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Total Internal Reflection Fluorescence Microscopy (TIRFM)
Physics 598 BPSpring 2015
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Contents
• Optical microscopy• TIRFM basics and principles• Optics – components and uses• Alignment tips and techniques
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Bright Field Fluorescence …
Epifluorescence TIRFM
Optical Microscopy
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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
Source: Mindy Hoffman: Hoffman-Single Enzym. Meth. Chapter figures PowerPoint slides
Dichroic Mirror
Emission Filter
CCD Camera
Microscope
Incoming Laser Beam
Emitted Light
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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
• Two configurations: prism method and objective lens method
www2.bioch.ox.ac.uk
http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html
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Epifluorescence vs TIRFM
Picture source: http://www.me.mtu.edu/~cchoi/practice.html
Epifluorescence TIRFM
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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/fluorescence/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
• They are also used to make sure that the beam passes through the beam path
Source: Thorlabs
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Irises• Alignment tips:
– 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
• Mirrors are mounted on the kinematic mount
• They are usually placed at corners of beam path
Source: Mindy Hoffman: Hoffman-Single Enzym. Meth. Chapter figures PowerPoint slides
Source: Thorlabs
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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
– First make sure the post holder and base are centered
– Then add the beam expander and center it in x and y direction
Source: http://www.edmundoptics.com/technical-support/lasers/beam-expanders/
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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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• To correctly align a laser, we need to adjust both the tilt and the position of the laser
Conclusion: Laser with Irises
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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)
http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.htmlhttp://www.labx.com/v2/spiderdealer2/vistasearchdetails.cfm?LVid=6853317
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