New TURF for TIRF Joel Schwartz Stowers Institute for Medical Research Imaging Center

New TURF for TIRF

Joel Schwartz

Stowers Institute for Medical Research

Imaging Center

• What is TIRF?• Why do we constantly use acronyms to

describe everything?• Microscope Configurations

– Prism vs Prismless• Biological Applications

– Brief Aside• Unique attributes to our system

– Calibrated TIRF planes– TIRF-FRET– TIRF-photoactivation

• Not ready for prime time players..

• What is TIRF?• Why do we constantly use acronyms to

describe everything?• Microscope Configurations

– Prism vs Prismless• Biological Applications

– Brief Aside• Unique attributes to our system

– Calibrated TIRF planes– TIRF-FRET– TIRF-photoactivation

• Not ready for prime time players..

Index of refraction “bends” light

Some refractive indices to know:

water 1.33

air 1.0003

glass 1.517

coverglass 1.523

immersion oil 1.516

cell cytosol 1.38

mount variable

At a specific critical angle [θcritical = sin-1(n1/n2)] light is totally reflected from the glass/water interface. The reflection generates a very thin electromagnetic field that has an identical frequency to that of the incident light, providing a means to selectively excite fluorophores within ≤ 100 nm of the coverslip.

The basics of imaging cells by TIRF microscopy

http://micro.magnet.fsu.edu/primer/java/tirf/penetration/index.html

note: d is only the depth at which the intensity of the evanescent wave is 37% of the initial intensity. Thus, can empirically determine the experimental depth at which fluorophores are visible using fluorescent beads (Keyel, Watkins, and Traub 2004 JBC)

Evanescent wave penetration

λ0 = 488; n2=1.52; n1=1.38 dempirical = 190 nmλ0 = 647; n2=1.52; n1=1.38 dempirical = 238 nmλ0 = 488; n2=1.78; n1=1.38 dempirical = 142 nm

The evanescent wave penetration (d) :d = λ0/4π (n2

2 sin2θ-n12)-1/2

TIRF selectively illuminates the cellular membrane

• What is TIRF?• Why do we constantly use acronyms to

describe everything?• Microscope Configurations

– Prism vs Prismless• Biological Applications

– Brief Aside• Unique attributes to our system

– Calibrated TIRF planes– TIRF-FRET– TIRF-photoactivation

• Not ready for prime time players..

Prism-based TIRF limit access to sample

Axelrod et al. Traffic 2001

Prism systems can be placed under a culture dish

Prism-based TIR on an upright microscope

Axelrod et al. Traffic 2001

Trapezoid TIR prism on condenser and the position of the beam is adjusted by moving external lens.

TIRF is commonly done inside the objective

The objective influences penetration depth

100X 1.65 NA objective:θc = sin -1(n1/n2) = 50.83º[calculated using n2 = 1.78 (RI coverglass and immersion liquid) and n1 = 1.38]

Maximum Angle θm from the optical axis that TIR will occur is:NA = n2 sin θm

60X 1.45 NA θm = 72.54º100X 1.65 NA θm = 67.97º

TIRF objectives are now starting to come with compensation collars for varying temperature and cover slip thickness

100X 1.45 NA objective:θc = sin-1(n1/n2) = 65.22º[calculated using n2 = 1.52 (RI coverglass and immersion liquid) and n1 = 1.38]

TIRF Comparison

Prism Method

1. “Purer” evanescent wave

2. Limited access to sample

3. Few commercial manufactures

4. Open laser systems

5. Typically lower NA objectives

Prism-less Method

1. Higher NA will allow confinement closer to surface

2. Not as pure an evanescent wave as prism

3. Commercial system readily available

• What is TIRF?• Why do we constantly use acronyms to

describe everything?• Microscope Configurations

– Prism vs Prismless• Biological Applications

– Brief Aside• Unique attributes to our system

– Calibrated TIRF planes– TIRF-FRET– TIRF-photoactivation

• Not ready for prime time players..

TIRF illumination enhances contrast

YFP on the Membrane

TIRF is more sensitive to Z-axial drift

Hogan, Biophotnics International May 2006 48-51

TIRF measures endocytosis of clathrin coated vesicles

Color Coded Motion

Red

Green

Blue

RGB

Clathrin coated pits are move in and out of the membrane

Membrane-localized fluorophores are difficult to separate from mitochondria

TIRF selectively visualizes the membrane localized fluorophores

Total Internal Reflection Fluorescence (TIRF) Microscopy is used to reduce background

•Selective visualization of cell/substrate contact regions.

•Visualization and spectroscopy of single molecule fluorescence near a surface.

•Tracking of secretory granules in intact cells before and during the secretory process.

•Micromorphological structures and dynamics on living cells.

•Long-term fluorescence movies of cells during development in culture.

•Comparison of membrane-proximal ionic transients with simultaneous transients deeper in the cytoplasm.

•Measurements of the kinetic rates of binding of extracellular and intracellular proteins to cell surface receptors and artificial membranes.

Applications of TIR microscopy

• What is TIRF?• Why do we constantly use acronyms to

describe everything?• Microscope Configurations

– Prism vs Prismless• Biological Applications

– Brief Aside• Unique attributes to our system

– Calibrated TIRF planes– TIRF-FRET– TIRF-photoactivation

• Not ready for prime time players..

The new rig

405, 440, 491, 561, 638AOTF operated

Environmental ChamberBack-thinned EM-CCD

Axiocam HS

BAD IDEA!

High tech TIRF calibration device

0 100 200 300 400 5000.0

0.2

0.4

0.6

0.8

1.0

Red =120nmGreen=88nm

Flu

ore

scen

cce

(Ep

i)

Distance (nm)

FRET measures protein proximity

TIRF enhances signal to noise measurements of membrane associated FRET

1 um z-axial~ 15 receptors

~ 75 associated proteins

100 nm

~ 15 receptors

~ 15 associated proteins

Excitation of CFP leads to some YFP excitation because YFP is ~5 fold brighter than CFP. CFP emission also bleeds into the YFP channel (i.e. there will always be some “FRET” signal).

We idealized the system to excite CFP for FRET measurements

The new TIRF scope is capable of specific membrane photoactivation

• What is TIRF?• Why do we constantly use acronyms to

describe everything?• Microscope Configurations

– Prism vs Prismless• Biological Applications

– Brief Aside• Unique attributes to our system

– Calibrated TIRF planes– TIRF-FRET– TIRF-photoactivation

• Not ready for prime time players..

Dickinson et al. Biotechniques. 31:1272 2001.

Spectral images separate overlaping spectra

The system is a linear transfer function similar to CT scanning and reconstruction

CTIS provides space and color information without any moving partsNOT YET AVAILABLE

CGH Disperser

Spectral Imaging: CTISSpace and Color in a Single Shot

Ford et al., Optics Express’01 (9) 444-453.

Raw Data on CCD

The CTIS images are deconvolved to generate the actual image

Color projection of final data stack

Thank You

• Imaging Center– Cameron Cooper– Paul Kulesa– Sarah Smith– Danny Stark– Jessica Teddy– Miranda Smith

• Adv. Inst. And Physics– Winfried Wiegraebe– Josef Huff– Amanda Combs

http://micro.magnet.fsu.edu/primer/java/tirf/evaintensity/index.html