optical_and_mechanical_alignment_on_ramm
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optical_and_mechanical_alignment_on_ramm [2017/08/25 20:34] – [Aligning the Multi-LED Illuminator] vik | optical_and_mechanical_alignment_on_ramm [2022/02/15 17:28] (current) – melissa | ||
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+ | ====== Optical and Mechanical Alignment of Epi-widefield and objective-TIRF on RAMM/MIM ====== | ||
+ | |||
+ | Correct adjustment of the illumination optics will depend upon the particular configuration of the RAMM/MIM microscope. | ||
+ | |||
+ | ===== General adjustment strategy ===== | ||
+ | |||
+ | - Align TIRF laser when in the “centered” position of the TIRF angle adjuster to the objective Back Focal Plane (BFP). | ||
+ | - Establish the correct zero position for the Port Switch mirror and other clocked devices. | ||
+ | - Center TIRF beam with adjusters on the main dichroic CUBE-III | ||
+ | - Focus TIRF beam roughly in BFP using the lens in the Cage assembly. | ||
+ | - Align and focus LED illuminator LEDs at the objective BFP. | ||
+ | - Center camera view on the epi-illumination field stop. | ||
+ | |||
+ | ===== Setting the C60-Port_Switch Zero Position ===== | ||
+ | |||
+ | This is usually the first adjustment for a newly assembled system. | ||
+ | The Port Switch is usually used with condenser lenses on each port that are designed to focus light into the objective BFP. With a TIRF illuminator, | ||
+ | The C60-PORT_SWITCH moves a mirror to select either port 1 or port 2 to be reflected to the common port. The geometry is defined by the figure below. | ||
+ | |||
+ | {{ : | ||
+ | |||
+ | To set the mirror location for port 1 we need to have the illumination source connected to port 1 turned on and roughly focused at the objective BFP. | ||
+ | Determine the name of the Port_Switch axis. For the TIGER controller the [[commands: | ||
+ | |||
+ | <wrap cmd> who </ | ||
+ | <wrap reply> At 30: Comm v3.17 TIGER_COMM Jun 01 2017: | ||
+ | At 31: X: | ||
+ | At 32: Z: | ||
+ | At 33: O: | ||
+ | At 37: L:Leds v3.16 TGLED_4 Oct 25 2016: | ||
+ | |||
+ | <wrap cmd> bu x </ | ||
+ | <wrap reply> TIGER_COMM \\ | ||
+ | Motor Axes: X Y Z __**M**__ O T L \\ | ||
+ | Axis Types: x x z __**h**__ o l i \\ | ||
+ | Axis Addr: 1 1 2 __**2**__ 3 3 7 \\ | ||
+ | Hex Addr: 31 31 32 32 33 33 37 \\ | ||
+ | Axis Props: | ||
+ | |||
+ | For the MS2000 controller the name is revealed by the [[commands: | ||
+ | |||
+ | <wrap cmd> bu x </ | ||
+ | <wrap reply> STD_XYZ_PSWTCH \\ | ||
+ | Motor Axes: X Y Z __**S**__ | ||
+ | Axis Types: x x z __**h**__ | ||
+ | CMDS: XYZSFTR \\ | ||
+ | BootLdr V:1 \\ | ||
+ | Hdwr REV.E \\ | ||
+ | LL COMMANDS \\ | ||
+ | RING BUFFER 50 \\ | ||
+ | SEARCH INDEX \\ | ||
+ | CLOCKED POSITIONS \\ | ||
+ | IN0_INT </ | ||
+ | |||
+ | We want to be able to manually rotate the mirror to the best position. To do that we need to connect the Port-Switch motor to the controller control knob using the [[commands: | ||
+ | |||
+ | |||
+ | For TIGER <wrap cmd> J M=22 </ | ||
+ | |||
+ | For MS2000 <wrap cmd> J S=4 </ | ||
+ | |||
+ | At this point you will be able to manually steer the port switch mirror using the knob on the controller. | ||
+ | |||
+ | ^ Tiger ^ MS2000 ^ ^ | ||
+ | | <wrap cmd> 2RESET </ | ||
+ | |||
+ | Check your work. Be sure you can now command the port switch to move the second position and back and that if comes back where you expect it to. | ||
+ | |||
+ | ^TIGER ^MS2000 ^ ^ | ||
+ | |<wrap cmd> M M=2 </ | ||
+ | |<wrap cmd> M M=1 </ | ||
+ | |||
+ | ===== Setting the Automated Objective Turret and/or Automated Cube Slider Zero Position ===== | ||
+ | |||
+ | |||
+ | The same procedures outlined above applies to setting the home/first position for other ASI clocked devices such as the automated objective turret or the automated filter cube slider. | ||
+ | |||
+ | For the objective turret it is best just to turn off the controller and manually move the nosepiece so that the first position is in place and the mechanism is centered on the detent for that position. | ||
+ | |||
+ | ^TIGER ^MS2000 ^ ^ | ||
+ | |<wrap cmd> 3RESET </ | ||
+ | |||
+ | Use the same procedure for the automated filter cube slider. | ||
+ | |||
+ | [{{ :: | ||
+ | |||
+ | |||
+ | |||
+ | ===== Steering the Beam by Adjusting the Dichroic Mirror ===== | ||
+ | |||
+ | The procedure above can only adjust the beam position in the axis that the mirror movement allows. | ||
+ | |||
+ | [{{ : | ||
+ | |||
+ | [{{ : | ||
+ | | ||
+ | Newer CUBEs use thumb screw adjusters, older CUBEs use a 3/32 Allen driver. | ||
+ | |||
+ | ===== Focusing the TIRF Laser ===== | ||
+ | |||
+ | The TIRF laser is focused at the objective BFP by sliding the condenser lens on the cage assembly. | ||
+ | |||
+ | [{{ : | ||
+ | |||
+ | Rough focus of the laser at the objective hole witness target is good enough for initial alignment. | ||
+ | |||
+ | ===== Aligning the Multi-LED Illuminator ===== | ||
+ | |||
+ | On systems that include a port switch and TIRF illuminator, | ||
+ | On systems with only the LED illuminator for epi-illumination, | ||
+ | In both cases, fine adjustment of each of the LED emitters should be done, one at a time, to best center and focus the emitter on the witness target at the objective aperture. | ||
+ | See the LED command for the syntax for turning on/off the LEDs. In our example with card #7 the LED control card, | ||
+ | |||
+ | <wrap cmd> 7LED x=0 y=0 z=0 f=0 </ | ||
+ | <wrap cmd> 7LED y=50 </ | ||
+ | |||
+ | ==== LED Focus ==== | ||
+ | |||
+ | The LED focus I achieved using the helical focus on the individual LED illuminators. | ||
+ | |||
+ | [{{ : | ||
+ | |||
+ | There are six set screws that hold the focus assembly, three in the helical grooves and three outside the grooves. | ||
+ | |||
+ | [{{ : | ||
+ | |||
+ | ==== LED Centering ==== | ||
+ | |||
+ | Once the LED is in focus, you can also center the emitter on the objective opening. | ||
+ | |||
+ | [{{ : | ||
+ | |||
+ | Loosen the three LED board mount screws and manipulate the LED board using the bolt heads of the mounting screws and the cable connector. | ||
+ | Repeat the LED focus and centering adjustments with each of the LEDs in the multi-LED illuminator. | ||
+ | |||
+ | |||
+ | |||
+ | {{tag> |
Address: 29391 W Enid Rd. Eugene, OR 97402, USA | Phone: +1 (541) 461-8181