Total Internal Reflection Fluorescence Microscopy (TIRFM)

Physics 598 BP Spring 2015

1 Contents

• Optical microscopy • TIRFM basics and principles • – 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 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

Emitted Light CCD

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 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 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 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