBH control box front switch on. Goes into standby mode.

Prior scan control unit for XY stage. Switch is on back right. It needs to be on (and may be left on at all times).

AOM on. (This is a laser.)

AOM on. (This is a laser.)

Touchpad on. Switch is at back right.
Wait for this to boot up with display on screen before proceeding to next step.

Big box at bottom of rack, power switch on front.

Log in to computer.

FV software.

Select whether to initialize Resonant Scanner &/or Piezo focusing.

Saving files

Each menu for image collection in FluoView has it's own save path and save names. Make sure you have all the menus populated correctly so you don't lose files.

Any files stored on Desktop or Drive C: may be deleted at any time without warning. DO NOT STORE ANYTHING ON THE DESKTOP OR DRIVE C: The only warning that files may be deleted is the warning you just read.

Where to store files:
Your lab should have a directory on drive D:
You should have a subdirectory in your lab's directory.
If we have a problem with the HD filling up, we will delete files moving roughly from oldest tonewest.
It is your repsonsibility to keep track of your data.

Drive E: is a solid state HD for high speed imaging. Do not store data here. As soon as you are done with imaging, copy your filed to D:.

If you are transferring data to an external hard drive or other USB device, BEFORE inserting into the computer, make sure the entire drive was scanned by anti-virus softtware, preferrably the insitution's. Plan ahead.

 

Filter sets for emitted fluorescence / harmonics

There is a total of four detectors.

Two are GaAsP detectors that are highly sensitive and you have to be careful not to overload them with too much light. They can be permanently damaged.

There are two PMTs that are less sensitive, but a lot more robust.

The filter sets are not on automated filter wheels; they need to be manually loaded.

The filters are paired as blue/green and red/far-red. There is also an option for green/red.

We have mirrors for the following:

You would install the filters to send the weakest signals to the GaAsP detectors. In general, don't send second harmonics to the GaAsP detectors.

Click here to see two example configurations for imaging blue, green, red, and far-red.

Considerations choosing detectors.
The GaAsP detectors are more sensitive, but more sensitive is not always better. They are higher contrast (narrow dynamic range) and can be harmed by excessive bright signal.

The PMTs (photo multiplier tubes) are less sensitive but allow for a wider range of signal.

In general, 2nd harmonics should always be sent to the PMTs because of the wider dynamic range, because 2nd harmonics tend to be brighter, and because you're unlikely to image the 2nd harmonics deep in the tissue.

When setting up channels in the software, each detector may be imaged twice for a total of 8 channels per scan. This is because each channel may be imaged by each laser line in sequence.

There are two lasers. You may excite simultaneously, in rapid sequence line-by-line, or per frame scan.

For instance, you may find it useful to image a red channel twice, once with excitation at 1130 nm and a second time with excitation at 930 nm. This should provide separation of mCherry and RFP respectively.

Why 1130 nm when 1150 to 1200 nm may generate a brighter fluorescent signal especually if also scanning for the far-red channel? If you scan greater than 1130 nm, then second harmonics are visible through the red filter set. To eliminate this, we recommend 1120 to 1130 nm.

 

Lasers, Insight X3-OL and MaiTai HP DS-OL

There are two lasers. Emission is turned on and turned off from the Tools > Configuration menu.

There is a floating window with controls for the wavelength. At the bottom is a beamsplitter choice of 690/1050 and 690/870. In general, leave it at 690/1050 for use with the MaiTai laser less than 1050 nm and the Insight greater than 1050 nm.

When you change the wavelength of a laser, wait for it to tune and then click the "Start active alignment" button in the fine adjustment pull down menu.

But when using two lasers, regardless whether in the same group or alternating groups, you may still have the following problem:
and click here for an example with real biology, cells expressing both RFP and GFP.
This is a single 4 um diameter multicolor bead and due to the laser paths at slight tilts, the excitation paths are not in register (not coaligned). To fix this, adjust the Tilt X and Tilt Y. You may want to go to a non-precious area and zoom in at 256 x 256 pixels and scan fast while adjusting. Adjust the Tilt, not the Shift, and this can be done with either or both of the lasers.

This setting may vary over time and based on room temperature.

 

Instrument shutdown

To turn off lasers, opposite of turning them on. Tools > Configuration > Lasers > Stop Emission

Set software to LSM (not aocular) mode.

Then quite software.

Touchmpad on table is two step process:
1. Off button in upper right of screen.
2. When only brand icon on screen, switch on right rear.

Roungly from bottom to top of rack:
Big controller box on bottom.
Two AOM boxes

CBH control box front switch on.

Prior stage switch is on back right.

Lamp-- hold power button for 3 seconds.

Computer: log out or shut down.