TIRF alignment guide - Revision history

Jelle at 14:38, 19 June 2025

2025-06-19T14:38:33Z

← Older revision		Revision as of 14:38, 19 June 2025
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	# Go "LiveAllCameras" and select the Fi3 view window		# Go "LiveAllCameras" and select the Fi3 view window
	# Press observation mode "Eyes only" on the front of the microscope (top button, out of 4) to direct all light to the Fi3 camera		# Press observation mode "Eyes only" on the front of the microscope (top button, out of 4) to direct all light to the Fi3 camera
	# Using the Fi3 camera window, adjust brightness/contrast of this view using the LUTs window, to clearly see the laser spot (if laser is not centered, move the TIRF angle ~~a bit~~ in the software, and then move it back to 0)		# Using the Fi3 camera window, adjust the screw on the Bertrand lens switch to focus on the incubator-box lights as good as possible
	# In the Ti2 E-TIRF Pad, hit "Align" and provide the system with the correct fully reflecting critical angle for about 5-6 different rotations (directions). The angle is correct when the laser spots look most similar, on opposite sides of the circular objective back aperture.		# Switch off the incubator box-lights
			# Ensure the correct laser is switched on, and substantially increase its intensity setting
			# Using the Fi3 camera window, adjust brightness/contrast of this view using the LUTs window, to clearly see the laser spot (if laser does not appear centered, move the TIRF angle slider in the software, and then move it back to 0)
			# In the Ti2 E-TIRF Pad, hit "Align" and provide the system with the correct fully reflecting critical angle for about 5-6 different rotations (directions). The angle is correct when the laser spots look most similar and are on opposite sides of the circular objective back aperture.
	# Press "compute zero from critical angles" and close window with "OK"		# Press "compute zero from critical angles" and close window with "OK"
	# (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser		# (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser

Jelle at 14:34, 19 June 2025

2025-06-19T14:34:55Z

← Older revision		Revision as of 14:34, 19 June 2025
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	# Using the Fi3 camera window, adjust brightness/contrast of this view using the LUTs window, to clearly see the laser spot (if laser is not centered, move the TIRF angle a bit in the software, and then move it back to 0)		# Using the Fi3 camera window, adjust brightness/contrast of this view using the LUTs window, to clearly see the laser spot (if laser is not centered, move the TIRF angle a bit in the software, and then move it back to 0)
	# In the Ti2 E-TIRF Pad, hit "Align" and provide the system with the correct fully reflecting critical angle for about 5-6 different rotations (directions). The angle is correct when the laser spots look most similar, on opposite sides of the circular objective back aperture.		# In the Ti2 E-TIRF Pad, hit "Align" and provide the system with the correct fully reflecting critical angle for about 5-6 different rotations (directions). The angle is correct when the laser spots look most similar, on opposite sides of the circular objective back aperture.
	# Press "compute ~~zeo~~ from critical angles" and close window with "OK"		# Press "compute zero from critical angles" and close window with "OK"
	# (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser		# (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser

Jelle: added image and formatted

2025-06-19T14:26:56Z

added image and formatted

← Older revision		Revision as of 14:26, 19 June 2025
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	=== Goal ===		=== Goal ===
	To ensure that laser critical angles are indeed critical, to reliably achieve ~~TIRF~~ at any desired laser direction		To ensure that while imaging, the laser critical angles are indeed critical, to reliably achieve '''total internal reflection''' at any desired laser rotational direction

			[[File:TIRF principle.png\|704x704px]]

	=== '''Setting up the microscope to prepare for TIRF alignment''' ===		=== '''Setting up the microscope to prepare for TIRF alignment''' ===
	~~=== [[File:Setting_up_TIRF_microscope_for_alignment.png\|1000x1000px]] ===~~

	# Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step		# Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step
	# Change side switch: From Eye -> Camera (rotate) (on the right of the microscope, under the eye pieces)		# Change side switch: From Eye -> Camera (rotate) (on the right of the microscope, under the eye pieces)
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	# Set Observation mode Camera L (left button out of 4 round buttons, front of the microscope)		# Set Observation mode Camera L (left button out of 4 round buttons, front of the microscope)
	# Move Condenser down (inside black box, above sample) as low as reasonably possible (not hitting the sample...) by adjusting one of the rotary buttons on either side of condenser		# Move Condenser down (inside black box, above sample) as low as reasonably possible (not hitting the sample...) by adjusting one of the rotary buttons on either side of condenser
			[[File:Setting_up_TIRF_microscope_for_alignment.png\|1000x1000px]]

	=== '''Placing the sample and finding it''' ===		=== '''Placing the sample and finding it''' ===

Jelle: small edits

2025-06-19T13:16:57Z

small edits

← Older revision		Revision as of 13:16, 19 June 2025
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	# Select TIRF Prime BSI mode, pick an Optical Configuration to be aligned		# Select TIRF Prime BSI mode, pick an Optical Configuration to be aligned
	# Go "LiveAllCameras" and select the Fi3 view window		# Go "LiveAllCameras" and select the Fi3 view window
	# Press "Eyes only" on the front of the microscope (top button ~~/ observation method~~) to direct all light to the Fi3 camera		# Press observation mode "Eyes only" on the front of the microscope (top button, out of 4) to direct all light to the Fi3 camera
	# Using the Fi3 window, adjust brightness/contrast of this view~~, done in~~ the LUTs window, to clearly ~~view~~ the laser spot (if laser is not centered, move the TIRF angle a bit in the software, and move it back to 0)		# Using the Fi3 camera window, adjust brightness/contrast of this view using the LUTs window, to clearly see the laser spot (if laser is not centered, move the TIRF angle a bit in the software, and then move it back to 0)
	# In the Ti2 E-TIRF Pad, hit Align and provide it with the correct fully reflecting critical angle for about 5-6 different rotations		# In the Ti2 E-TIRF Pad, hit "Align" and provide the system with the correct fully reflecting critical angle for about 5-6 different rotations (directions). The angle is correct when the laser spots look most similar, on opposite sides of the circular objective back aperture.
	# Press "compute zeo from critical angles" and close window with "OK"		# Press "compute zeo from critical angles" and close window with "OK"
	# (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser		# (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser

Jelle at 13:05, 19 June 2025

2025-06-19T13:05:33Z

← Older revision		Revision as of 13:05, 19 June 2025
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	# Select the lens (e.g. 100X)		# Select the lens (e.g. 100X)
	# Lower the lens with ESC, hold ESC to make that the current position		# Lower the lens with ESC, then hold ESC to make that the current position
	# Put oil on the lens		# Put oil on the lens
	# Center the sample holder		# Center the sample holder
	# Place the sample on the ~~holde~~		# Place the sample on the holder
	# Switch on an illumination mode (e.g. brightfield, or epifluorescence in the software)		# Switch on an illumination mode (e.g. brightfield, or epifluorescence in the software)
	# Find focus on the sample (lens up, until image = sharp)		# Find focus on the sample (lens up, until image = sharp)

Jelle at 08:52, 19 June 2025

2025-06-19T08:52:59Z

← Older revision		Revision as of 08:52, 19 June 2025
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	=== Goal ===		=== Goal ===
	To ensure that laser critical angles are ~~always~~ critical, ~~independent of chosen~~ laser direction ~~(rotation orientation)~~		To ensure that laser critical angles are indeed critical, to reliably achieve TIRF at any desired laser direction

	=== '''Setting up the microscope to prepare for TIRF alignment''' ===		=== '''Setting up the microscope to prepare for TIRF alignment''' ===
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	=== '''Placing the sample and finding it''' ===		=== '''Placing the sample and finding it''' ===
	~~1) Select the lens (e.g. 100X)~~

	2) Lower the lens with ESC, hold ESC to make that the current position		# Select the lens (e.g. 100X)
			# Lower the lens with ESC, hold ESC to make that the current position
			# Put oil on the lens
			# Center the sample holder
			# Place the sample on the holde
			# Switch on an illumination mode (e.g. brightfield, or epifluorescence in the software)
			# Find focus on the sample (lens up, until image = sharp)
			# (optional) switch-on PFS in the software to keep this focus

	~~3) Put oil on~~ the ~~lens~~		=== '''Performing TIRF alignment using the NIS Elements software''' ===

	~~4) Center the sample holder~~		# Select TIRF Prime BSI mode, pick an Optical Configuration to be aligned
			# Go "LiveAllCameras" and select the Fi3 view window
	~~5) Place the sample on the holde~~		# Press "Eyes only" on the front of the microscope (top button / observation method) to direct all light to the Fi3 camera
			# Using the Fi3 window, adjust brightness/contrast of this view, done in the LUTs window, to clearly view the laser spot (if laser is not centered, move the TIRF angle a bit in the software, and move it back to 0)
	~~6) Switch on an illumination mode (e.g. brightfield, or epifluorescence in the software)~~		# In the Ti2 E-TIRF Pad, hit Align and provide it with the correct fully reflecting critical angle for about 5-6 different rotations
			# Press "compute zeo from critical angles" and close window with "OK"
	~~7) Find focus on the sample (lens up, until image = sharp)~~		# (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser

	~~8) (optional) switch-on PFS in the software to keep this focus~~

	~~=== '''Performing the TIRF alignment using the NIS Elements software''' ===~~
	1) Select TIRF Prime BSI mode, pick an OC to be aligned

	2) Go "LiveAllCameras" and select the Fi3 view window

	3) Press "Eyes only" on the front of the microscope (top button / observation method) to direct all light to the Fi3 camera

	4) Using the Fi3 window, adjust brightness/contrast of this view, done in the LUTs window, to clearly view the laser spot (if laser is not centered, move the TIRF angle a bit in the software, and move it back to 0)

	5) In the Ti2 E-TIRF Pad, hit Align and provide it with the correct fully reflecting critical angle for about 5-6 different rotations

	6) Press "compute zeo from critical angles" and close window with "OK"

	7) (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser

Jelle at 08:50, 19 June 2025

2025-06-19T08:50:23Z

← Older revision		Revision as of 08:50, 19 June 2025
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	=== '''Setting up the microscope to prepare for TIRF alignment''' ===		=== '''Setting up the microscope to prepare for TIRF alignment''' ===
			=== [[File:Setting_up_TIRF_microscope_for_alignment.png\|1000x1000px]] ===

			# Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step
	~~[[File:Setting_up_TIRF_microscope_for_alignment.png\|none\|thumb\|1200x1200px]]~~		# Change side switch: From Eye -> Camera (rotate) (on the right of the microscope, under the eye pieces)
	1) Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step		# Switch Box-lights on (proximity sensor button top right of big incubator box on microscope)
			# Set Observation mode Camera L (left button out of 4 round buttons, front of the microscope)
	2) Change side switch: From Eye -> Camera (rotate) (on the right of the microscope, under the eye pieces)		# Move Condenser down (inside black box, above sample) as low as reasonably possible (not hitting the sample...) by adjusting one of the rotary buttons on either side of condenser

	3) Switch Box-lights on (proximity sensor button top right of big incubator box on microscope)

	4) Set Observation mode Camera L (left button out of 4 round buttons, front of the microscope)

	5) Move Condenser down (inside black box, above sample) as low as reasonably possible (not hitting the sample...) by adjusting one of the rotary buttons on either side of condenser

	=== '''Placing the sample and finding it''' ===		=== '''Placing the sample and finding it''' ===

Jelle at 08:47, 19 June 2025

2025-06-19T08:47:50Z

← Older revision		Revision as of 08:47, 19 June 2025
Line 5:		Line 5:

	=== '''Setting up the microscope to prepare for TIRF alignment''' ===		=== '''Setting up the microscope to prepare for TIRF alignment''' ===
	[[File:~~Setting up TIRF microscope for alignment~~.png\|thumb]]

			[[File:Setting_up_TIRF_microscope_for_alignment.png\|none\|thumb\|1200x1200px]]
	1) Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step		1) Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step

Jelle: added instructional pictures

2025-06-19T08:44:48Z

added instructional pictures

Jelle: formatting

2025-06-19T08:04:02Z

formatting

← Older revision		Revision as of 08:04, 19 June 2025
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	Aligning TIRF:		== '''Aligning TIRF lasers''' ==

	~~Setting up the microscope to prepare for TIRF alignment~~		=== Goal ===
			To ensure that laser critical angles are always critical, independent of chosen laser direction (rotation orientation)

			=== '''Setting up the microscope to prepare for TIRF alignment''' ===
	1) Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step		1) Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step

	2) Change side switch: From Eye -> Camera (rotate) (on the right of the microscope, under the eye pieces)		2) Change side switch: From Eye -> Camera (rotate) (on the right of the microscope, under the eye pieces)

	3) Switch Box-lights on (proximity sensor button top right of big incubator box on microscope)		3) Switch Box-lights on (proximity sensor button top right of big incubator box on microscope)

	4) Change Observation mode: Camera L + Eyes (bottom button out of 4 round buttons, front of the microscope)		4) Change Observation mode: Camera L + Eyes (bottom button out of 4 round buttons, front of the microscope)

	5) Move Condenser down (inside black box, above sample) as low as reasonably possible (not hitting the sample...) by adjusting one of the rotary buttons on either side of condenser		5) Move Condenser down (inside black box, above sample) as low as reasonably possible (not hitting the sample...) by adjusting one of the rotary buttons on either side of condenser

	Placing the sample and finding it		=== '''Placing the sample and finding it''' ===
			1) Select the lens (e.g. 100X)

	~~1) Select the lens (e.g. 100X)~~
	2) Lower the lens with ESC, hold ESC to make that the current position		2) Lower the lens with ESC, hold ESC to make that the current position

	3) Put oil on the lens		3) Put oil on the lens

	4) Center the sample holder		4) Center the sample holder
	5) Place the sample on the ~~holder~~
			5) Place the sample on the holde

	6) Switch on an illumination mode (e.g. brightfield, or epifluorescence in the software)		6) Switch on an illumination mode (e.g. brightfield, or epifluorescence in the software)
	~~7) Find focus on the sample (lens up until image = sharp)~~
	~~8) (optional) switch on PFS to keep this focus~~

	~~TIRF alignment~~ in the software		7) Find focus on the sample (lens up, until image = sharp)

			8) (optional) switch-on PFS in the software to keep this focus

			=== '''Performing the TIRF alignment using the NIS Elements software''' ===
	1) Select TIRF Prime BSI mode, pick an OC to be aligned		1) Select TIRF Prime BSI mode, pick an OC to be aligned

	2) Go "LiveAllCameras" and select the Fi3 view window		2) Go "LiveAllCameras" and select the Fi3 view window
	3) Press "Eyes only" on the front of the microscope (top button / observation method)
			3) Press "Eyes only" on the front of the microscope (top button / observation method) to direct all light to the Fi3 camera

	4) Using the Fi3 window (should probably adjust brightness/contrast of this view, done in the LUTs window), align the laser belonging to the desired OC you are configuring (step 5)		4) Using the Fi3 window (should probably adjust brightness/contrast of this view, done in the LUTs window), align the laser belonging to the desired OC you are configuring (step 5)

	5) In the Ti2 E-TIRF Pad, hit Align and give it the correct fully reflecting critical angle for about 5-6 different rotations		5) In the Ti2 E-TIRF Pad, hit Align and give it the correct fully reflecting critical angle for about 5-6 different rotations

	6) Press "compute zeo from critical angles" and close window with "OK"		6) Press "compute zeo from critical angles" and close window with "OK"

	7) (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser		7) (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser

← Older revision		Revision as of 08:44, 19 June 2025
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	=== '''Setting up the microscope to prepare for TIRF alignment''' ===		=== '''Setting up the microscope to prepare for TIRF alignment''' ===
			[[File:Setting up TIRF microscope for alignment.png\|thumb]]
	1) Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step		1) Bertrand lens switch: Rotate to "B" (counterclockwise) - will need to adjust small rotatory button on this switch in a later step

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	3) Switch Box-lights on (proximity sensor button top right of big incubator box on microscope)		3) Switch Box-lights on (proximity sensor button top right of big incubator box on microscope)

	4) ~~Change~~ Observation mode: Camera L ~~+ Eyes~~ (~~bottom~~ button out of 4 round buttons, front of the microscope)		4) Set Observation mode Camera L (left button out of 4 round buttons, front of the microscope)

	5) Move Condenser down (inside black box, above sample) as low as reasonably possible (not hitting the sample...) by adjusting one of the rotary buttons on either side of condenser		5) Move Condenser down (inside black box, above sample) as low as reasonably possible (not hitting the sample...) by adjusting one of the rotary buttons on either side of condenser
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	3) Press "Eyes only" on the front of the microscope (top button / observation method) to direct all light to the Fi3 camera		3) Press "Eyes only" on the front of the microscope (top button / observation method) to direct all light to the Fi3 camera

	4) Using the Fi3 window ~~(should probably~~ adjust brightness/contrast of this view, done in the LUTs window), ~~align~~ the laser ~~belonging~~ to ~~the desired OC you are configuring (step 5~~)		4) Using the Fi3 window, adjust brightness/contrast of this view, done in the LUTs window, to clearly view the laser spot (if laser is not centered, move the TIRF angle a bit in the software, and move it back to 0)

	5) In the Ti2 E-TIRF Pad, hit Align and ~~give~~ it the correct fully reflecting critical angle for about 5-6 different rotations		5) In the Ti2 E-TIRF Pad, hit Align and provide it with the correct fully reflecting critical angle for about 5-6 different rotations

	6) Press "compute zeo from critical angles" and close window with "OK"		6) Press "compute zeo from critical angles" and close window with "OK"

	7) (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser		7) (optional) move on to another Optical Configuration, and repeat steps 1-6 for that particular laser