Total Internal Reflection
Definitions, Applications and Uses in Microscopy
Total internal reflection refers to the complete
reflection of a ray of light within a given medium from the surrounding
surface. Here, the ray of light continues to be reflected within the medium
(glass, water etc.) without being refracted off. In order to get a good
understanding of what total internal reflection really is, it is important to
Different media have different densities. For
instance, the densities of water, air and crown glass are 1g/cm3, 1.225kg/m3
and 2.59g/cm3 respectively. As a result, they also have different index of
refraction (water-1.333, air-1.0003, crown glass-1.52).
Because of the
different properties between different media, light travelling from one media
to another not only changes speed (travelling differently in different media)
but is also refracted (bent). The
image below will help understand refraction better.
n1 – water
– angle of incident ray (Incident ray is the ray that strikes the surface)
angle of refracted ray (refracted ray is the ray that is transmitted to the
the ray of light moves from water (incident ray) to air (refracted ray) it is
possible to see in the image above that the ray of light is bent at an angle.
The dotted light in the image forms a 90 degrees angle with the surface between
the two media.
As a result of the differences in density and thus the
refractive index between the two media, the angle of incidence and the angle of
refraction are always different. In this case, the angle of incidence is
smaller (due to higher density of water) while the angle of refraction tends to
* In the diagram, the dotted line
forms a 90 degree angle with the surface between the two media. The angles of
incident and refraction and the angles formed between the rays and the dotted
line (also referred to as the normal)
understood how refraction occurs, it is now possible to understand total
By increasing the incident angle (ᶱ1) in the
image above, the refracted angle is also increased further. As the incident
angle continues to increase, there reaches a point where the refracted angle
forms a 90 degrees angle with the dotted line (where the refracted ray is
parallel with the surface between the two media). This is referred to as the
critical angle. At this point, the refracted ray is not transmitted in to the
second medium (in this case air) but rather forms between the surface of the
water and the air.
If the incident angle is increased further at this point,
then the refracted angle moves from the surface and back to the first media
(water) given that it is now reflected. Here, it is no longer the refracted
ray, but a reflected ray of light. As a result, total internal reflection
Here, it is important to know the difference
between the refracted ray and a reflected ray. Whereas a refracted ray of light
is transmitted from one medium to another (from a dense medium to a less dense
medium) a reflected ray of light is not transmitted to the second medium.
Rather, it is reflected back into the same medium.
* Critical angle - this is the angle of incidence
beyond which the ray of light passing through the denser medium to the surface
of the less dense medium and is no longer refracted, but totally reflected.
* For water and air, the critical angle is 48.6
degrees (angle of incidence or ᶱ1) beyond this angle (say 50) then total internal reflection
The critical angle (θc) is easily obtained using
Snell's law which states that:
n1 sinθi = n2 sin θt
n1 - refractive index of media 1
sin θi - sin of angle of incidence
n2 - refractive index of media 2
sin θt - sin of refracted angle (90degrees at critical angle)
Here, the goal is to find the value of θi when θt if at 90 degrees (sinθt = 1). By obtaining the
angle of incidence, it becomes possible to get the critical angle.
To get the critical angle therefore:
θc = θi = inverse of sin of n2/n1
The refraction index of air is 1.00 while that
of acrylic glass is about 1.50. If a ray of light is travelling from the glass
into air, then getting the critical angle would involve the following
θc = θi = inverse sin of
Following calculation, the critical angle
obtained from this is 41.8 degrees. Here, it is worth noting that this is
simply the critical angle at which the refracted ray forms a 90 degree angle
with the surface between the two medium. By increasing this critical angle from
41.8 to above (say 42, 43 or 45 etc) then total internal reflection occurs.
Total internal reflection is very useful. As a
result, it has a number of applications that include:
Use in right angled isosceles prism - These
prisms can turn light through 90 and 180 degrees based on internal reflection.
right angled isosceles prism are mostly used in various optical instruments.
Diamonds - Essentially, faces of a diamond are cut in a manner that allows
the light entering to fall at a given angle, which in turn results in multiple,
total internal reflections at various angles while at the same time remaining
within the diamond. This is what results in the spackles.
Fiber Optics - An optic fiber is as thick as human hair.
they are composed of fine quality glass or quartz fiber and coated with a thin
layer of material with lower refractive index. This has a number of uses, which
include fiber optic diagnostic tools, fiber optic cables used in
telecommunications as well as in endoscopes where fiber optics are used by
physicians to view inside the body.
Reflectors - reflectors are typically fixed on various
roads where they are used to indicate bending roads etc. They reflect light
from headlights from cars guiding the driver along the path.
Total internal reflection also finds use in:
- Single-lens reflex camera
Uses in Microscopy
Total Internal Reflection Fluorescence Microscopy (TIRF)
TIRF is a type of microscopy that allows for the
imaging of various fluorescent molecules that are located near the glass/water
or glass/specimen interface. This technique was developed in the early 80s and
delivers very high axial resolution, which allows technicians and other users
to be able to observe processes associated with the membrane.
How it Works
To observe the fluorescent molecules, an
evanescent wave is employed for the excitation of fluorophores rather than
using direct illumination through light delivered from such sources as LEDs.
Here, the evanescent field occurs when the incident light is totally reflected
at the interface between the two transparent media with varying refractive
indices. With biological applications, the incident light is laser light while
the interface is usually the glass of a cover slip and film of aqueous solution
between the cover slip and adherent cells.
Here, as the energy of the evanescent field
decreases exponentially with distance to the interface, it is only the
fluorosophores in given proximities to the cover slip that are excited. This
results in images with excellent signal-to-noise ratio since the fluorophores
in the other parts of the cell are barely excited.
With outstanding high axial
resolution of below 100nm obtained through this technique, it becomes possible
to observe such membrane related processes like cell adhesion, binding of
hormones, transport of molecules as well as various exocytic and endocytic
processes like the release and uptake of neurotransmitters.
* Essentially, this technique
was developed in order to restrict background fluorescence and thus increase
signal-to-noise ration in the resulting image. Light is used to create an
evanescent wave (field) at limited range within the sample and beyond an
interface of two substrates with varying refractive index.
Prism-Based and Objective Based Total Internal Reflection
Prism-based and objective-based approaches are two of the main approaches used to
achieve total internal reflection.
prism is attached to the surface of the cover slip. This then directs a focused
beam of light towards the cover slip (medium interface). Here, the prism allows
for the angle of penetrating light to be adjusted to a critical angle. With the
development of the objective-based approach, prism-based total internal
reflection has become less popular.
* One of
the main problems with the prism-based method is that the prism significantly
limits access to the specimen. This makes it rather difficult to do certain
things like changing media, add drugs or other substances or even carry out
the other disadvantages of the prism-based system include:
- It is more complicated to align the laser and use different angles
- It presents laser safety concerns
objective-based total internal reflection, light is directed to the specimen
via the objective (the objective also collects the emitted fluorescence light).
Here, it is essential that the objective have a very high numerical aperture
(NA) of over 1.45. (achieved by using immersion oil or other
specialized liquid immersion media). This allows for an angle
of incidence to be greater than the critical angle in order to form total
The objective-based total internal reflection has been
shown to be more effective compared to the prism approach. This is due to the
fact that the specimen is accessible while the incidence angle can be
easily changed. Here, the user can simply place the laser spot in different
areas in the back focal place of the objective and choose the angle of
incidence of the laser light thereby changing penetration depth of the wave.
objective-based system also has an advantage over prism given that the laser
system is directly coupled in to the microscope itself which helps reduce laser
internal reflection fluorescence microscopy is typically used for imaging
events occurring at the surface of the specimen (cell). However, it works well
with adherent cells given that this technique only illuminates the region near
the cover slip (part that has adhered to the cover slip) and cannot be used for
imaging non-adherent cells.
With some cells, it becomes necessary to coat the
cover slip with such extracellular matrix molecules as polylysine to enhance
cell adherence. It is also important to ensure that the refractive index of the
cell is below the NA of the objective for analysis. If cells have a higher
numerical aperture, as is the case with chromaffin cells, it becomes difficult
to obtain TIRF images using the standard objectives.
technique is particularly ideal for imaging of live cells. In the event that
the cells are fixed, then they have to be mounted in a media with low
refractive index (such as PBS). Here, it might be important to avoid:
- Mounting media that hardens
or contains glycerol given that they have high refractive index which render
Some of the dyes like FM4-64 and DiI that obscure
internal reflection fluorescence microscopy achieves what both widefield and
confocal imaging techniques are unable to through its imaging capabilities. As
such, it has been used for:
- Selectively imaging bursts of actin activities on plasma membrane during enodcytosis
- Mapping the flow of
information in cell signaling networks (where it follows the transport of signaling
proteins from cytosol to plasma membrane)
- Imaging of a large number
of cells simultaneously
- Study of the secretion of
newly synthesized proteins
- Observation of detailed
kinetics of individual exocytotic events
- Live cell imaging while
minimizing photobleaching and phototoxic effects to the entire specimen
to other techniques like confocal microscopy, TIRF microscopy has an advantage
in that technicians can be able to observe smaller sections of a specimen
(100nm compared to 600 mn using confocal microscopy). Moreover, it has been
shown to be the ideal technique for other applications that require restricted
illumination and consequently reduced cell damage.
On the other hand, this
technique has been shown to be more economical during configuration given that
it does not really require complex scanning galvanometer systems. As such, it
can be easily applied to ordinary research grade laboratory microscopes.
Related Articles: Fluorescence Microscopy and Immunofluorescence Microscopy as well understanding the Green Fluorescent Protein.
Return from Total Internal Reflection to MicroscopeMaster Research Home
Axelrod, Daniel (1 November 2001). "Total
Internal Reflection Fluorescence Microscopy in Cell Biology". Traffic. 2
Giancoli, D.C. (2000) Physics for Scientists and
Engineers 3rd edition, Prentice Hall, New Jersey.
Richard Fitzpatrick (2007) Total Internal