Total Internal Reflection Fluorescence Microscopy (TIRFM)
Physics 598 BP Spring 2015
1 Contents
• Optical microscopy • TIRFM basics and principles • Optics – components and uses • Alignment tips and techniques
2 Optical Microscopy
Bright Field Fluorescence …
Epifluorescence TIRFM
3 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
4 Fluorescence Microscopy
Incoming Laser Beam Dichroic Mirror
Emission Filter Microscope
Emitted Light CCD Camera
Source: Mindy Hoffman: Hoffman-Single Enzym. Meth. Chapter figures PowerPoint slides 5 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
http://www.microscopyu.com/articles/fluore6 scence/tirf/tirfintro.html Epifluorescence vs TIRFM
Epifluorescence TIRFM
Picture source: http://www.me.mtu.edu/~cchoi/practice.html
7 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
8 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
9 10 11 12 13 14 15 Optical Components: Posts, Post Holders and Bases
16 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
17 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 Source: Thorlabs away from each other along the direction of the beam path
– Adjust mirrors/ lasers so that the beam passes through both irises 18 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 Source: Thorlabs coincident to any line
• Mirrors are mounted on the kinematic mount
• They are usually placed at corners of beam path
19 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
20 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
21 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
22 Alignment Technique: Laser with Irises
23 Alignment Technique: Laser with Irises
24 Alignment Technique: Laser with Irises
25 Alignment Technique: Laser with Irises
26 Alignment Technique: Laser with Irises
27 Conclusion: Laser with Irises
• To correctly align a laser, we need to adjust both the tilt and the position of the laser
28 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
29 Alignment Technique: Mirror with Irises
30 Alignment Technique: Mirror with Irises
31 Alignment Technique: Mirror with Irises
32 Alignment Technique: Mirror with Irises
33 Alignment Technique: Mirror with Irises
34 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)
35 http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html http://www.labx.com/v2/spiderdealer2/vistasearchdetails.cfm?LVid=6853317