Total Internal Reflection
Fluorescence Microscopy (TIRFM)
Physics 598 BP
Spring 2015
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Contents
• Optical microscopy
• TIRFM basics and principles
• Optics – components and uses
• Alignment tips and techniques
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Optical Microscopy
Bright Field
Fluorescence
Epifluorescence
…
TIRFM
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Fluorescence Microscopy
• Use of fluorophores
– Absorb and emit at unique
wavelengths
• Laser used to excite fluorophores
• Dichroic mirror reflects light at
excitation wavelength while
allowing emitted light to pass
through
• Emission filter allows emitted light
to pass through while absorbing
lights at other wavelengths
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Fluorescence Microscopy
Incoming
Laser Beam
Dichroic Mirror
Emission Filter
Microscope
CCD
Camera
Emitted Light
Source: Mindy Hoffman: Hoffman-Single Enzym. Meth. Chapter figures PowerPoint slides
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TIRFM Basics and Principles
• Make use of total internal
reflection at the interface
between glass coverslip
and specimen buffer
• Principles: evanescent
wave formation
• Advantages:
– Low fluorescence
background
– Penetration depth: ~ 100
nm
www2.bioch.ox.ac.uk
• Two configurations: prism
method and objective lens
method
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http://www.microscopyu.com/articles/fluore
scence/tirf/tirfintro.html
Epifluorescence vs TIRFM
Epifluorescence
TIRFM
Picture source: http://www.me.mtu.edu/~cchoi/practice.html
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Alignment Goal
• Beam needs to pass
through the center
of the front and back
focal plane of the
objective (before the
TIR lens is added)
http://www.microscopyu.com/articles/fluore
scence/tirf/tirfintro.html
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General Strategy for Alignment
• Draw beam path
• Add and align laser, shutter and neutral
density filter
• Add and align appropriate mirrors at
respective corners
• Add and align beam expander
• Add and align TIR lens
• Image in TIR
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Optical Components: Posts, Post Holders and Bases
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Irises
• Two irises are used to
make sure that the
beam line is parallel to
the optical table
Source: Thorlabs
• They are also used to
make sure that the
beam passes through
the beam path
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• Alignment tips:
Irises
– First adjust the height of
the iris to the same height
as the microscope laser
input port
– Place two irises as far
away from each other
along the direction of the
beam path
– Adjust mirrors/ lasers so
that the beam passes
through both irises
Source: Thorlabs
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Mirrors
• Mirrors help direct beams
• One mirror can direct a beam
to any point
• Two mirrors can direct a beam
so that it is parallel and
coincident to any line
Source: Thorlabs
• Mirrors are mounted on the
kinematic mount
• They are usually placed at
corners of beam path
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Source: Mindy Hoffman: Hoffman-Single Enzym. Meth. Chapter
figures PowerPoint slides
Mirrors
• Alignment tips:
– First align the post holders
(Note: center of the post
holder is offset from the
mirror surface).
– Once post holders are aligned
and secured, place the mirrors
such that the beam hits the
center of the mirror
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Beam Expander
• Purpose:
– Expand beam (5x,10x, 20x)
– Collimate beam  parallel
output beam
• Alignment tips:
– Beam needs to pass through
the center of expander
Source: http://www.edmundoptics.com/technical-support/lasers/beam-expanders/
– First make sure the post
holder and base are
centered
– Then add the beam
expander and center it in x
and y direction
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TIR Lens
• Focuses beam onto the back focal plane of the objective
• Translation of the TIR lens will cause the beam to enter
the objective at an angle, allowing total internal
reflection to be achieved
• Alignment tips:
– Place the TIR lens onto a three axes translation stage
– Position the lens (x and y direction) such that the beam passes
through its center
– Position the lens (z direction) such that the beam is converging
through the objective
– Fine adjust the translation stage such that the laser beam
(through the objective) is projecting upward onto the ceiling and
its image is circular in shape
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Alignment Technique: Laser with Irises
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Alignment Technique: Laser with Irises
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Alignment Technique: Laser with Irises
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Alignment Technique: Laser with Irises
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Alignment Technique: Laser with Irises
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Conclusion: Laser with Irises
• To correctly align a laser, we need to
adjust both the tilt and the position of the
laser
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Alignment Technique: Mirrors with Irises
• Two mirrors can align the laser beam with the
center line of the irises
• Mirror 1 (further from the irises) is responsible
for the position of the beam onto the second
mirror. It is adjusted to center the beam on the
first iris
• Mirror 2 (closer to the irises) is responsible for
the tilt of the beam towards the first iris. It is
adjusted to center the beam on the second iris
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Alignment Technique: Mirror with Irises
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Alignment Technique: Mirror with Irises
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Alignment Technique: Mirror with Irises
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Alignment Technique: Mirror with Irises
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Alignment Technique: Mirror with Irises
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Alignment Technique: Alignment through
Objective
• The last alignment with mirrors is to
align the beam through the objective
• The strategy is the same as aligning
mirrors through irises. In place of the
irises, we now have the front and
back focal plane of the objective
• Use the center of the eyepiece and
center of the crosshair to align
through the front and back focal
plane respectively
• Use mirror 1 (further from objective)
to align through crosshair (back) and
mirror 2 (closer to objective) to align
through the eyepiece (front)
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http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html
http://www.labx.com/v2/spiderdealer2/vistasearchdetails.cfm?LVid=6853317