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tirf_alignment [2019/07/22 16:53]
gary
tirf_alignment [2019/07/23 22:20] (current)
melissa minor changes -mg
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 </​WRAP>​ </​WRAP>​
  
-[{{ ::​tirf1.jpg?​direct&​400 |TIRF assembly shown with micrometer option}}]+[{{ ::​tirf1.jpg?​direct&​400 |TIRF assembly shown with manual ​micrometer option}}]
  
-[{{ ::​tirf2.jpg?​direct&​400 |Manual and MA12 the motorized ​actuators ​options}}] ​+[{{ ::​tirf2.jpg?​direct&​400 |Manual and motorized ​actuator ​options}}] ​
  
 The assembly consists of a  The assembly consists of a 
   - Fiber launch ​   - Fiber launch ​
-  - Actuator to steer the beam,  +  - Actuator to position ​the fiber tip  
-  - Beam expander ​and  +  - Beam expander  
-  - Lens cage assembly. The lens cage assembly lets users focus the laser onto the Back Focal Plane (BFP) of the objective+  - Lens cage assembly ​- allows ​users to focus the beam onto the Back Focal Plane (BFP) of the objective ​
  
 ====== Assembling the TIRF ====== ====== Assembling the TIRF ======
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 For safer shipping, ASI might ship the TIRF disassembled. ​ For safer shipping, ASI might ship the TIRF disassembled. ​
  
-[{{ ::​tirf3.jpg?​direct&​400 | TIRF assembly ​and the ring with tube lens}}]+[{{ ::​tirf3.jpg?​direct&​400 |TIRF assembly ​with the ring-mounted focusing ​lens removed from the cage system}}]
  
-[{{ ::​tirf4.jpg?​direct&​400 |insert ​with the collar of the ring facing out or towards the RAMM}}]+[{{ ::​tirf4.jpg?​direct&​400 |Install the focusing lens with the collar of the ring facing out towards the RAMM}}]
  
-Attach ​the lens ring to the cage by lining up the four holes with the four stainless steel rods and slide it in with the collar of the ring facing out or towards the RAMM. The curved side of the lens now points towards the objective. The ring has four 1.5mm setscrews, ​might have to loosen them so they can slide on the rods. Leave the setscrews ​loose for now. +Install ​the focusing ​lens by lining up the four holes in the lens' ring mount with the four stainless steel rods and slide it in with the collar of the ring facing out towards the RAMM. The curved side of the lens now points towards the objective. The ring has four 1.5 mm set screws; you might have to loosen them so they can slide on the rods. Leave the set screws ​loose for now. 
  
-The picture above shows a TIRF with 200mm lens, depending ​on the configuration of your particular RAMM or microscope ASI might ship you a different ​tube lens. +The picture above shows a TIRF with 200 mm lens. Depending ​on the configuration of your particular RAMM or microscopeASI might ship you a different ​focal length ​lens. 
  
-[{{ ::​tirf5.jpg?​direct&​400 | Four star screws ​holding ​the back cover in place }}]+[{{ ::​tirf5.jpg?​direct&​400 |Four Phillips head screws ​hold the back cover in place}}]
  
-If the actuator is shipped separately ​then attach it by removing the plastic cover on the fiber launch by unscrewing the four screws with a star/Phillips head screwdriver.+If the actuator is shipped separatelyattach it by removing the plastic cover on the fiber launch by unscrewing the four Phillips head screws.
  
 [{{ ::​tirf6.jpg?​direct&​400 |Slide the actuator in and lock it in place with the setscrew}}] [{{ ::​tirf6.jpg?​direct&​400 |Slide the actuator in and lock it in place with the setscrew}}]
  
-Insert the actuator all the way. On the micrometer ​orient it such that the graduation markings are easy to read. Then lock it in place by tightening the setscrew ​with a 1.5mm Allen hex wrench. ​Then replace ​the cover. ​+Insert the actuator all the way. Orient ​the micrometer such that the graduation markings are easy to read. Then lock it in place by tightening the set screw with a 1.5 mm hex wrench. ​Replace ​the cover. ​
  
  
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 [{{ :​tirf30.gif?​direct&​400 |Fiber launch at upper limit of MA12's travel}}] [{{ :​tirf30.gif?​direct&​400 |Fiber launch at upper limit of MA12's travel}}]
  
-A note of caution for TIRF with the motorized actuator, the [[http://​www.asiimaging.com/​products/​manipulation-injection/​ma-12-motorized-actuator/​|MA12 actuator]] sold with the TIRF assembly has over 12mm travel. It's more travel than needed ​to steer the beam. At the upper limit, the fiber launch block is pushed to an extreme position overextending the springs. Over time the springs will lose their elasticity, and the fiber launch block won't spring back when the actuator is withdrawn.  ​+A note of caution for TIRF with the motorized actuator, the [[http://​www.asiimaging.com/​products/​manipulation-injection/​ma-12-motorized-actuator/​|MA12 actuator]] sold with the TIRF assembly has over 12 mm travel. It's more travel than needed ​for this application. At the upper limit, the fiber launch block is pushed to an extreme position overextending the springs. Over time the springs will lose their elasticity, and the fiber launch block won't spring back when the actuator is withdrawn.  ​
  
-[{{ :​tirf31.jpg?​direct&​400 |Limiting ​MA12's travel}}]+[{{ :​tirf31.jpg?​direct&​400 |Limit MA12's travel}}]
  
-We suggest limiting the MA12's travel range by setting the soft upper limit at a position that doesn'​t overextend the springs. Move the fiber launch to the position shown in the above image, about 1-2mm from the edge of the cutout and set the upper limit by issuing serial command [[commands:​setup|SU [axis]+]]. The current position is set as the upper limit, the controller remembers this position between restarts. ​On [[commands:​reset|reset command]] or button pressthe controller ​reverts ​to default travel. ​+We suggest limiting the MA12's travel range by setting the soft upper limit at a position that doesn'​t overextend the springs. Move the fiber launch to the position shown in the above image, about 1-2 mm from the edge of the cutoutand set the upper limit by issuing serial command [[commands:​setup|SU [axis]+]]. The current position is set as the upper limit, the controller remembers this position between restarts. ​[[commands:​reset|reset ​serial ​command]] or reset button press will cause the controller to revert back to the default travel ​range
  
 ====== Attaching the TIRF assembly to RAMM ====== ====== Attaching the TIRF assembly to RAMM ======
  
-TIRF is commonly connected to a Cube 2, Cube 3 or a motorized cube. The cubes have a ring with four holes for the cage rods already installed. ​+TIRF is commonly connected to a Cube 2, Cube 3or a motorized cube. The cubes have a ring with four holes for the cage rods already installed. ​
  
-[{{ ::​tirf8.jpg?​direct&​400 |Undoing ​the first screw holding the C support clamp closed}}]+[{{ ::​tirf8.jpg?​direct&​400 |Loosen ​the first screw holding the C support clamp closed}}]
  
-[{{ ::​tirf9.jpg?​direct&​400 |Second ​screw holding the C support clamp closed}}]+[{{ ::​tirf9.jpg?​direct&​400 |Loosen the second ​screw holding the C support clamp closed}}]
  
-First, open the C clamp support by unscrewing the two screws shown in the above image with a 2.5mm Allen hex wrench.+First, open the C clamp support by unscrewing the two screws shown in the above image with a 2.5 mm hex wrench.
  
-[{{ ::​tirf10.jpg?​direct&​400 |Attaching ​the lens cage assembly }}]+[{{ ::​tirf10.jpg?​direct&​400 |Attach ​the lens cage assembly}}]
  
-[{{ ::​tirf11.jpg?​direct&​400 |Push it in place}}]+[{{ ::​tirf11.jpg?​direct&​400 |Push it into place}}]
  
 Next, slide the assembly thru it, and line up the post with the corresponding holes and push it in.  Next, slide the assembly thru it, and line up the post with the corresponding holes and push it in. 
  
-[{{ ::​tirf12.jpg?​direct&​400 |Securing ​the lens cage assembly to the cube by tightening ​setscrew ​1 and 2}}]+[{{ ::​tirf12.jpg?​direct&​400 |Secure ​the lens cage assembly to the cube by tightening ​set screws ​1 and 2}}]
  
  
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 [{{ ::​tirf14.jpg?​direct&​400 |Setscrew 4}}] [{{ ::​tirf14.jpg?​direct&​400 |Setscrew 4}}]
  
-Then secure the stainless steel posts to the ring by tightening the four setscrews ​with a 1.5mm Allen hex wrench. Location of the four setscrews ​in the above picture+Then secure the stainless steel posts to the ring by tightening the four set screws ​with a 1.5 mm hex wrench. Location of the four set screws shown in the above pictures
  
 [{{ ::​tirf15.jpg?​direct&​400 |First screw to close the C support clamp}}] [{{ ::​tirf15.jpg?​direct&​400 |First screw to close the C support clamp}}]
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 Then put the support clamp back together so that the TIRF assembly is well supported. ​ Then put the support clamp back together so that the TIRF assembly is well supported. ​
  
-[{{ ::​tirf17.jpg?​direct&​400 |Setscrew location, loosening it will let you rotate the fiber launch}}]+[{{ ::​tirf17.jpg?​direct&​400 |This set screw will allow you to rotate the fiber launch}}]
  
 [{{ ::​tirf18.jpg?​direct&​400 |Orient the actuator to be parallel to the air table}}] [{{ ::​tirf18.jpg?​direct&​400 |Orient the actuator to be parallel to the air table}}]
  
-Lastly, the actuator should be oriented on the left side and angled such that its parallel to the table or optical bench. Loosen the fiber launch ​setscrew ​with a 2mm Allen hex wrench and orient it. This is not an absolute rule.  It is best to have the TIRF beam tilt away from the operator for laser safety.  ​+Lastly, the actuator should be oriented on the left side and angled such that its parallel to the table or optical bench. Loosen the fiber launch ​set screw with a 2 mm hex wrench and orient it. This is not an absolute rule; it is safest ​to have the collimated ​beam exiting the objective ​tilt away from users.  ​
  
 ====== Aligning the TIRF ====== ====== Aligning the TIRF ======
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 Connect the laser to the fiber launch. ​ Connect the laser to the fiber launch. ​
  
-[{{ ::​tirf20.jpg?​direct&​400 |Remove the cover by undoing ​the 3 black thumb screws}}]+[{{ ::​tirf20.jpg?​direct&​400 |Remove the cover by unscrewing ​the 3 black thumb screws}}]
  
-To verify the laser is correctly coupled to the fiber launch, ​undo the three black thumb screws and remove CUBE's cover. Careful to not look at the laser directly check for its presence. If you do not see the laser exit the cube, there might be a problem with your fiber or the fiber launch on the light source. ​+To verify the laser is correctly coupled to the fiber launch, ​unscrew ​the three black thumb screws and remove ​the CUBE's cover. Careful to not look at the laser directly, but safely ​check for its presence. If you do not see the laser exit the cube, there might be a problem with your fiber or the fiber launch on the light source. ​
  
-If you haven'​t ​done this already, now is a good time to mount the TIRF objective and install any dichroic into the cube and cover the cube up again. With the right dichroic, we direct the laser towards the microscope objective. ​+If you haven'​t already, now is a good time to mount the TIRF objective and install any dichroic into the cube and re-install the cover on the cube. With the right dichroic, we direct the beam towards the microscope objective. ​
  
-[{{ ::​tirf21.jpg?​direct&​400 |Steer the beam with the micrometer or motorized actuator}}]+[{{ ::​tirf21.jpg?​direct&​400 |Position ​the fiber tip at the central/​neutral position ​with the micrometer or motorized actuator}}]
  
-[{{ ::​tirf22.jpg?​direct&​400 |Adjusting ​the tilt of the dichroic with the 3 stainless steel screws}}]+[{{ ::​tirf22.jpg?​direct&​400 |Adjust ​the tilt of the dichroic with the 3 stainless steel screws}}]
  
-[{{ ::​tirf23.jpg?​direct&​400 |When aligned ​laser shoots out vertically from the objective, can be seen on the ceiling as a dim blur}}]+[{{ ::​tirf23.jpg?​direct&​400 |When well alignedshoots out vertically from the objective, can be seen on the ceiling as a dim blur}}]
  
-Align the laser, so it shoots vertically out of the microscope objective and onto the ceiling. To do this steer the fiber launch ​by rotating ​the micrometer. Begin by first positioning the fiber launch at about center of the tube. If you have a motorized actuator, assign the actuator to a manual input device (joystick or knob) with the [[commands:​joystick|Command:​JOYSTICK (J)]] command. With the manual input device, you can now steer the fiber launch. ​+Position ​the fiber launch ​at the center of the tube. If you have a motorized actuator, assign the actuator to a manual input device (joystick or knob) with the [[commands:​joystick|Command:​JOYSTICK (J)]] command. With the manual input device, you can now steer the fiber launch.
  
-Adjusting ​the tilt of the dichroic by rotating the stainless steel thumb screws ​can also help you steer the beam so that it exits the objective vertically. You might be going back and forth between adjusting ​the fiber launch and tilt of the dichroic to get this aligned correctly+Adjust ​the tilt of the dichroic by rotating the stainless steel thumb screws ​so that the beam exits the objective vertically and hits the ceiling right above the objective.
  
-Be careful and do not look at the objective directlyas you make adjustments the beam exits the objective at odd angles. ​+Be careful and do not look at the objective directlyas you make adjustments the beam may exit the objective at odd angles. ​
  
-[{{ ::​tirf24.jpg?​direct&​400 |Focus the beam by sliding the lens ring}}]+[{{ ::​tirf24.jpg?​direct&​400 |Focus the beam into the BFP by sliding the caged lens along the rails}}]
  
-[{{ ::​tirf25.jpg?​direct&​400 |Focused ​beam appears as a small bright dot on the ceiling}}]+[{{ ::​tirf25.jpg?​direct&​400 |Collimated ​beam appears as a small bright dot on the ceiling}}]
  
-Initially, the beam would be dim and defocusedTo focus the laser, use the lens in the lens cage assembly ​to focus the beam onto the BFP of the objective. ​adjust ​the position of the tube lens in the cage assembly until you see the focused laser or a small bright dot. When satisfied, lock the position of the lens by tightening one or two of the four setscrews in the lens ring with a 1.5mm Allen hex wrench. ​+Initially, the beam will probably ​be dim and broadUse the caged lens to focus the beam onto the BFP of the objective. ​Adjust ​the position of the tube lens in the cage assembly until you see a small bright dot. When satisfied, lock the position of the lens by tightening one or two of the four setscrews in the lens ring with a 1.5 mm hex wrench. ​
  
-The steps so far covered basic TIRF alignmentthe steps below get into TIRF mode. For this, we need a sample, place it on the stage, and bring your objective ​to focus. ​+The steps so far covered basic alignmentthe steps below get into TIRF mode. For this, we need a sample on the stage with the objective ​at focus. ​
  
-[{{ ::​tirf27.jpg?​direct&​400 |A Petric ​dish with water and dye from a highlighter pen. A beam shoots out vertically}}]+[{{ ::​tirf27.jpg?​direct&​400 |A Petri dish with water and dye from a highlighter pen; the beam shoots out vertically}}]
  
-Initially, the beam exits the objective and thru the sample vertically. ​+Initially, the beam exits the objective and goes through ​the sample vertically. ​
  
 <WRAP center round info 60%> <WRAP center round info 60%>
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 [{{ ::​tirf21.jpg?​direct&​400 |Steer the beam by rotating the micrometer or moving the MA12 motorized actuator }}] [{{ ::​tirf21.jpg?​direct&​400 |Steer the beam by rotating the micrometer or moving the MA12 motorized actuator }}]
  
-[{{ ::​tirf28.jpg?​direct&​400 |Beam starting to tilt and reflect off the interface}}]+[{{ ::​tirf28.jpg?​direct&​400 |Beam starting to angle}}]
  
-As you continue to steer the fiber launch by rotating the micrometer or by moving the motorized actuator, the laser beam exits the microscope at an angle. At one point, you might see the beam reflect ​of the 2nd interface (water to air in this case). ​+As you continue to move the fiber launch ​from its central position ​by rotating the micrometer or by moving the motorized actuator, the laser beam exits the microscope at an increasing ​angle. At one point, you might see the beam reflect ​off the 2nd interface (water to air in this case). ​
  
-[{{ ::​tirf29.jpg?​direct&​400 |Beam shing out horizontally}}]+[{{ ::​tirf29.jpg?​direct&​400 |Beam shooting ​out horizontally}}]
  
-Continue ​steering ​the fiber launch, and eventuallyyou see the beam exiting ​the objective perpendicular to the objectiveThe beam illuminates ​only the sample close to the first interface (glass to water in this case)this is the TIRF mode. +Continue ​moving ​the fiber launch, and eventually you see the beam stops passing through ​the petri dish and instead reflects back down from the upper surface of the petri glassNow only the part of the sample ​extremely ​close to the first interface (glass to water in this case) is illuminated; ​this is TIRF mode. 
  
 Motorized TIRF users might want to save this position either by [[commands:​zero|zeroing]] or loading it into a [[ring_buffer|ring buffer]]. Another tip, users can move past this point a bit and set that position as a soft upper limit by issuing serial command [[commands:​setup|SU [axis]+]].  ​ Motorized TIRF users might want to save this position either by [[commands:​zero|zeroing]] or loading it into a [[ring_buffer|ring buffer]]. Another tip, users can move past this point a bit and set that position as a soft upper limit by issuing serial command [[commands:​setup|SU [axis]+]].  ​
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tirf_alignment.txt · Last modified: 2019/07/23 22:20 by melissa