TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPE

1. TIRF MICROSCOPE
Supriya Pan
Sukhjovan Singh Gill
Ajay Baro

2. SmallIntroductionof TIRFM
 The idea of using total internal reflection to illuminate cells contacting the surface of glass was first described by E.J.
Ambrose in 1956.This idea was then extended by Daniel Axelrod at the University of Michigan, Ann Arbor in the early
1980s as TIRFM
 Total internal reflection fluorescence microscopy (TIRFM) exploits the unique properties of an induced field in a limited
specimen region immediately adjacent to the interface between two media having different refractive indices.
 This surface electromagnetic field, called the 'evanescent wave', can selectively excite fluorescent molecules in the
liquid near the interface, most commonly the contact area between a specimen and a glass coverslip or tissue culture
container.
 TIRF examination of cell/surface contacts dramatically reduces background fluorescence from fluorophores either in
the bulk solution or inside the cells (i.e. auto fluorescence and debris). Moreover, because TIRF minimizes the
exposure of the cell interior to light, the healthy survival of the culture during imaging procedures is much enhanced
relative to standard epi- (or trans-) illumination.
 Generally total internal reflection illumination has potential benefits in any application requiring imaging of minute
structures or single molecules in specimens having large numbers of fluorophores located outside of the optical plane
of interest, such as molecules in solution in Brownian motion, vesicles undergoing endocytosis or exocytosis, or single
protein trafficking in cells.
 An ideal candidate for application of the technique is the study of neurotransmitter release and uptake from single
vesicles in primary culture of neurons and astrocytes.

3. Physical Basisof TIRFM
 Total Internal Reflection:
Whenever light encounters the interface of two transparent media with different refractive indices, it will be partially
diffracted and partially reflected. At a certain angle of incidence, the so called critical angle, the light will be completely
reflected and a phenomenon called total internal reflection occurs,
• If the light travels from a medium with a higher refractive index (n1) to a medium with a lower refractive index (n2)
• The critical angle (ΘC) of incident light, at which total internal reflection occurs, can be determined by Snell’s law as
θc = sin-1 (n2/n1)

4.  Evanescent Field:
On occurrence of total internal reflection, a portion of the energy of
the incident light will be converted to an electromagnetic field and
pass through the interface to form an evanescent wave originating at
the interface.
The emerging evanescent wave has the same frequency as the incident
light and its amplitude decays exponentially with depth of penetration,
the field extends at most a few hundred nanometres into the specimen
in the z direction (normal to the interface).
Evanescent means disappearing. Within a limited region near the interface, it is capable of exciting
fluorophores. The range over which excitation is possible is limited by the exponential decay of the evanescent
wave energy in the z direction (perpendicular to the interface). The following equation defines this energy as a
function of distance from the interface:
E(z) = E(0)exp(-z/d)
where E(z) is the energy at a perpendicular distance z from the interface, and E(0) is the energy at the
interface.

5. The penetration depth (d) is dependent upon the wavelength of the incident illumination
(λ(i)), the angle of incidence, and the refractive indices of the media at the interface,
according to the equation:
d = λ(i)/4π × (n(1)2sin2θ(1) - n(2)2)-1/2
The penetration depth of this field typically ranges from 60 to 100 nm but can go up to
200 nm.
Increasing the angle of incidence of the light leads to a reduced penetration depth and
a higher wavelength of the incident light leads to an increased penetration depth.
The refractive index of the medium behind the interface (e.g. the specimen) also has
an influence on the penetration depth, as a higher refractive index increases the
evanescent wave’s penetration depth.
It should also be stated that in TIRF microscopy high power laser light, which is
stronger than the laser light usually employed in confocal systems, is used to form an
evanescent wave with sufficient energy.
In practice, however, scatter, reflections, and refractions produce undesirable rays of
light, collectively termed “stray light.” Stray light contaminates the exponential decay
of EW, excites the bulk of specimen, and deteriorates the TIRF effect, as shown in left.
This is especially serious problem in the case of objective-type TIRF.

6.  Detection:
In a typical experimental setup, fluorophores located in the vicinity of the glass-liquid or plastic-liquid surface can be excited
by the evanescent field, provided they have potential electronic transitions at energies within or very near the wavelength
bandwidth of the illuminating beam. Because of the exponential falloff of evanescent field intensity, the excitation of
fluorophores is restricted to a region that is typically less than 100 nanometers in thickness. By comparison, this optical
section thickness is approximately one-tenth that produced by confocal fluorescence microscopy techniques. Because
excitation of fluorophores in the bulk of the specimen is avoided, confining the secondary fluorescence emission to a very
thin region, a much higher signal-to-noise ratio is achieved compared to conventional widefield epifluorescence
illumination. This enhanced signal level makes it possible to detect single-molecule fluorescence by the TIRFM method
 With adjustment of the laser excitation incidence angle to a value greater
than the critical angle, the illuminating beam is entirely reflected back into the
microscope slide upon encountering the interface, and an evanescent field is
generated in the specimen medium immediately adjacent to the interface.
The fluorophores nearest the glass surface are selectively excited by
interaction with the evanescent field, and secondary fluorescence from these
emitters can be collected by the microscope optics.
 Although TIRFM is limited to imaging at the interface of two different media
having suitable refractive indices, a great number of applications are ideally
suited to the technique. One of the most active areas of research interest is in
the biomedical arena, in which many compelling questions involve processes
that take place at the cell surface or plasma membrane — appropriate
interfaces for TIRFM investigation.
TIRFM Specimen Illumination Configurations

7. BasicInstrumentalApproachesto TIRFM
There are two basic approaches to configuring an instrument for total internal
reflection fluorescence microscopy
 Prism Based TIRF Microscopy
 Objective Based TIRF Microscopy
 Lightguide-based TIRF Microscopy

9.  Prism Based TIRF Microscopy
 Prism used to attain critical angle
 Purer evanescent wave
 Easier to set up than prism-less system
 Laser focused to spot size about equal to field of view
 Access to sample can be restricted depending on prism position
 No commercially available system
 Prism-based scheme ensures the cleanest TIRF effect with the best
signal-to-background ratio , the highest contrast (for reliable detection
of single molecules)
 Uses: pTIRF can be used for a variety of applications, including analysis
of biomolecular interactions, characterizing of antibody-based and
nucleic acid-based assays, real-time microarrays, membrane
biophysics, the dynamics of lipid rafts, and many other.
 Prism-TIRF is so efficient that allows for using even low-cost, moderate
sensitivity CCD cameras for detecting single molecules
 pTIRF Systems are compatible with dry, water- and oil-immersion
objectives. In puTIRF total internal reflection occurs at the interface
between a slide/coverslip and water/aqueous solution. The TIRF prism
and slide are brought in optical contact by a droplet of refractive-index-
matching fluid. For excitation light, the prism and the slide represent
continuous optical medium. A thin layer of aqueous solution and
an optical window separate the TIRF surface from the objective.

11.  Objective Based TIRF Microscopy
Modern TIRF microscopy systems are usually objective-based.
 The light, usually laser light, is directed to the specimen through the
objective, which also collects the emitted fluorescence light.
 It is mandatory that objectives for TIRF microscopy feature an extremely
high numerical aperture (NA) (> 1.45 NA) which allows an angle of
incidence greater than the critical angle.
 The higher the NA of the objective, the lower the possible penetration
depth of the evanescent field, as the angle of incidence of the light can be
more flat.
 Compared to the prism type method, the objective lens method is more
convenient to use as the specimen is well accessible and the angle of
incidence of the laser light can be changed easily.
 By placing the laser spot in different areas in the back focal plane of the
objective, the user can choose the angle of incidence of the laser light and
therefore change the penetration depth of the evanescent wave
Disadvantage: In oTIRF, the excitation light uses the emission channel optics,
which generates large intensity of stray light.
Undesirable stray light excites the bulk of the specimen and
deteriorates the TIRF effect

13.  Lightguide-based TIRF Microscopy
TIRF is a sensible alternative to o-TIRF. It is a flexible geometry available with
open perfusion chambers on inverted microscopes, and closed flow cells
 It features the excitation path, which is naturally independent from the emission
channel
 It yields a superior signal-to-background ratio
 It can be used with dry-, water-, and oil-immersion objectives
 It is a factory-aligned system that is well-suited for multicolor TIRF experiments
 It uses glass or silica coverslips, or Petri dishes with optical bottom
 It platform also implements Shallow Angle Fluorescence Microscopy (SAFM)
 It uses fiber-coupled illuminators connected by ~2-m optical fiber cable
 It provides a reproducible intensity of the evanescent wave
 It can be used with UV excitation, which is not available in objective-TIRF
 It allows for precision control of the penetration depth by using optical traps
 It takes no time to install/uninstall It, and to switch between TIRF, SAFM, micro-
spot excitation, epi-fluorescence, transmittance, and other methods
Uses: lgTIRF is a novel powerful tool for single molecule detection, cell membrane,
real-time microarray, and other studies that require the excitation of fluorescence
confined in space.

14. There are three versions of TIRF excitation light launchers that differ by the geometry of coupling light:
(i) from the end of the coverslip;
(ii) from the top, and
(iii) from the bottom of the coverslip.
Different light launchers are
used for TIRFing :
Petri dishes,
rectangular coverslips, and
other formats of specimen
substrate.

15. Advantagesand Disadvantages Of Different Approaches
 In prism-based TIRF microscopy systems the prism strongly limits the access to the specimen and makes it difficult to e.g.
change media, add drugs or carry out physiological measurements
 Prism-based is good for low magnification and water immersion objectives. But for high magnification and aperture its
better to use Object based method.
 Prism-based and lightguide based are not so stable mechanism like object based.
 Object based works fine with either of collimated laser beam, optical fibres or conventional arc sources whereas prism
based is easiest to use with collimated free laser sources and so is lightguide based.
 The prism-based scheme provides the best signal-to-background ratio , but is difficult to use with open perfusion chamber
on inverted microscopes.
 The objective-based scheme collects the maximal amount of emitted fluorescence, but TIRF effect is compromised due to
large intensity of stray light.
 Lightguide-based geometry (lgTIRF) offers superior signal-to-background ratio and is exceptionally well-suited for
multicolor TIRF, including FRET for single molecule detection, cell membrane, real-time microarray, and other
studies. lgTIRF can be used with dry, water-, and oil-immersion objectives. It provides a reproducible intensity of the
evanescent wave in one experiment and between experiments. lgTIRF can be used with UV excitation, a feature which is
not available in objective-based TIRF

17. EPI TIRF

19. What can we do with it?

28. Applications Of TIRFM :
Membrane research
• Diffusion of molecules (e.g. Sytaxin)
• Kinetic of transporters p
• Membrane fusion
• Cell/Cell interaction
• Cytoskeletal dynamics
Vesicle transport
• Understanding of transport processes
• Localization of molecules
• Endocytosis and Exocytosis
Single molecule detection

29. Objective-based TIRF with NA=1.65
GFD-marked chromaffin granules

30. Objective-based TIRF with NA=1.65
dil-labeled chromaffin cell culture

31. TIRF with an Arc Lamb source:

32. TIRF microscopy in life science research
 TIRF microscopy is an excellent technique for combining kinetic studies with spatial information in live
samples or even in vitro. It is routinely used for investigating molecule trafficking as it occurs e.g. in
cytoskeleton assembly.
 .The rapid image acquisition and the outstanding background elimination in TIRF microscopy provide superb
conditions for observing dynamic events like the recruitment of proteins to the plasma membrane. For
example: It is also possible to track whole organelles like mitochondria using TIRF microscopy. For
investigations in cell-cell interaction, special structures like focal adhesions of cells can easily be visualized
with TIRF microscopy to observe.
 By combining mathematical models (e.g. centroid tracking methods) with the unparalleled signal-to-noise ratio and z-
resolution of TIRF microscopy, subdiffraction-limited localization of single molecules is achievable with a precision of
1 nm. This is possible as the fluorophore excitation by an evanescent wave produces low background fluorescence from
out-of-focus fluorophores, resulting in a low signal-to-noise-ratio in a defined volume of the sample (e.g. the penetration
depth of the evanescent wave multiplied by the area of the field of view). In a conventional lamp-based fluorescence
system all fluorophores in the beam path are simultaneously excited and detected without any information about their z-
position.

33. TIRF microscopy in life science research
 The spatial proximity between fluorescence spots in TIRF images in x- and y-direction is relatively low as the
light emitted from fluorophores from other z-planes is not overlaying the detected signal. If mathematical
models (e.g. centroid tracking methods) are then applied to calculate the center of mass of the detected
fluorescing molecule, subdiffraction-limited localization of single molecules is possible with a precision of
1 nm.
 Another large field of application of TIRF microscopy is the examination of membrane-fusion processes
such as vesicle trafficking. As the evanescent wave only excites fluorophores close to the plasma
membrane, it is possible to monitor the formation of endocytotic vesicles as well as the fusion of
secretory vesicles with the plasma membrane. For this purpose, vesicles can be labeled by tagging
exocytosis cargo proteins with fluorescent proteins like GFP.
 Another important process that takes place at the plasma membrane is cellular signaling (e.g. signaling of G-
protein-coupled receptors). Here e.g. the recruitment or movement of single molecules (e.g. G-proteins) in a
signaling cascade can be observed.

34. Widefield image of tubulin expressing TIRF image of tubulin,YFP,penetration depth 120mm
TIRF image of tubulin,YFP,penetration depth 90mm
TIRF image of tubulin,YFP,penetration depth 70mm

35. Light scattering and interference fringes
1.Scattering of excitation light in the objectives.
2.Scattering by refractive index discontinuities in
the sample.
3.Less excited TIRF produces interference fringes.

36. Scattering in the Objective:
Measurement of actual evanescent field profile

37. Actual intensity profile:
For through the lens TIRF NA=1.45

38. Scattering of barely supercritical
evanescent light by inhomogeneities in the
sample

39. Elimination of interference fringes by
azimuthal spinning

41. Bibliography:
• http://www.leica-microsystems.com/science-lab/applications-of-tirf-
microscopy-in-life-science-research/
• https://www.microscopyu.com/techniques/fluorescence/total-
internal-reflection-fluorescence-tirf-microscopy
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540339/
• https://en.wikipedia.org/wiki/Total_internal_reflection_fluorescence
_microscope#Background