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.